Patent application title: INSECTICIDAL PROTEINS
Inventors:
IPC8 Class: AC07K14325FI
USPC Class:
1 1
Class name:
Publication date: 2016-10-20
Patent application number: 20160304568
Abstract:
Compositions and methods for controlling plant pests are disclosed. In
particular, novel engineered hybrid insecticidal proteins (eHIPs) having
toxicity to at least corn rootworm are provided. By fusing unique
combinations of complete or partial variable regions and conserved blocks
of at least two different Bacillus thuringiensis (Bt) Cry proteins or a
modified Cry proteins an eHIP having activity against corn rootworm is
designed. Nucleic acid molecules encoding the novel eHIPs are also
provided. Methods of making the eHIPs and methods of using the eHIPs and
nucleic acids encoding the eHIPs of the invention, for example in
transgenic plants to confer protection from insect damage are also
disclosed.Claims:
1. A method of making an engineered hybrid insecticidal protein (eHIP),
comprising: (a) obtaining a first Bacillus thuringiensis (Bt) Cry protein
consisting essentially of a Cry3A protein or a modified Cry3A protein;
(b) obtaining a second Bacillus thuringiensis (Bt) Cry protein consisting
essentially of a Cry1Aa protein or a Cry1Ab protein; (c) fusing in an
N-terminus to C-terminus direction an N-terminal region of the first
Bacillus thuringiensis (Bt) Cry protein to a C-terminal region of the
second Bt Cry protein, wherein at least one crossover position between
the first and the second Bt Cry protein is located in conserved block 2,
conserved block 3, variable region 4 or conserved block 4; and optionally
inserting i. at the N-terminus a peptidyl fragment; or ii. at the
C-terminus a protoxin tail region of a Bt Cry protein; or iii. both (i)
and (ii), wherein the engineered hybrid insecticidal protein has at least
80% identity to SEQ ID NO:64 and has activity against western corn
rootworm (Diabrotica virgifera).
2. A method of making an engineered hybrid insecticidal protein (eHIP), comprising: (a) obtaining a first nucleic acid that encodes a Bacillus thuringiensis (Bt) Cry3A protein or a modified Cry3A protein; (b) obtaining a second nucleic acid that encodes a Bt Cry1Aa protein or a Bt Cry1Ab protein; (c) fusing in an 5' to 3' direction a 5' portion of the first nucleic acid that encodes an N-terminal region of the Bt Cry3A protein or the modified Cry3A protein to a 3' portion of the second nucleic acid that encodes a C-terminal region of the Bt Cry1Aa protein or the Bt Cry1Ab protein, wherein at least one crossover position between the first and the second nucleic acids is located in the nucleotides that encode conserved block 2, conserved block 3, variable region 4 or conserved block 4 to make a hybrid nucleic acid that encodes an eHIP that has at least 80% identity to SEQ ID NO:64 and has activity against western corn rootworm; and optionally fusing i. to the 5' end of the hybrid nucleic acid a nucleotide sequence that encodes a peptidyl fragment resulting in a 5' extension; or ii. to the 3' end of the hybrid nucleic acid a nucleotide sequence that encodes a protoxin tail region of a Bt Cry protein resulting in a 3' extension; or iii. both (i) and (ii); (d) inserting the hybrid nucleic acid with or without one or both of the 5' or 3' extensions into an expression cassette; and (e) transforming the expression cassette into a host cell, resulting in the host cell producing an engineered hybrid insecticidal protein that has at least 80% identity to SEQ ID NO:64 and has activity against western corn rootworm (Diabrotica virgifera).
3. The method according to either of claims 1 or 2, wherein the protoxin tail region is from a Cry1Aa or Cry1Ab.
4. The method according to claim 3, wherein said protoxin tail region comprises at least 38 amino acids.
5. The method according to claim 4, wherein the protoxin tail region comprises an amino acid sequence that corresponds to amino acids 611-648 of SEQ ID NO: 72.
6. The method according to claim 5, wherein the protoxin tail region comprises amino acids 611-648 of SEQ ID NO: 72.
7. The method according to either of claims 1 or 2, wherein the peptidyl fragment comprises the amino acid sequence YDGRQQHRG (SEQ ID NO: 132) or the amino acid sequence TSNGRQCAGIRP (SEQ ID NO: 133).
8. The method according to claim 7, wherein the peptidyl fragment is selected from the group consisting of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132.
9. The method according to either of claims 1 or 2, wherein the Cry3A is a Cry3Aa or a modified Cry3Aa.
10. The method according to claim 9, wherein (a) the Cry3Aa comprises SEQ ID NO:68 or SEQ ID NO:135, or (b) the modified Cry3Aa comprises SEQ ID NO:70 or SEQ ID NO:72.
11. The method according to either of claims 1 or 2, wherein the crossover position is located in conserved block 3 immediately following Ser450, Phe454, or Leu468 of SEQ ID NO:70.
12. The method according to either of claims 1 or 2, wherein the eHIP comprises at least two crossover positions between an amino acid sequence from a Cry3A protein and an amino acid sequence from a Cry1Aa or a Cry1Ab protein.
13. The method according to claim 12, wherein (a) the first crossover position is located in conserved block 2 and the second crossover position is located in conserved block 3; or (b) the first crossover position is located in conserved block 3 and the second crossover position is located in conserved block 4.
14. The method according to claim 13, wherein the Cry3A is a Cry3Aa or a modified Cry3Aa.
15. The method according to claim 14, wherein (a) the first crossover position between Cry3Aa and Cry1Aa or Cry1Ab or modified Cry3Aa and Cry1Aa or Cry1Ab is located in conserved block 2 immediately following an amino acid corresponding to Asp232 of SEQ ID NO: 70 and a second crossover position between Cry1Aa or Cry1Ab and Cry3Aa or Cry1Aa or Cry1Ab and modified Cry3Aa is located in conserved block 3 immediately following an amino acid corresponding to Leu476 of SEQ ID NO: 72; or (b) the first crossover position between Cry3Aa and Cry1Aa or Cry1Ab or modified Cry3Aa and Cry1Aa or Cry1Ab is located in conserved block 3 immediately following an amino acid corresponding to Leu468 of SEQ ID NO: 70 and the second crossover position between Cry1Aa or Cry1Ab and Cry3Aa or Cry1Aa or Cry1Ab and modified Cry3Aa is located in conserved block 4 immediately following an amino acid corresponding to Ile527 of SEQ ID NO: 72.
16. The method according to claim 14, wherein (a) the first crossover position between Cry3Aa and Cry1Aa or Cry1Ab or modified Cry3Aa and Cry1Aa or Cry1Ab is located in conserved block 2 immediately following Asp232 of SEQ ID NO: 70 and the second crossover position between Cry1Aa or Cry1Ab and Cry3Aa or Cry1Aa or Cry1Ab and modified Cry3Aa is located in conserved block 3 immediately following Leu476 of SEQ ID NO: 72; or (b) the first crossover position between Cry3Aa and Cry1Aa or Cry1Ab or modified Cry3Aa and Cry1Aa or Cry1Ab is located in conserved block 3 immediately following Leu468 of SEQ ID NO: 70 and the second crossover position between Cry1Aa or Cry1Ab and Cry3Aa or Cry1Aa or Cry1Ab and modified Cry3Aa is located in conserved block 4 immediately following Ile527 of SEQ ID NO: 72.
17. The method according to either of claims 1 or 2, wherein the insecticidal protein is additionally active against northern corn rootworm (Diabrotica undecimpunctata) or Mexican corn rootworm (D. zea).
18. The method according to either of claims 1 or 2, wherein the engineered hybrid insecticidal protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 62; SEQ ID NO: 64, SEQ ID NO: 147, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 160.
19. The method according to claim 2, wherein the engineered hybrid nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 61; SEQ ID NO: 63, SEQ ID NO: 146, SEQ ID NO: 152, SEQ ID NO: 154 and SEQ ID NO: 158.
Description:
CROSS-REFERENCE
[0001] This application is a divisional of, and claims priority to, U.S. patent application Ser. No. 13/623,921, filed Sep. 21, 2012, which is a divisional of U.S. patent application Ser. No. 12/529,246, now U.S. Pat. No. 8,309,516, filed Aug. 31, 2009, which is a .sctn.371 of PCT/US08/58182, filed Mar. 26, 2008, which claims priority to U.S. Provisional Application 60/920,493, filed Mar. 28, 2007, all of which are incorporated herein by reference in their entirety.
BACKGROUND
[0002] The present invention relates to the fields of protein engineering, plant molecular biology and pest control. More particularly the invention relates to novel engineered hybrid proteins having insecticidal activity, nucleic acids whose expression results in the insecticidal proteins, and methods of making and methods of using the insecticidal proteins and corresponding nucleic acids to control insects.
[0003] Insect pests are a major cause of crop losses. In the US alone, billions of dollars are lost every year due to infestation by various genera of insects. In addition to losses in field crops, insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and they are a nuisance to gardeners and homeowners.
[0004] Species of corn rootworm are considered to be the most destructive corn pests. In the United States, the three important species are Diabrotica virgifera virgifera, the western corn rootworm, D. longicornis barberi, the northern corn rootworm and D. undecimpunctata howardi, the southern corn rootworm. Only western and northern corn rootworms are considered primary pests of corn in the US Corn Belt. An important corn rootworm pest in the Southern US is the Mexican corn rootworm, Diabrotica virgifera zeae. Corn rootworm larvae cause the most substantial plant damage by feeding almost exclusively on corn roots. This injury has been shown to increase plant lodging, to reduce grain yield and vegetative yield as well as alter the nutrient content of the grain. Larval feeding also causes indirect effects on corn by opening avenues through the roots for bacterial and fungal infections which lead to root and stalk rot diseases. Adult corn rootworms are active in cornfields in late summer where they feed on ears, silks and pollen, thus interfering with normal pollination.
[0005] Corn rootworms are mainly controlled by intensive applications of chemical pesticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or cause death. Good corn rootworm control can thus be reached, but these chemicals can sometimes also affect other, beneficial organisms. Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect varieties. Yet another problem is due to the fact that corn rootworm larvae feed underground thus making it difficult to apply rescue treatments of insecticides. Therefore, most insecticide applications are made prophylactically at the time of planting. This practice results in a large environmental burden. This has been partially alleviated by various farm management practices, but there is an increasing need for alternative pest control mechanisms.
[0006] Biological pest control agents, such as Bacillus thuringiensis (Bt) strains expressing pesticidal toxins like .delta.-endotoxins (delta-endotoxins; also called crystal toxins or Cry proteins), have also been applied to crop plants with satisfactory results against primarily lepidopteran insect pests. The .delta.-endotoxins are proteins held within a crystalline matrix that are known to possess insecticidal activity when ingested by certain insects. The various .delta.-endotoxins have been classified based upon their spectrum of activity and sequence homology. Prior to 1990, the major classes were defined by their spectrum of activity with the Cry1 proteins active against Lepidoptera (moths and butterflies), Cry2 proteins active against both Lepidoptera and Diptera (flies and mosquitoes), Cry3 proteins active against Coleoptera (beetles) and Cry4 proteins active against Diptera (Hofte & Whitely, 1989, Microbiol. Rev. 53:242-255). A new nomenclature was developed in 1998 which systematically classifies the Cry proteins based on amino acid sequence homology rather than insect target specificities (Crickmore et al. 1998, Microbiol. Molec. Biol. Rev. 62:807-813).
[0007] The spectrum of insecticidal activity of an individual .delta.-endotoxin from Bt is quite narrow, with a given .delta.-endotoxin being active against only a few species within an Order. For instance, a Cry3A toxin is known to be very toxic to the Colorado potato beetle, Leptinotarsa decemlineata, but has very little or no toxicity to related beetles in the genus Diabrotica (Johnson et al., 1993, J. Econ. Entomol. 86:330-333). According to Slaney et al. (1992, Insect Biochem. Molec. Biol. 22:9-18) a Cry3A toxin is at least 2000 times less toxic to southern corn rootworm larvae than to the Colorado potato beetle. It is also known that Cry3A has little or no toxicity to the western corn rootworm or northern corn rootworm.
[0008] Specificity of the .delta.-endotoxins is the result of the efficiency of the various steps involved in producing an active toxic protein and its subsequent interaction with the epithelial cells in an insect mid-gut. To be insecticidal, most known .delta.-endotoxins must first be ingested by the insect and proteolytically activated to form an active toxin. Activation of the insecticidal crystal (Cry) proteins is a multi-step process. After ingestion, the crystals must first be solubilized in the insect gut. Once solubilized, the .delta.-endotoxins are activated by specific proteolytic cleavages. The proteases in the insect gut can play a role in specificity by determining where the .delta.-endotoxin is processed. Once the .delta.-endotoxin has been solubilized and processed it binds to specific receptors on the surface of the insects' mid-gut epithelium and subsequently integrates into the lipid bilayer of the brush border membrane. Ion channels then form disrupting the normal function of the midgut eventually leading to the death of the insect.
[0009] In Lepidoptera, which have alkaline pH guts, gut proteases process .delta.-endotoxins for example, Cry1Aa, Cry1Ab, Cry1Ac, Cry1B and Cry1F, from 130-140 kDa protoxins to toxic proteins of approximately 60-70 kDa. Processing of the protoxin to toxin has been reported to proceed by removal of both N- and C-terminal amino acids with the exact location of processing being dependent on the specific .delta.-endotoxin and the specific insect gut fluids involved (Ogiwara et al., 1992, J. Invert. Pathol. 60:121-126). Thus activation requires that the entire C-terminal protoxin tail region be cleaved off. This proteolytic activation of a .delta.-endotoxin can play a significant role in determining its specificity.
[0010] Coleopteran insects have guts that are more neutral to acidic and coleopteran-specific .delta.-endotoxins are similar to the size of the activated lepidopteran-specific toxins. Therefore, the processing of coleopteran-specific .delta.-endotoxins was formerly considered unnecessary for toxicity. However, data suggests that coleopteran-active .delta.-endotoxins are solubilized and proteolyzed to smaller toxic polypeptides. A 73 kDa Cry3A .delta.-endotoxin protein produced by B. thuringiensis var. tenebrionis is readily processed in the bacterium at the N-terminus, losing 49-57 residues during or after crystal formation to produce the commonly isolated 67 kDa form (Carroll et al., 1989, Biochem. J. 261:99-105). McPherson et al., (1988, Biotechnology 6:61-66) also demonstrated that a native cry3A coding sequence contains two functional translational initiation codons in the same reading frame, one coding for a 73 kDa protein and the other coding for a 67 kDa protein starting at Met-1 and Met-48 respectively, of the deduced amino acid sequence. Both proteins then can be considered naturally occurring full-length Cry3A proteins.
[0011] As more knowledge has been gained as to how the .delta.-endotoxins function, attempts to engineer .delta.-endotoxins to have new activities have increased. Engineering .delta.-endotoxins was made more possible by solving the three dimensional structure of Cry3A in 1991 (Li et al., 1991, Nature 353:815-821). Li et al. determined that a Cry3A protein has three structural domains: the N-terminal domain I, from residues 58-290, consists of 7 .alpha.-helices, domain II, from residues 291-500, contains three .beta.-sheets in a so-called Greek key-conformation, and the C-terminal domain III, from residues 501-644, is a .beta.-sandwich in a so-called jellyroll conformation. The three dimensional structure for the lepidopteran active Cry1Aa toxin has also been solved (Grochulski et al., 1995, J. Mol. Biol. 254:447-464). The Cry1Aa toxin also has three domains: the N-terminal domain I, from residues 33-253, domain II from residues 265-461, and domain III from residues 463-609 with an additional outer strand in one of the .beta.-sheets from by residues 254-264. If the Cry3A and Cry1Aa structures are projected on other Cry1 sequences, domain I runs from about amino acid residue 28 to 260, domain II from about 260 to 460 and domain III from about 460 to 600. See, Nakamura et al., Agric. Biol. Chem. 54(3): 715-724 (1990); Li et al., Nature 353: 815-821 (1991); Ge et al., J. Biol. Chem. 266(27): 17954-17958 (1991); and Honee et al., Mol. Microbiol. 5(11): 2799-2806 (1991); each of which are incorporated herein by reference. Thus, it is now known that based on amino acid sequence homology, known Bt .delta.-endotoxins have a similar three-dimensional structure comprising three domains.
[0012] The toxin portions of Bt Cry proteins are also characterized by having five conserved blocks across their amino acid sequence numbered CB1 to CB5 from N-terminus to C-terminus, respectively (Hofte & Whiteley, supra). Conserved block 1 (CB1) comprises approximately 29 amino acids, conserved block 2 (CB2) comprises approximately 67 amino acids, conserved block 3 (CB3) comprises approximately 48 amino acids, conserved block 4 (CB4) comprises approximately 10 amino acids and conserved block 5 (CB5) comprises approximately 12 amino acids. The sequences before and after these five conserved blocks are highly variable and thus are designated the "variable regions," V1-V6. Domain I of a Bt .delta.-endotoxin typically comprises variable region 1, conserved block 1, variable region 2, and the N-terminal 52 amino acids of conserved block 2. Domain II typically comprises approximately the C-terminal 15 amino acids of conserved block 2, variable region 3, and approximately the N-terminal 10 amino acids of conserved block 3. Domain III typically comprises approximately the C-terminal 38 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, and conserved block 5. The Cry1 lepidopteran active toxins, among other delta-endotoxins, have a variable region 6 with approximately 1-3 amino acids lying within domain III.
[0013] Many Bt strains and .delta.-endotoxins are active against different insect species and nematodes. However, relatively few of these strains and toxins have activity against coleopteran insects. Further, most of the now known native coleopteran-active .delta.-endotoxins, for example Cry3A, Cry3B, Cry3C, Cry7A, Cry8A, Cry8B, and Cry8C, have insufficient oral toxicity against corn rootworm to provide adequate field control if delivered, for example, through microbes or transgenic plants. Therefore, other approaches for producing novel toxins active against corn rootworm need to be explored.
[0014] Lepidopteran-active .delta.-endotoxins have been engineered in attempts to improve specific activity or to broaden the spectrum of insecticidal activity. For example, the silk moth (Bombyx mori) specificity domain from a Cry1Aa protein was moved to a Cry1Ac protein, thus imparting a new insecticidal activity to the resulting hybrid Bt protein (Ge et al. 1989, PNAS 86: 4037-4041). Also, Bosch et al. 1998 (U.S. Pat. No. 5,736,131, incorporated herein by reference) describes Bacillus thuringiens is hybrid toxins comprising at their C-terminus domain III of a first Cry protein and at its N-terminus domains I and II of a second Cry protein. Such hybrid toxins were shown to have altered insecticidal specificity against lepidopteran insects. For example, the H04 hybrid toxin, which is also described in De Maagd et al., Appl. Environ. Microbiol. 62(5): 1537-1543 (1996), comprises at its N-terminus domains I and II of a Cry1Ab and at its C-terminus domain III of a Cry1C. H04 is reportedly highly toxic to the lepidopteran insect Spodoptera exigua (beet armyworm) compared with the parental Cry1Ab toxin and significantly more toxic than the Cry1C parental toxin. It has also been shown that substitution of domain III of toxins, which are not active against the beet armyworm such as Cry1E and Cry1Ab, by domain III of Cry1C, which is active against beet armyworm, can produce hybrid toxins that are active against this insect. All of the hybrids disclosed in Bosch et al. use domains from lepidopteran active Cry proteins to make new toxins with lepidopteran activity. The results do suggest that domain III of Cry1C is an important determinant of specificity for beet armyworm. See also, Bosch et al., FEMS Microbiology Letters 118: 129-134 (1994); Bosch et al., Bio/Technology 12: 915-918 (1994); De Maagd et al., Appl. Environ. Microbiol. 62(8): 2753-2757 (1996); and De Maagd et al., Mol. Microbiol. 31(2): 463-471 (1999); each of which is incorporated herein by reference.
[0015] Several attempts at engineering the coleopteran-active 6-endotoxins have been reported. Chen and Stacy (U.S. Pat. No. 7,030,295, herein incorporated by reference) successfully created a corn rootworm active toxin by inserting a non-naturally occurring protease recognition site in domain I, domain III, or both domains I and III of a Cry3A protein. One of the resulting modified Cry3A proteins, designated Cry3A055, having a protease recognition site inserted in domain I, was active against several species of Diabrotica. Van Rie et al., 1997, (U.S. Pat. No. 5,659,123) engineered Cry3A by randomly replacing amino acids, thought to be important in solvent accessibility, in domain II with the amino acid alanine. Several of these random replacements confined to domain II were reportedly involved in increased western corn rootworm toxicity. However, others have shown that some alanine replacements in domain II of Cry3A result in disruption of receptor binding or structural instability (Wu and Dean, 1996, J. Mol. Biol. 255: 628-640). English et al., 1999, (Intl. Pat. Appl. Publ. No. WO 99/31248) reported amino acid substitutions in Cry3Bb that caused increases in toxicity to southern and western corn rootworm. However, of the 35 reported Cry3Bb mutants, only three, with mutations primarily in domain II and the domain I-domain II interface, were active against western corn rootworm. Further, the variation in toxicity of wild-type Cry3Bb against western corn rootworm in the same assays appear to be greater than any of the differences between the mutated Cry3Bb toxins and the wild-type Cry3Bb. Shadenkov et al. (1993, Mol. Biol. 27:586-591), made a hybrid protein by fusing amino acids 48-565 of a Cry3A protein to amino acids 526-725 of a Cry1Aa protein. Therefore, the cross-over between Cry3A and Cry1Aa sequence was in conserved block 4 located in domain III. Cry3A is very active against the Colorado potato beetle (Leptinotarsa decemlineata). However, the hybrid protein disclosed by Shadenkov et al. was not active against Colorado potato beetle even though more than 75% of the hybrid protein was made up of Cry3A sequence. Thus, the addition of only 25% of Cry1Aa sequence destroyed activity against a coleopteran insect that the parent Cry3A was active against. This suggests that hybrid proteins made by fusing portions of a coleopteran-active Cry protein, e.g. Cry3A, and a lepidopteran-active Cry protein, e.g. Cry1A, would not have activity against coleopteran insects, particularly a coleopteran insect that is not naturally susceptible to Cry3A like corn rootworm.
[0016] In view of the above discussion, there remains a need to design new and effective pest control agents that provide an economic benefit to farmers and that are environmentally acceptable. Particularly needed are proteins that are toxic to Diabrotica species, a major pest of corn, that have a different mode of action than existing insect control products as a way to mitigate the development of resistance. Furthermore, delivery of control agents through products that minimize the burden on the environment, as through transgenic plants, are desirable.
SUMMARY
[0017] In view of these needs, it is an object of the present invention to provide novel engineered hybrid insecticidal proteins (eHIPs). Such novel eHIPs are made by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins and optionally including a protoxin tail region from a Bt Cry protein at the C-terminus or an N-terminal peptidyl fragment or both. For example, without limitation, by combining complete or partial variable regions and conserved blocks from a first Cry protein that has coleopteran activity with complete or partial variable regions and conserved blocks from a second Cry protein that has lepidopteran activity, and is different from the first Cry protein, and optionally including a protoxin tail region from a lepidopteran active Bt Cry protein, or an N-terminal peptidyl fragment or both, new engineered hybrid insecticidal proteins are created that have activity against a spectrum of insects different from the first or second parent Cry proteins or both. Such novel eHIPs may comprise complete or partial variable regions, conserved blocks or domains from a modified Cry3A protein and a Cry protein different from the modified Cry3A protein. The peptidyl fragment may confer insecticidal activity upon the eHIP, or may increase the insecticidal activity of the eHIP over an eHIP without the peptidyl fragment, or make the eHIP more stable than an eHIP without the peptidyl fragment. The eHIPs of the invention have surprising and unexpected toxicity to corn rootworm (Diabrotica sp.). The invention is further drawn to nucleic acids that encode the eHIPs or which is complementary to one which hybridizes under stringent conditions with the recombinant hybrid nucleic acids according to the invention.
[0018] Also included in the invention are vectors containing such recombinant (or complementary thereto) nucleic acids; a plant or micro-organism which includes, and enables expression of such nucleic acids; plants transformed with such nucleic acids, for example transgenic corn plants; the progeny of such plants which contain the nucleic acids stably incorporated and hereditable in a Mendelian manner, and/or the seeds of such plants and such progeny.
[0019] The invention also includes compositions and formulations containing the eHIPs, which are capable of inhibiting the ability of insect pests to survive, grow and reproduce, or of limiting insect-related damage or loss to crop plants, for example applying the eHIPs or compositions or formulations to insect-infested areas, or to prophylactically treat insect-susceptible areas or plants to confer protection against the insect pests.
[0020] The invention is further drawn to a method of making the eHIPs and to methods of using the nucleic acids, for example in microorganisms to control insects or in transgenic plants to confer protection from insect damage.
[0021] The novel eHIPs described herein are highly active against insects. For example, the eHIPs of the present invention can be used to control economically important insect pests such as western corn rootworm (Diabrotica virgifera virgifera) northern corn rootworm (D. longicornis barberi) and Mexican corn rootworm (D. virgifera zeae). Certain eHIPs may also be used to control European corn borer (Ostrinia nubilalis) and other lepidopteran insects. The eHIPs can be used singly or in combination with other insect control strategies to confer maximal pest control efficiency with minimal environmental impact.
[0022] Other aspects and advantages of the present invention will become apparent to those skilled in the art from a study of the following description of the invention and non-limiting examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0024] FIG. 1A-1E shows a sequence alignment of some eHIP embodiments with parental Cry proteins or modified Cry 3A used to construct the eHIPs, including, a Cry3A, Cry1Ab, and Cry3A055, and indicates percent identity. N-terminal peptidyl fragments are underlined. The 5 conserved blocks are labeled CB1-CB5. Location of junctions between domains I, II and III are designated by a vertical dashed line. A cathepsin G protease recognition sequence, AAPF, is in bold.
[0025] FIG. 2A-2E shows an alignment of eHIP embodiments that are active against at least western corn rootworm and indicates percent identity compared to the 8AF eHIP. N-terminal peptidyl fragments are single underlined. C-terminal protoxin tail regions are double underlined. The 5 conserved blocks are labeled CB1-CB5. Locations of junctions between domains I, II and III are indicated by ".dwnarw." and labeled accordingly. Locations of crossover positions are indicted by a ".diamond-solid.". A cathepsin G protease recognition sequence, AAPF, is in bold.
[0026] FIG. 3 shows a map of recombinant vector 12207 used to transform corn comprising an expression cassette with a maize ubiquitin promoter operably linked to a FRCG coding sequence operably linked to a NOS terminator.
[0027] FIG. 4 shows a map of recombinant vector 12161 used to transform corn comprising an expression cassette with a maize ubiquitin promoter operably linked to a FR8a coding sequence operably linked to a NOS terminator.
[0028] FIG. 5 shows a map of recombinant vector 12208 used to transform corn comprising an expression cassette with a cestrum yellow leaf curling virus promoter (cmp) operably linked to a FRCG coding sequence operably linked to a NOS terminator.
[0029] FIG. 6 shows a map of recombinant vector 12274 used to transform corn comprising an expression cassette with a cestrum yellow leaf curling virus promoter (cmp) operably linked to a FR8a coding sequence operably linked to a NOS terminator.
[0030] FIG. 7 shows a map of recombinant vector 12473 used to transform corn comprising an expression cassette with a maize ubiquitin promoter (ubi) operably linked to a FRD3 coding sequence operably linked to a NOS terminator.
[0031] FIG. 8 shows a map of recombinant vector 12474 used to transform corn comprising an expression cassette with a cestrum yellow leaf curling virus promoter (cmp) operably linked to a FRD3 coding sequence operably linked to a NOS terminator.
BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
[0032] SEQ ID NO: 1 is the 2OL-8a nucleotide sequence.
[0033] SEQ ID NO: 2 is the 2OL-8a encoded by SEQ ID NO: 1.
[0034] SEQ ID NO: 3 is the FR8a nucleotide sequence.
[0035] SEQ ID NO: 4 is the FR8a encoded by SEQ ID NO: 3.
[0036] SEQ ID NO: 5 is the FRCG nucleotide sequence.
[0037] SEQ ID NO: 6 is the FRCG encoded by SEQ ID NO: 5.
[0038] SEQ ID NO: 7 is the FR8a-9F nucleotide sequence.
[0039] SEQ ID NO: 8 is the FR8a-9F encoded by SEQ ID NO: 7.
[0040] SEQ ID NO: 9 is the FR-9F-catg nucleotide sequence.
[0041] SEQ ID NO: 10 is the FR-9F-catg encoded by SEQ ID NO: 9.
[0042] SEQ ID NO: 11 is the FR8a-12AA nucleotide sequence.
[0043] SEQ ID NO: 12 is the FR8a-12AA encoded by SEQ ID NO: 11.
[0044] SEQ ID NO: 13 is the WR-9mut nucleotide sequence.
[0045] SEQ ID NO: 14 is the WR-9mut encoded by SEQ ID NO: 13.
[0046] SEQ ID NO: 15 is the FRD3 nucleotide sequence.
[0047] SEQ ID NO: 16 is the FRD3 encoded by SEQ ID NO: 15.
[0048] SEQ ID NO: 17 is the FR-12-cg-dm3 nucleotide sequence.
[0049] SEQ ID NO: 18 is the FR-12-cg-dm3 encoded by SEQ ID NO: 17.
[0050] SEQ ID NO: 19 is the 9F-cg-de6 nucleotide sequence.
[0051] SEQ ID NO: 20 is the 9F-cg-del6 encoded by SEQ ID NO: 19.
[0052] SEQ ID NO: 21 is the FR-cg-dm3 nucleotide sequence.
[0053] SEQ ID NO: 22 is the FR-cg-dm3 encoded by SEQ ID NO: 21.
[0054] SEQ ID NO: 23 is the 9F-cg-dm3 nucleotide sequence.
[0055] SEQ ID NO: 24 is the 9F-cg-dm3 encoded by SEQ ID NO:23.
[0056] SEQ ID NO: 25 is the B8a nucleotide sequence.
[0057] SEQ ID NO: 26 is the B8a encoded by SEQ ID NO: 25.
[0058] SEQ ID NO: 27 is the 5*B8a nucleotide sequence.
[0059] SEQ ID NO: 28 is the 5*B8a encoded by SEQ ID NO: 27.
[0060] SEQ ID NO: 29 is the V3A nucleotide sequence.
[0061] SEQ ID NO: 30 is the V3A encoded by SEQ ID NO: 29.
[0062] SEQ ID NO: 31 is the V4F nucleotide sequence.
[0063] SEQ ID NO: 32 is the V4F encoded by SEQ ID NO: 31.
[0064] SEQ ID NO: 33 is the 5*V4F nucleotide sequence.
[0065] SEQ ID NO: 34 is the 5*V4F encoded by SEQ ID NO: 33.
[0066] SEQ ID NO: 35 is the 2OL-7 nucleotide sequence.
[0067] SEQ ID NO: 36 is the 2OL-7 encoded by SEQ ID NO: 35.
[0068] SEQ ID NO: 37 is the T7-2OL-7 nucleotide sequence.
[0069] SEQ ID NO: 38 is the T7-2OL-7 encoded by SEQ ID NO: 37.
[0070] SEQ ID NO: 39 is the 5*2OL-7 nucleotide sequence.
[0071] SEQ ID NO: 40 is the 5*2OL-7 encoded by SEQ ID NO: 39.
[0072] SEQ ID NO: 41 is the 2OL-10 nucleotide sequence.
[0073] SEQ ID NO: 42 is the 2OL-10 encoded by SEQ ID NO: 41.
[0074] SEQ ID NO: 43 is the 5*2OL-10 nucleotide sequence.
[0075] SEQ ID NO: 44 is the 5*2OL-10 encoded by SEQ ID NO: 43.
[0076] SEQ ID NO: 45 is the 2OL-12A nucleotide sequence.
[0077] SEQ ID NO: 46 is the 2OL-12A encoded by SEQ ID NO: 45.
[0078] SEQ ID NO: 47 is the 2OL-13 nucleotide sequence.
[0079] SEQ ID NO: 48 is the 2Ol-13 encoded by SEQ ID NO: 47.
[0080] SEQ ID NO: 49 is the V5&6 nucleotide sequence.
[0081] SEQ ID NO: 50 is the V5&6 encoded by SEQ ID NO: 49.
[0082] SEQ ID NO: 51 is the 5*V5&6 nucleotide sequence.
[0083] SEQ ID NO: 52 is the 5*V5&6 encoded by SEQ ID NO: 51.
[0084] SEQ ID NO: 53 is the 88A-dm3 nucleotide sequence.
[0085] SEQ ID NO: 54 is the 88A-dm3 encoded by SEQ ID NO: 53.
[0086] SEQ ID NO: 55 is the FR(1Fa) nucleotide sequence.
[0087] SEQ ID NO: 56 is the FR(1Fa) encoded by SEQ ID NO: 55.
[0088] SEQ ID NO: 57 is the FR(1Ac) nucleotide sequence.
[0089] SEQ ID NO: 58 is the FR(1Ac) encoded by SEQ ID NO: 57.
[0090] SEQ ID NO: 59 is the FR(1Ia) nucleotide sequence.
[0091] SEQ ID NO: 60 is the FR(1Ia) encoded by SEQ ID NO: 59.
[0092] SEQ ID NO: 61 is the DM23A nucleotide sequence.
[0093] SEQ ID NO: 62 is the DM23A encoded by SEQ ID NO: 61.
[0094] SEQ ID NO: 63 is the 8AF nucleotide sequence.
[0095] SEQ ID NO: 64 is the 8AF encoded by SEQ ID NO: 63.
[0096] SEQ ID NO: 65 is the 5*cry3A055 nucleotide sequence.
[0097] SEQ ID NO: 66 is the 5*Cry3A055 encoded by SEQ ID NO: 65.
[0098] SEQ ID NO: 67 is a maize optimized cry3A nucleotide sequence.
[0099] SEQ ID NO: 68 is the Cry3A encoded by SEQ ID NO: 67.
[0100] SEQ ID NO: 69 is the cry3A055 nucleotide sequence.
[0101] SEQ ID NO: 70 is the Cry3A055 encoded by SEQ ID NO: 69.
[0102] SEQ ID NO: 71 is a maize optimized cry1Ab nucleotide sequence.
[0103] SEQ ID NO: 72 is the Cry1Ab encoded by SEQ ID NO: 71.
[0104] SEQ ID NO: 73 is a maize optimized crylBa nucleotide sequence.
[0105] SEQ ID NO: 74 is the Cry1Ba encoded by SEQ ID NO: 73.
[0106] SEQ ID NO: 75 is a maize optimized cry1Fa nucleotide sequence.
[0107] SEQ ID NO: 76 is the Cry1Fa encoded by SEQ ID NO: 75.
[0108] SEQ ID NO: 77 is a cry8Aa nucleotide sequence.
[0109] SEQ ID NO: 78 is the Cry8Aa encoded by SEQ ID NO: 77.
[0110] SEQ ID NO: 79 is a cry1Ac nucleotide sequence.
[0111] SEQ ID NO: 80 is the Cry1Ac encoded by SEQ ID NO: 79.
[0112] SEQ ID NO: 81 is a cry1Ia nucleotide sequence.
[0113] SEQ ID NO: 82 is the Cry1Ia encoded by SEQ ID NO: 81.
[0114] SEQ ID NOs 83-125 are primer sequences useful in the present invention.
[0115] SEQ ID NOs 126-134 are N-terminal peptidyl fragments.
[0116] SEQ ID NO: 135 is a full-length Cry3A protein.
[0117] SEQ ID NO: 136-143 are primer sequences useful in the present invention.
[0118] SEQ ID NO: 144 is the T7-8AF coding sequence.
[0119] SEQ ID NO: 145 is the T7-8AF encoded by ASEQ ID NO: 144.
[0120] SEQ ID NO: 146 is the -catG8AF coding sequence.
[0121] SEQ ID NO: 147 is the -CatG8AF encoded by SEQ ID NO: 146.
[0122] SEQ ID NO: 148 is the 8AFdm3 coding sequence.
[0123] SEQ ID NO: 149 is the 8AFdm3 encoded by SEQ ID NO: 148.
[0124] SEQ ID NO: 150 is the 8AFlongdm3 coding sequence.
[0125] SEQ ID NO: 151 is the 8AFlongdm3 encoded by SEQ ID NO: 150.
[0126] SEQ ID NO: 152 is the cap8AFdm3 coding sequence.
[0127] SEQ ID NO: 153 is the cap8AFdm3 encoded by SEQ ID NO: 152.
[0128] SEQ ID NO: 154 is the 8AFdm3T coding sequence.
[0129] SEQ ID NO: 155 is the 8AFdm3T encoded by SEQ ID NO: 154.
[0130] SEQ ID NO: 156 is the 8AFlongdm3T coding sequence.
[0131] SEQ ID NO: 157 is the 8AFlongdm3T encoded by SEQ ID NO: 156.
[0132] SEQ ID NO: 158 is the cap8AFdm3T coding sequence.
[0133] SEQ ID NO: 159 is the cap8AFdm3T encoded by SEQ ID NO: 158.
[0134] SEQ ID NO: 160 is the FR8a+34 eHIP.
DEFINITIONS
[0135] For clarity, certain terms used in the specification are defined and presented as follows:
[0136] "Activity" of the eHIPs of the invention is meant that the eHIPs function as orally active insect control agents, have a toxic effect, or are able to disrupt or deter insect feeding, which may or may not cause death of the insect. When an eHIP of the invention is delivered to the insect, the result is typically death of the insect, or the insect does not feed upon the source that makes the eHIP available to the insect.
[0137] "Associated with/operatively linked" refer to two nucleic acids that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence that codes for RNA or a protein if the two sequences are operatively linked, or situated such that the regulatory DNA sequence will affect the expression level of the coding or structural DNA sequence.
[0138] In the context of the present invention, a "chimeric insecticidal protein" (CIP) is an insecticidal protein comprising a peptidyl fragment fused to the N-terminus of an eHIP. The peptidyl fragment may confer insecticidal activity upon the eHIP, may increase the insecticidal activity of the eHIP over an eHIP without the peptidyl fragment, or may make the eHIP more stable than an eHIP without the peptidyl fragment, particularly against at least western corn rootworm. The peptidyl fragment is an amino acid sequence that is typically heterologus to (not derived from) a Bt Cry protein but may be derived from a Bt Cry protein. Such peptidyl fragments extend from the N-terminus of the insecticidal protein and do not naturally occur at the N-terminus of Bt Cry proteins. One example of an N-terminal peptidyl fragment has the amino acid sequence MTSNGRQCAGIRP (SEQ ID NO: 129) which is not derived from a Bt Cry protein.
[0139] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.
[0140] In the context of the present invention, "connecting" nucleic acids means to join two or more nucleic acids together using any means known in the art. For example, without limitation, the nucleic acids may be ligated together using for example, DNA ligase, or may be annealed using PCR. The nucleic acids may also be joined by chemically synthesizing a nucleic acid using the sequence of two or more separate nucleic acids.
[0141] To "control" insects means to inhibit, through a toxic effect, the ability of insect pests to survive, grow, feed, and/or reproduce, or to limit insect-related damage or loss in crop plants. To "control" insects may or may not mean killing the insects, although it preferably means killing the insects.
[0142] In the context of the present invention, "corresponding to" means that when the amino acid sequences of certain proteins (for example Bt Cry proteins or modified Cry3A proteins) are aligned with each other, the amino acids that align with certain enumerated positions in for example, but not limited to, a Cry3A toxin (either SEQ ID NO: 68 or SEQ ID NO: 134); a Cry3A055 toxin (SEQ ID NO: 70); or a Cry1Ab toxin (SEQ ID NO: 72), but that are not necessarily in these exact numerical positions relative to the reference amino acid sequence, particularly as it relates to identification of domains I, II and III, and/or the conserved blocks and variable regions, these amino acid positions "correspond to" each other. For example, in delineating Domain I of a hybrid protein, amino acids 11-244 of a Cry3A055 protein (SEQ ID NO: 70) correspond to amino acids 58-290 of a native Cry3A toxin (SEQ ID NO: 135) or to amino acids 11-243 of a native Cry3A toxin (SEQ ID NO: 68) or to amino acids 33-254 of a native Cry1Ab toxin.
[0143] In the context of the present invention the words "Cry protein" can be used interchangeably with the words "delta-endotoxin" or ".delta.-endotoxin."
[0144] In the context of the present invention, an "engineered hybrid insecticidal protein" (eHIP) is an insecticidal protein created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins. Such novel eHIPs may comprise complete or partial variable regions, conserved blocks or domains from a modified Cry3A protein and a Cry protein different from the modified Cry3A protein. The eHIPs of the invention may optionally include a protoxin tail region from a Bt Cry protein or an N-terminal peptidyl fragment or both. For example without limitation, an eHIP is created by combining in an N-terminal to C-terminal direction, amino acids 1-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5 and variable region 6, and a 38 amino acid region of a Cry1Ab protoxin tail. An eHIP that comprises an N-terminal peptidyl fragment may also be designated as a "chimeric insecticidal protein (CIP)."
[0145] To "deliver" an eHIP means that the eHIP comes in contact with an insect, resulting in a toxic effect and control of the insect. The eHIP may be delivered in many recognized ways, e.g., through a transgenic plant expressing the eHIP, formulated protein composition(s), sprayable protein composition(s), a bait matrix, or any other art-recognized toxin delivery system.
[0146] "Effective insect-controlling amount" means that concentration of an eHIP that inhibits, through a toxic effect, the ability of insects to survive, grow, feed and/or reproduce, or to limit insect-related damage or loss in crop plants. "Effective insect-controlling amount" may or may not mean killing the insects, although it preferably means killing the insects.
[0147] "Expression cassette" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may have at least one of its components heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development.
[0148] A "gene" is a defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acids responsible for the control of the expression, that is to say the transcription and translation, of the coding portion. A gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns. The regulatory nucleic acid sequence of the gene may not normally be operatively linked to the associated nucleic acid sequence as found in nature and thus would be a chimeric gene.
[0149] "Gene of interest" refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability. The "gene of interest" may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant.
[0150] A "heterologous" nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence. A heterologous amino acid sequence is one that is not naturally associated with a native amino acid sequence, for example an amino acid sequence of a Cry protein.
[0151] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.
[0152] "Homologous recombination" is the reciprocal exchange of nucleic acid fragments between homologous nucleic acid molecules.
[0153] "Identity" or "percent identity" refers to the degree of similarity between two nucleic acid or protein sequences. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
[0154] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).
[0155] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
[0156] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
[0157] Another widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nuc. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between a 200 and a 400 amino acid proteins, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.
[0158] Another indication that two nucleic acids are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
[0159] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions" a probe will hybridize to its target subsequence, but to no other sequences.
[0160] The T.sub.mis the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T.sub.m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42.degree. C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72.degree. C. for about 15 minutes. An example of stringent wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6.times.SSC at 40.degree. C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30.degree. C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2.times. (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
[0161] The following are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree. C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0162] A further indication that two nucleic acids or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.
[0163] "Insecticidal" is defined as a toxic biological activity capable of controlling insects, preferably by killing them.
[0164] A nucleic acid sequence is "isocoding with" a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.
[0165] An "isolated" nucleic acid molecule or an isolated toxin is a nucleic acid molecule or toxin that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or toxin may exist in a purified form or may exist in a non-native environment such as, for example without limitation, a recombinant microbial cell, plant cell, plant tissue, or plant.
[0166] A "modified Cry3A toxin" or "Cry3A055" of this invention refers to a Cry3A-derived toxin having at least one additional protease recognition site that is recognized by a gut protease of a target insect, which does not naturally occur in a Cry3A toxin, as described in U.S. Pat. No. 7,030,295, herein incorporated by reference.
[0167] A "modified cry3A coding sequence" according to this invention can be derived from a native cry3A coding sequence or from a synthetic cry3A coding sequence and comprises the coding sequence of at least one additional protease recognition site that does not naturally occur in an unmodified cry3A gene.
[0168] A "nucleic acid molecule" or "nucleic acid sequence" is a segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is typically a segment of DNA.
[0169] A "plant" is any plant at any stage of development, particularly a seed plant.
[0170] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
[0171] "Plant cell culture" means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
[0172] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
[0173] A "plant organ" is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
[0174] "Plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
[0175] A "promoter" is an untranslated DNA sequence upstream of the coding region that contains the binding site for RNA polymerase and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.
[0176] "Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
[0177] "Transformation" is a process for introducing heterologous nucleic acid into a host cell or organism. In particular, "transformation" means the stable integration of a DNA molecule into the genome of an organism of interest.
[0178] "Transformed/transgenic/recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
[0179] Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise indicated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; l), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
DESCRIPTION
[0180] This invention relates to novel engineered hybrid insecticidal proteins (eHIPS), created to have activity against at least western corn rootworm, and may further include northern corn rootworm, Mexican corn rootworm, and/or Colorado potato beetle. Some eHIPs have activity against the lepidopteran pest, European corn borer. Such novel eHIPs are made by fusing unique combinations of complete or partial variable regions and conserved blocks of at least two different Cry proteins and optionally include a protoxin tail region from a Bt Cry protein at the C-terminus or an N-terminal peptidyl fragment or both. For example, without limitation, by combining complete or partial variable regions and conserved blocks from a first Cry protein that has coleopteran activity with complete or partial variable regions and conserved blocks from a second Cry protein that has lepidopteran activity and is different from the first Bt Cry protein, and optionally including a protoxin tail region from a Bt Cry protein at the C-terminus or an N-terminal peptidyl fragment or both, new engineered hybrid insecticidal proteins that have activity against a spectrum of insects that is different from the first or second parent Cry protein, or both, is created. Such novel eHIPs may also comprise complete or partial variable regions, conserved blocks or domains from a modified Cry3A protein and a Cry protein different from the modified Cry3A protein. The N-terminal peptidyl fragment or protoxin tail region may confer insecticidal activity upon the eHIP, may increase the insecticidal activity of an eHIP over an eHIP without the peptidyl fragment or protoxin tail region, and/or may make the eHIP more stable than an eHIP without the peptidyl fragment or protoxin tail region, particularly against at least western corn rootworm. The amino acid sequence of the peptidyl fragment typically is heterologous to (i.e. not derived from) a Bt Cry protein. However, based on the teaching disclosed herein, the skilled person will recognize that an N-terminal peptidyl fragment may be generated using an amino acid sequence derived from a Bt Cry protein. The eHIPs of the invention have surprising and unexpected toxicity to corn rootworm, particularly to western, northern and Mexican corn rootworm. The present invention also relates to nucleic acids whose expression results in eHIPs, and to the making and using of the eHIPs to control insect pests. The expression of the nucleic acids results in eHIPs that can be used to control coleopteran insects such as western, northern or Mexican corn rootworm, or used to control lepidopteran insects such as European corn borer, particularly when expressed in a transgenic plant such as a transgenic corn plant.
[0181] In one embodiment, the invention encompasses an engineered hybrid insecticidal protein comprising an amino acid sequence from a first Bacillus thuringiensis (Bt) Cry protein comprising complete or partial variable regions and conserved blocks of the first Cry protein fused to an amino acid sequence from a second Bt Cry protein different from the first Bt Cry protein comprising complete or partial variable regions and conserved blocks of the second Cry protein, and optionally comprising: (a) a protoxin tail region of a Bt Cry protein located at the C-terminus; or (b) an N-terminal peptidyl fragment; or both (a) and (b), wherein the eHIP has activity against at least western corn rootworm.
[0182] In another embodiment, the present invention encompasses an eHIP comprising an N-terminal region of a first Bt Cry protein fused to a C-terminal region of a second Bt Cry protein different from the first Bt Cry protein, wherein at least one crossover position between the first and the second Bt Cry protein is located in conserved block 2, conserved block 3, variable region 4 or conserved block 4, and optionally comprising: (a) a protoxin tail region of a Bt Cry protein located at the C-terminus; or (b) an N-terminal peptidyl fragment; or both (a) and (b), wherein the eHIP has insecticidal activity against at least western corn rootworm.
[0183] In another embodiment, an eHIP according to the invention comprises from N-terminus to C-terminus variable region 1 or a C-terminal portion of variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and an N-terminal portion of conserved block 3 of a first Bt Cry protein fused to a C-terminal portion of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6 of a second Bt Cry protein.
[0184] In another embodiment, an eHIP of the invention comprises at least two crossover positions between an amino acid sequence from the first Bt Cry protein and an amino acid sequence from the second Bt Cry protein. In one embodiment, a first crossover position is located in conserved block 2 and a second crossover position is located in conserved block 3. In another embodiment, a first crossover junction is located in conserved block 3 and a second crossover position is located in conserved block 4.
[0185] In another embodiment, an eHIP of the invention comprises at the C-terminus a protoxin tail region of a Bt Cry protein. The protoxin tail region may confer insecticidal activity upon the eHIP, meaning that without the protoxin tail region the eHIP would not be active, may increase activity of the eHIP over an eHIP without the protoxin tail region, or may make the eHIP more stable than an eHIP without the protoxin tail region. In one embodiment, the protoxin tail region is from a lepidopteran active Bt Cry protein. In another embodiment, the protoxin tail region is from a Cry1A protein. In yet another embodiment, the protoxin tail region is from a Cry1Aa or a Cry1Ab protein. The protoxin tail region of the invention may comprise an entire protoxin tail of a Bt Cry protein or any fragment thereof. In one aspect of this embodiment, the protoxin tail region of an eHIP comprises at least 38 amino acids from the N-terminus of a protoxin tail of a Cry1Ab protein. In another aspect of this embodiment, the protoxin tail region comprises an amino acid sequence corresponding to amino acids 611-648 of SEQ ID NO: 72. In still another aspect of this embodiment, the protoxin tail region comprises amino acids 611-648 of SEQ ID NO: 72.
[0186] In still another embodiment, an eHIP comprises an N-terminal peptidyl fragment. The N-terminal peptidyl fragment may confer insecticidal activity upon the eHIP, meaning that without the N-terminal peptidyl fragment the protein does not have insecticidal activity, or the N-terminal peptidyl fragment may increase the insecticidal activity of the eHIP over an eHIP without the N-terminal peptidyl fragment, or the N-terminal peptidyl fragment may make the eHIP more stable than an eHIP without an N-terminal peptidyl fragment. In one aspect of this embodiment, the peptidyl fragment comprises an amino acid sequence that is heterologous to (i.e. not derived from) a Bt Cry protein. In another aspect of this embodiment, the N-terminal peptidyl fragment comprises at lest 9 amino acids. In yet another aspect of this embodiment, the peptidyl fragment comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90 or 100 amino acids. In another aspect of this embodiment, the peptidyl fragment comprises greater than 100 amino acids. In still another aspect of this embodiment, the N-terminal peptidyl fragment comprises the amino acid sequence YDGRQQHRG (SEQ ID NO: 133) or TSNGRQCAGIRP (SEQ ID NO: 134). In yet another aspect of this embodiment, the N-terminal peptidyl fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132.
[0187] In yet another embodiment, the variable regions and conserved blocks of a first Cry protein active against coleopteran insects are used to make the eHIP of the invention in combination with variable regions and conserved blocks of a second Cry protein active against a lepidopteran insect. Coleopteran active Cry proteins include but are not limited to Cry3, Cry7, Cry8, and Cry34/Cry35. The lepidopteran active Cry proteins include but are not limited to Cry1 and Cry9. In one aspect of this embodiment, the first Cry protein is a Cry3A and the second Cry protein is a Cry1A. In another aspect, the Cry3A protein can be replaced with a modified Cry3A, for example without limitation, the Cry3A055 protein disclosed in U.S. Pat. No. 5,659,123, which is herein incorporated by reference. In still another aspect of this embodiment, the Cry3A protein is a Cry3Aa and the Cry1A protein is a Cry1Aa or a Cry1Ab. In another aspect of this embodiment, the Cry3Aa is selected from the following group and has the indicated GenBank Accession Number: Cry3Aa1 (M22472), Cry3Aa2 (J02978), Cry3Aa3 (Y00420), Cry3Aa4 (M30503), Cry3Aa5 (M37207), Cry3Aa6 (U10985), Cry3Aa7 (AJ237900), Cry3Aa8 (AAS79487), Cry3Aa9 (AAW05659), Cry3Aa10 (AAU29411), and Cry3Aa1 (AY882576). In another aspect of this embodiment the Cry1Aa is selected from the following group and has the indicated GenBank Accession Number: Cry1Aa1 (M11250), Cry1Aa2 (M10917), Cry1Aa3 (D00348), Cry1Aa4 (X13535), Cry1Aa5 (D17518), Cry1Aa6 (U43605), Cry 1 Aa7 (AF081790), Cry 1 Aa8 (126149), Cry 1 Aa9 (AB026261), Cry1Aa10 (AF154676), Cry1Aa1l (Y09663), Cry1Aa12 (AF384211), Cry1Aa13 (AF510713), Cry1Aa14(AY197341), and Cry1Aa15 (DQ062690). In still another aspect of this embodiment, the Cry1Ab is selected from the following group and has the indicated GenBank Accession Number: Cry1Ab1 (M13898), Cry1Ab2 (M12661), Cry1Ab3 (M15271), Cry1Ab4 (D00117), Cry1Ab5 (X04698), Cry1Ab6 (M37263), Cry1Ab7 (X13233), Cry1Ab8 (M16463), Cry1Ab9 (X54939), Cry1Ab10 (A29125), Cry1Ab11 (112419), Cry1Ab12 (AF059670), Cry1Ab13 (AF254640), Cry1Ab14 (U94191), Cry1Ab15 (AF358861),Cry1Ab16 (AF37560), Cry1Ab17 (AAT46415), Cry1Ab18 (AAQ88259), Cry1Ab19 (AY847289), Cry1Ab20 (DQ241675), Cry1Ab21 (EF683163), and Cry1Ab22 (ABW87320). In yet another aspect of this embodiment, the first Cry protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 135, and the second Cry protein comprises an amino acid sequence set forth in SEQ ID NO: 72.
[0188] In one embodiment, the present invention encompasses an eHIP of the invention comprising at least one crossover position between the N-terminal region of the first Cry protein and the C-terminal region of the second Cry protein located in conserved block 3, variable region 4, or conserved block 4. In one aspect of this embodiment, the crossover position in conserved block 3 is located immediately following an amino acid corresponding to Ser451, Phe454, or Leu468 of SEQ ID NO: 70. In another aspect of this embodiment, the crossover position is located in conserved block 3 immediately following Ser451, Phe454, or Leu468 of SEQ ID: 70 or Ser450, Phe453, or Leu467 of SEQ ID NO: 68; or Ser497, Phe100, Leu114 of SEQ ID NO: 135. The crossover positions in certain Cry3A/Cry1Ab eHIP embodiments or modified Cry3A/Cry1Ab eHIP embodiments according to the invention are indicated on FIG. 2, which indicates percent identity.
[0189] In another embodiment, an eHIP of the invention comprises at least two crossover positions between an amino acid sequence from a first Bt Cry protein and an amino acid sequence from the second Bt Cry protein. In one aspect of this embodiment, a crossover position between a Cry3A or modified Cry3A and a Cry1Ab or a Cry1Aa is located in conserved block 2 immediately following an amino acid corresponding to Asp232 of SEQ ID NO: 70 and a second crossover position between Cry1Ab and Cry3A or modified Cry3A is located in conserved block 3 immediately following an amino acid corresponding to Leu476 of SEQ ID NO: 72. In another aspect of this embodiment, a crossover position between a Cry3A or modified Cry3A and a Cry1Ab or a Cry1Aa is located in conserved block 2 immediately following Asp232 of SEQ ID NO: 70, or Asp231 of SEQ ID NO: 68, or Asp278 of SEQ ID NO: 135, and a second crossover position between Cry1Ab and Cry3A or modified Cry3A is located in conserved block 3 immediately following Leu476 of SEQ ID NO: 72.
[0190] In still another aspect of this embodiment, a first crossover position between a Cry3A or modified Cry3A and a Cry1Ab is located in conserved block 3 immediately following an amino acid corresponding to Leu468 of SEQ ID NO: 70 and a second crossover position between a Cry1Ab and a Cry3A or modified Cry3A is located in conserved block 4 immediately following an amino acid corresponding to Ile527 of SEQ ID NO: 72. In another aspect of this embodiment, the first crossover position between a Cry3A or modified Cry3Aa and a Cry1Ab is located in conserved block 3 immediately following an Leu468 of SEQ ID NO: 70, or Leu467 of SEQ ID NO: 68, or Leu114 of SEQ ID NO: 135, and the second crossover position between a Cry1Ab and a Cry3A or modified Cry3A is located in conserved block 4 immediately following Ile527 of SEQ ID NO: 72. In yet another aspect of this embodiment, the eHIP comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 34.
[0191] In one embodiment, the present invention encompasses an eHIP wherein the first Cry protein is a Cry3A or a modified Cry3A and the second Cry protein is a Cry1Aa or Cry1Ab and wherein the eHIP comprises an amino acid sequence that has at least 80% identity to SEQ OD NO: 64. In another embodiment the eHIP comprises an amino acid sequence that has at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 64. An alignment of certain eHIP embodiments of the invention with SEQ ID NO: 64 is shown in FIG. 2, which indicates percent identity.
[0192] In another embodiment, the present invention encompasses an eHIP wherein the first Cry protein is a Cry3A or a modified Cry3A and the second Cry protein is a Cry1Aa or Cry1Ab and wherein the eHIP comprises an amino acid sequence that has at least 75% identity to SEQ OD NO: 70. In another embodiment the eHIP comprises an amino acid sequence that has at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 70. An alignment of certain eHIP embodiments of the invention with SEQ ID NO: 70 is shown in FIG. 1, which indicates percent identity.
[0193] In another embodiment, the present invention encompasses an eHIP having a first crossover position between Cry3A or modified Cry3A and Cry1Aa or Cry1Ab in conserved block 2 and a second crossover position between Cry1Aa or Cry1Ab and Cry3A or modified Cry3A in conserved block 3 and wherein the eHIP comprises an amino acid sequence that has at least 56% identity to SEQ OD NO: 64. In one aspect of this embodiment, the eHIP has at least 60, 70 or 80% identity to SEQ ID NO: 64. In another aspect of this embodiment, the eHIP has at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 64.
[0194] In yet another embodiment, the present invention encompasses an eHIP comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 62; SEQ ID NO: 64, SEQ ID NO: 147, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 160.
[0195] In one embodiment, the eHIPs of the invention have activity against other insect pests including but not limited to northern corn rootworm, Mexican corn rootworm, Colorado potato beetle, and/or European corn borer.
[0196] In another embodiment, the present invention encompasses a nucleic acid molecule comprising a nucleotide sequence that encodes an eHIP of the invention. In one aspect of this embodiment, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 61; SEQ ID NO: 63, SEQ ID NO: 146, SEQ ID NO: 152, SEQ ID NO: 154 and SEQ ID NO: 158. Specifically exemplified teachings of methods to make nucleic acid molecules that encode eHIPs can be found in Examples 1-41. Those skilled in the art will recognize that modifications can be made to the exemplified methods to make eHIPs encompassed by the present invention.
[0197] The present invention further encompasses expression cassettes comprising the nucleic acid molecules, and recombinant vectors and transgenic non-human host cells, such as bacterial cells or plant cells, comprising the expression cassettes of the invention.
[0198] The present invention also encompasses recombinant vectors comprising the nucleic acids of this invention. In such vectors, the nucleic acids are preferably comprised in expression cassettes comprising regulatory elements for expression of the nucleotide sequences in a host cell capable of expressing the nucleotide sequences. Such regulatory elements usually comprise promoter and termination signals and preferably also comprise elements allowing efficient translation of polypeptides encoded by the nucleic acids of the present invention. Vectors comprising the nucleic acids are may be capable of replication in particular host cells, preferably as extrachromosomal molecules, and are therefore used to amplify the nucleic acids of this invention in the host cells. In one embodiment, host cells for such vectors are microorganisms, such as bacteria, in particular Bacillus thuringiensis or E. coli. In another embodiment, host cells for such recombinant vectors are endophytes or epiphytes. In yet another embodiment, such vectors are viral vectors and are used for replication of the nucleotide sequences in particular host cells, e.g. insect cells or plant cells. Recombinant vectors are also used for transformation of the nucleotide sequences of this invention into host cells, whereby the nucleotide sequences are stably integrated into the DNA of a transgenic host. In one embodiment, the transgenic host is plant such as corn plant.
[0199] The eHIPs of the present invention have insect control activity when tested against insect pests in bioassays. In one embodiment, the eHIPs of the invention are active against coleopteran insects or lepidopteran insects or both. In one aspect of this embodiment, the eHIPs of the invention are active against western corn rootworm, northern corn rootworm, Mexican corn rootworm and/or Colorado potato beetle. In another aspect of this embodiment, the eHIPs of the invention are active against European corn borer. The insect controlling properties of the eHIPs of the invention are further illustrated in Examples 43, 45 and 46.
[0200] The present invention also encompasses a composition comprising an effective insect-controlling amount of an eHIP according to the invention.
[0201] In another embodiment, the invention encompasses a method of producing a eHIP that is active against insects, comprising: (a) obtaining a host cell comprising a gene, which itself comprises a heterologous promoter sequence operatively linked to a nucleic acid molecule of the invention; and (b) growing the transgenic host cell in such a manner to express an eHIP that is active against insects.
[0202] In yet another embodiment, the invention encompasses a method of producing an insect-resistant transgenic plant, comprising introducing a nucleic acid molecule of the invention into the transgenic plant, wherein the nucleic acid molecule causes the expression of an eHIP in the transgenic plant in an effective amount to control insects. In one aspect of this embodiment, the insects are coleopteran insects or lepidopteran insects. In another aspect of this embodiment, the coleopteran insects are western corn rootworm, northern corn rootworm, Mexican corn rootworm and/or Colorado potato beetle. In still another aspect of this embodiment, the lepidopteran insects are European corn borer.
[0203] In yet a further embodiment, the invention encompasses a method of controlling insects, comprising delivering to the insects an effective amount of an eHIP of the invention. In one aspect of this embodiment, the insects are coleopteran insects or lepidopteran insects. In another aspect of this embodiment, the coleopteran insects are western corn rootworm, northern corn rootworm, Mexican corn rootworm and/or Colorado potato beetle. In still another aspect of this embodiment, the lepidopteran insects are European corn borer. Typically, the eHIP is delivered to the insects orally. In one aspect, the eHIP is delivered orally through a transgenic plant comprising a nucleic acid sequence that expresses an eHIP of the present invention.
[0204] The present invention further encompasses a method of controlling insects wherein the transgenic plant further comprises a second nucleic acid molecule or groups of nucleic acid molecules that encode a second pesticidal principle. Examples of such second nucleic acids are those that encode a Bt Cry protein, those that encode a Vegetative Insecticidal Protein, disclosed in U.S. Pat. Nos. 5,849,870 and 5,877,012, incorporated herein by reference, or those that encode a pathway for the production of a non-proteinaceous principle.
[0205] The present invention also encompasses a method of making an engineered hybrid insecticidal protein (eHIP), comprising: (a) obtaining a first Bt Cry protein or modified Bt Cry protein; (b) obtaining a second Bt Cry protein which is different from the first Bt Cry protein or modified Bt Cry protein; (c) combining complete or partial variable regions and conserved blocks of the first Bt Cry protein or modified Bt Cry protein with complete or partial variable regions and conserved blocks of the second Bt Cry protein to make an eHIP that has activity against at least western corn rootworm; and optionally (d) inserting a peptidyl fragment at the N-terminus or a protoxin tail region of a Bt Cry protein at the C-terminus of the eHIP, or both, wherein the N-terminal peptidyl fragment or the C-terminal protoxin region or both confers activity upon the eHIP, or increases the insecticidal activity of the eHIP or makes the eHIP more stable than an eHIP without the peptidyl fragment or protoxin tail region or both.
[0206] In another embodiment, the present invention encompasses a method of making an engineered hybrid insecticidal protein (eHIP) comprising: (a) obtaining a first nucleic acid encoding a first Bt Cry protein or modified Bt Cry protein and a second nucleic acid encoding a second Cry protein different from the first Cry protein or modified Bt Cry protein; (b) isolating from the first and second nucleic acids, a nucleotide sequence that encodes complete or partial variable regions and conserved blocks of the first Bt Cry protein or modified Bt Cry protein and the second Bt Cry protein; (c) connecting together the resulting isolated nucleic acids of step (b) in such a way as to make a new hybrid nucleic acid that encodes a protein, and optionally fusing to a 5' end of said hybrid nucleic acid a nucleic acid that encodes a peptidyl fragment resulting in a 5' extension or fusing to a 3' end of said hybrid nucleic acid a nucleic acid that encodes a protoxin tail region of a Bt Cry protein resulting in a 3' extension, or both; (d) inserting the hybrid nucleic acid with or without one or both of the 5' or 3' extensions into an expression cassette; (e) transforming the expression cassette into a host cell, resulting in said host cell producing an eHIP; and (f) bioassaying the eHIP against at least western corn rootworm, which results in insecticidal activity against western corn rootworm.
[0207] In further embodiments of the methods of the invention, the first Bt Cry protein or modified Bt Cry protein is a Cry3A or modified Cry3A and the second Bt Cry protein is A Cry1Aa or Cry1Ab.
[0208] In another embodiment of the methods of the invention, the N-terminal peptidyl fragment comprises at 9 amino acids. In one aspect of this embodiment the N-terminal peptidyl fragment comprises the amino acid sequence YDGRQQHRG (SEQ ID NO: 132) or the amino acid sequence TSNGRQCAGIRP (SEQ ID NO: 133). In another aspect of this embodiment the N-terminal peptidyl fragment is selected from the group consisting of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132.
[0209] In still another embodiment of the methods of the invention, the protoxin tail region is from a Cry1Aa or Cry1Ab. In one aspect of this embodiment, the protoxin tail region comprises at least 38 amino aacids. In another aspect of this embodiment, the protoxin tail region comprises an amino acid sequence that corresponds to amino acids 611-648 of SEQ ID NO: 72. In yet another aspect of this embodiment, the protoxin tail region comprises amino acids 611-648 of SEQ ID NO: 72.
[0210] Specifically exemplified teachings of the methods of making the hybrid nucleic acids and the eHIPs of the invention can be found in Examples 1-41.
[0211] In further embodiments, the nucleotide sequences of the invention, particularly a sequence encoding the peptidyl fragment, the protoxin tail, and/or conserved blocks 2, 3, and 4, can be further modified by incorporation of random mutations in a technique known as in vitro recombination or DNA shuffling. This technique is described in Stemmer et al., Nature 370:389-391 (1994) and U.S. Pat. No. 5,605,793, which are incorporated herein by reference. Millions of mutant copies of a nucleotide sequence are produced based on an original nucleotide sequence of this invention and variants with improved properties, such as increased insecticidal activity, enhanced stability, or different specificity or ranges of target-insect pests are recovered. The method encompasses forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide comprising a nucleotide sequence of this invention, wherein the template double-stranded polynucleotide has been cleaved into double-stranded-random fragments of a desired size, and comprises the steps of adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded template polynucleotide; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide; and repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide. In a preferred embodiment, the concentration of a single species of double-stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of the total DNA. In a further preferred embodiment, the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides. In another preferred embodiment, the size of the double-stranded random fragments is from about 5 by to 5 kb. In a further preferred embodiment, the fourth step of the method comprises repeating the second and the third steps for at least 10 cycles.
[0212] As biological insect control agents, the eHIPs are produced by expression of the nucleic acids in heterologous host cells capable of expressing the nucleic acids. In one embodiment, B. thuringiensis cells comprising modifications of a nucleic acid of this invention are made. Such modifications encompass mutations or deletions of existing regulatory elements, thus leading to altered expression of the nucleic acid, or the incorporation of new regulatory elements controlling the expression of the nucleic acid. In another embodiment, additional copies of one or more of the nucleic acids are added to Bacillus thuringiensis cells either by insertion into the chromosome or by introduction of extrachromosomally replicating molecules containing the nucleic acids.
[0213] In another embodiment, at least one of the nucleic acids of the invention is inserted into an appropriate expression cassette, comprising a promoter and termination signal. Expression of the nucleic acid is constitutive, or an inducible promoter responding to various types of stimuli to initiate transcription is used. In another embodiment, the cell in which the eHIP is expressed is a microorganism, such as a virus, bacteria, or a fungus. In yet another embodiment, a virus, such as a baculovirus, contains a nucleic acid of the invention in its genome and expresses large amounts of the corresponding insecticidal protein after infection of appropriate eukaryotic cells that are suitable for virus replication and expression of the nucleic acid. The insecticidal protein thus produced is used as an insecticidal agent. Alternatively, baculoviruses engineered to include the nucleic acid are used to infect insects in vivo and kill them either by expression of the insecticidal toxin or by a combination of viral infection and expression of the insecticidal toxin.
[0214] Bacterial cells are also hosts for the expression of the nucleic acids of the invention. In one embodiment, non-pathogenic symbiotic bacteria, which are able to live and replicate within plant tissues, so-called endophytes, or non-pathogenic symbiotic bacteria, which are capable of colonizing the phyllosphere or the rhizosphere, so-called epiphytes, are used. Such bacteria include bacteria of the genera Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Envinia, Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas. Symbiotic fungi, such as Trichoderma and Gliocladium are also possible hosts for expression of the inventive nucleic acids for the same purpose.
[0215] Techniques for these genetic manipulations are specific for the different available hosts and are known in the art. For example, the expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in E. coli, either in transcriptional or translational fusion, behind the tac or trc promoter. For the expression of operons encoding multiple ORFs, the simplest procedure is to insert the operon into a vector such as pKK223-3 in transcriptional fusion, allowing the cognate ribosome binding site of the heterologous genes to be used. Techniques for overexpression in gram-positive species such as Bacillus are also known in the art and can be used in the context of this invention (Quax et al. In:Industrial Microorganisms:Basic and Applied Molecular Genetics, Eds. Baltz et al., American Society for Microbiology, Washington (1993)). Alternate systems for overexpression rely for example, on yeast vectors and include the use of Pichia, Saccharomyces and Kluyveromyces (Sreekrishna, In:Industrial microorganisms:basic and applied molecular genetics, Baltz, Hegeman, and Skatrud eds., American Society for Microbiology, Washington (1993); Dequin & Barre, Biotechnology L2:173-177 (1994); van den Berg et al., Biotechnology 8:135-139 (1990)).
[0216] In one embodiment, at least one of the eHIPs of the invention is expressed in a higher organism such as a plant. In this case, transgenic plants expressing effective amounts of the eHIP protect themselves from insect pests. When the insect starts feeding on such a transgenic plant, it also ingests the expressed eHIP. This will deter the insect from further biting into the plant tissue or may even harm or kill the insect. A nucleic acid of the present invention is inserted into an expression cassette, which may then be stably integrated in the genome of the plant. In another embodiment, the nucleic acid is included in a non-pathogenic self-replicating virus. Plants transformed in accordance with the present invention may be monocots or dicots and include, but are not limited to, corn, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous and deciduous trees.
[0217] Once a desired nucleic acid has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.
[0218] A nucleic acid of this invention is preferably expressed in transgenic plants, thus causing the biosynthesis of the corresponding eHIP in the transgenic plants. In this way, transgenic plants with enhanced resistance to insects, particularly corn rootworm, are generated. For their expression in transgenic plants, the nucleic acids of the invention may require other modifications and optimization. Although in many cases genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants may result from microbial nucleic acids having codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleic acids described in this invention can be changed to conform with plant preferences, while maintaining the amino acids encoded thereby. Furthermore, high expression in plants is best achieved from coding sequences that have at least about 35% GC content, preferably more than about 45%, more preferably more than about 50%, and most preferably more than about 60%. Microbial nucleic acids that have low GC contents may express poorly in plants due to the existence of ATTTA motifs that may destabilize messages, and AATAAA motifs that may cause inappropriate polyadenylation. Although preferred gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17:477-498 (1989)). In addition, the nucleic acids are screened for the existence of illegitimate splice sites that may cause message truncation. All changes required to be made within the nucleic acids such as those described above are made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods described in the published patent applications EP 0 385 962, EP 0 359 472, and WO 93/07278.
[0219] In one embodiment of the invention an eHIP coding sequence and/or a parent Bt Cry protein coding sequence is/are made according to the procedure disclosed in U.S. Pat. No. 5,625,136, herein incorporated by reference. In this procedure, maize preferred codons, i.e., the single codon that most frequently encodes that amino acid in maize, are used. The maize preferred codon for a particular amino acid might be derived, for example, from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is found in Murray et al., Nucleic Acids Research 17:477-498 (1989), the disclosure of which is incorporated herein by reference.
[0220] In this manner, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.
[0221] For efficient initiation of translation, sequences adjacent to the initiating methionine may require modification. For example, they can be modified by the inclusion of sequences known to be effective in plants. Joshi has suggested an appropriate consensus for plants (NAR 15:6643-6653 (1987)) and Clonetech suggests a further consensus translation initiator (1993/1994 catalog, page 210). These consensuses are suitable for use with the nucleic acids of this invention. The sequences are incorporated into constructions comprising the nucleic acids, up to and including the ATG (whilst leaving the second amino acid unmodified), or alternatively up to and including the GTC subsequent to the ATG (with the possibility of modifying the second amino acid of the transgene).
[0222] Expression of the nucleic acids in transgenic plants is driven by promoters that function in plants. The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. Thus, expression of the nucleic acids of this invention in leaves, in stalks or stems, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/or seedlings is preferred. In many cases, however, protection against more than one type of insect pest is sought, and thus expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleic acids in the desired cell.
[0223] In one embodiment promoters are used that are expressed constitutively including the actin or ubiquitin or cmp promoters or the CaMV 35S and 19S promoters. The nucleic acids of this invention can also be expressed under the regulation of promoters that are chemically regulated. This enables the eHIPs to be synthesized only when the crop plants are treated with the inducing chemicals. Preferred technology for chemical induction of gene expression is detailed in the published application EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a promoter.
[0224] In another embodiment a category of promoters which is wound inducible can be used. Numerous promoters have been described which are expressed at wound sites and also at the sites of phytopathogen infection. Ideally, such a promoter should only be active locally at the sites of infection, and in this way the eHIPs only accumulate in cells that need to synthesize the eHIPs to kill the invading insect pest. Preferred promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), and Warner et al. Plant J. 3:191-201 (1993).
[0225] Tissue-specific or tissue-preferential promoters useful for the expression of genes encoding eHIPs in plants, particularly corn, are those which direct expression in root, pith, leaf or pollen, particularly root. Such promoters, e.g. those isolated from PEPC or trpA, are disclosed in U.S. Pat. No. 5,625,136, or MTL, disclosed in U.S. Pat. No. 5,466,785. Both U. S. patents are herein incorporated by reference in their entirety.
[0226] Further embodiments are transgenic plants expressing the nucleic acids in a wound-inducible or pathogen infection-inducible manner.
[0227] In addition to promoters, a variety of transcriptional terminators are also available for use in hybrid nucleic acid construction using the eHIP genes of the present invention. Transcriptional terminators are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators and those that are known to function in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase (NOS) terminator, the pea rbcS E9 terminator and others known in the art. These can be used in both monocotyledons and dicotyledons. Any available terminator known to function in plants can be used in the context of this invention.
[0228] Numerous other sequences can be incorporated into expression cassettes described in this invention. These include sequences that have been shown to enhance expression such as intron sequences (e.g. from Adhl and bronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).
[0229] It may be preferable to target expression of the nucleic acids of the present invention to different cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle may be preferred. Subcellular localization of transgene-encoded enzymes is undertaken using techniques well known in the art. Typically, the DNA encoding the target peptide from a known organelle-targeted gene product is manipulated and fused upstream of the nucleic acid. Many such target sequences are known for the chloroplast and their functioning in heterologous constructions has been shown. The expression of the nucleic acids of the present invention is also targeted to the endoplasmic reticulum or to the vacuoles of the host cells. Techniques to achieve this are well known in the art.
[0230] Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construction of interest may be preferred. In the case of direct gene transfer, transformation with a single DNA species or co-transformation can be used (Schocher et al. Biotechnology 4:1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transfer, transformation is usually (but not necessarily) undertaken with a selectable marker that may provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (basta). Plant transformation vectors comprising the eHIP genes of the present invention may also comprise genes (e.g. phosphomannose isomerase; PMI) which provide for positive selection of the transgenic plants as disclosed in U.S. Pat. Nos. 5,767,378 and 5,994,629, herein incorporated by reference. The choice of selectable marker is not, however, critical to the invention.
[0231] In another embodiment, a nucleic acid of the present invention is directly transformed into the plastid genome. A major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial codon optimization, and plastids are capable of expressing multiple open reading frames under control of a single promoter. Plastid transformation technology is extensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al. (1994) Proc. Nati. Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Nati. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub, J. M., and Maliga, P. (1993) EMBO J. 12, 601-606). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransf erase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein. In a preferred embodiment, a nucleic acid of the present invention is inserted into a plastid-targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleic acid of the present invention are obtained, and are preferentially capable of high expression of the nucleic acid.
[0232] The eHIPs of the invention can be used in combination with other pesticidal principles (e.g. Bt Cry proteins) to increase pest target range. Furthermore, the use of the eHIPs of the invention in combination with modified Cry3A toxins, Bt Cry proteins, or other CRW-active principles, such as an RNAi, which have a different mode of action or target a different receptor in the insect gut, has particular utility for the prevention and/or management of corn rootworm resistance. Other insecticidal principles include, but are not limited to, lectins, .alpha.-amylase, peroxidase, and cholesterol oxidase. Vip genes, as disclosed in U.S. Pat. No. 5,889,174 and herein incorporated by reference, are also useful in combination with the eHIPs of the present invention.
[0233] This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by making a single recombinant vector comprising coding sequences of more than one insecticidal principle in a so called molecular stack and genetically engineering a plant to contain and express all the insecticidal principles in the transgenic plant. Such molecular stacks may be also be made by using mini-chromosomes as described, for example in U.S. Pat. No. 7,235,716. Alternatively, a transgenic plant comprising one nucleic acid encoding a first insecticidal principle can be re-transformed with a different nucleic acid encoding a second insecticidal principle and so forth. Alternatively, a plant, Parent 1, can be genetically engineered for the expression of genes of the present invention. A second plant, Parent 2, can be genetically engineered for the expression of a supplemental insect control principle. By crossing Parent 1 with Parent 2, progeny plants are obtained which express all the genes introduced into Parents 1 and 2.
[0234] Transgenic seed of the present invention can also be treated with an insecticidal seed coating as described in U. S. Pat. Nos. 5,849,320 and 5,876,739, herein incorporated by reference. Where both the insecticidal seed coating and the transgenic seed of the invention are active against the same target insect, the combination is useful (i) in a method for enhancing activity of a eHIP of the invention against the target insect and (ii) in a method for preventing development of resistance to a eHIP of the invention by providing a second mechanism of action against the target insect. Thus, the invention provides a method of enhancing activity against or preventing development of resistance in a target insect, for example corn rootworm, comprising applying an insecticidal seed coating to a transgenic seed comprising one or more eHIPs of the invention.
[0235] Even where the insecticidal seed coating is active against a different insect, the insecticidal seed coating is useful to expand the range of insect control, for example by adding an insecticidal seed coating that has activity against lepidopteran insects to the transgenic seed of the invention, which has activity against coleopteran insects, the coated transgenic seed produced controls both lepidopteran and coleopteran insect pests.
EXAMPLES
[0236] The invention will be further described by reference to the following detailed examples. These examples are provided for the purposes of illustration only, and are not intended to be limiting unless otherwise specified. Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by J. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001); by T. J. Silhavy, M. L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular Biology, New York, John Wiley and Sons Inc., (1988), Reiter, et al., Methods in Arabidopsis Research, World Scientific Press (1992), and Schultz et al., Plant Molecular Biology Manual, Kluwer Academic Publishers (1998).
Example 1
Parental Coding Sequences
[0237] Maize optimized cry3Aa, cry1Ab, cry1Ba, and cry 1Fa coding sequences; designated herein mocry3Aa, mocry1Ab, mocry1Ba and mocry1Fa, respectively, were made according to the procedure disclosed in U.S. Pat. No. 5,625,136, herein incorporated by reference in its entirety.
[0238] The cry3A055 (SEQ ID NO: 67) coding sequence, which encodes a Cry3A055 protein (SEQ ID NO: 68) was made by modifying the mocry3A coding sequence by inserting a nucleotide sequence that encodes a Cathepsin G protease recognition site into domain I according to U.S. Pat. No. 7,030,295, herein incorporated by reference in its entirety.
[0239] The mocry3Aa (SEQ ID NO: 67), which encodes the protein set forth in SEQ ID NO: 68, cry3A055 (SEQ ID NO: 69), which encodes the protein set forth in SEQ ID NO: 70 , mocry1Ab (SEQ ID NO: 71), which encodes the protein set forth in SEQ ID NO: 72, mocry1Ba (SEQ ID NO: 73), which encodes the protein set forth in SEQ ID NO: 74, mocry1Fa (SEQ ID NO: 75), which encodes the protein set forth in SEQ ID NO: 76, cry8Aa (SEQ ID NO: 77), which encodes the protein set forth in SEQ ID NO: 78, cry1Ac (SEQ ID NO: 79), which encodes the protein set forth in SEQ ID NO: 80, and cry1Ia (SEQ ID NO: 81), which encodes the protein set forth in SEQ ID NO: 82, were used in the construction of the hybrid nucleic acids and the proteins which they encode and described in the following Examples.
Example 2
Use of PCR Primers to Construct Hybrid Nucleic Acids
[0240] Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (See Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki, Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim
[1985] "Enzymatic Amplification of .beta.-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia," Science 230:1350-1354.). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3' ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5' ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated.
[0241] The chimeric coding sequences described in the following examples were constructed using various combinations of the exemplified primers shown in Table 1. The PCR reaction mixes and PCR thermocycling protocols used in the experiments are listed in Tables 2 and 3, respectively. In each of the examples that follow, the PCR primers are referred to by name and "SEQ ID NO:" and the PCR reaction mixes and PCR thermocycling protocols are referred to by their respective numbers. It will be recognized by the skilled person that other PCR primers and PCR reaction conditions can be used to construct the chimeric coding sequences of the invention and by listing the exemplified primers and PCR conditions that were used in the instant invention is not meant to be limiting in any way.
TABLE-US-00001 TABLE 1 Primers used to construct the coding sequences encoding eHIPs. Primer Name Sequence SEQ ID NO: 5'3A-1-bam 5'-CCGGATCCATGACGGCCGACAACAACACCGAGGC-3' SEQ ID NO: 83 C3-3A-6 5'-CAGGGGCAGCTGGGTGATCT-3' SEQ ID NO: 84 C3-1Ab-3 5'-AGATCACCCAGATCCCCCTG-3' SEQ ID NO: 85 1Ab-6-sac 5'-CCGAGCTCAGCTCCTACACCTGATCGATGTGGTAGTCGG-3' SEQ ID NO: 86 8A-atg-delRI 5'-CCGGATCCACCATGACTAGTAACGGCCGCCAGTGTGCTGGTATTCGCCCTTATGAC-3' SEQ ID NO: 87 C2-3A-4 5'-GTCCAGCACGGTCAGGGTCA-3' SEQ ID NO: 88 reverse 5'-GCGTGCAGTCAAGTCAGATC-3' SEQ ID NO: 89 FR8a-OL-1 5'-GGTGTTGTTGTCGGCCGTCATAGGGCGAATACCAGCAC-3' SEQ ID NO: 90 FR8a-OL-2 5'-GCCGACAACAACACCGAGGCCCTGGACAGCAGCACCACC-3' SEQ ID NO: 91 C1-3A-2 5'-CAGGTGGGTGTTGGCGGCCTGGGCGTA-3' SEQ ID NO: 92 5'FR8a 5'-GGATCCACCATGACTAGTAAC-3' SEQ ID NO: 93 5'FR8a-12aa 5'-CCGGATCCACCATGTATGACGGCCGACAACAACACC-3' SEQ ID NO: 94 C2-3A-3 5'-TGACCCTGACCGTGCTGGAC-3' SEQ ID NO: 95 3'1Ab-dm3 5'-GAGCTCCTAGGTCACCTCGGCGGGCAC-3' SEQ ID NO: 96 5'FR-del6 5'-GGATCCACCATGTGTGCTGGTATTCGCCCTAT-3' SEQ ID NO: 97 5'1Ab-bam 5'-CCGGATCCATGGACAACAACCCCAACATCAAC-3' SEQ ID NO: 98 C3-3A-8 5'-GATGTCGCCGCCGGTGAAGC-3' SEQ ID NO: 99 C3-3A-7 5'-GCTTCACCGGCGGCGACATC-3' SEQ ID NO: 100 1B-5 5'-CCGCCGCGACCTGACCCTGGGCGTGCTGGAC-3' SEQ ID NO: 101 1B-10 5'-CCGAGCTCCTAGAACAGGGCGTTCAC-3' SEQ ID NO: 102 3A-22 5'-GGCCTTCACCAGGGGCAGCTGGGTGAT-3' SEQ ID NO: 103 1B-7 5'-ATCACCCAGATCCCCATGGTGAAGGCC-3' SEQ ID NO: 104 C3-1Ab-2 5'-CAGGGGGATCTGGGTGATCT-3' SEQ ID NO: 105 C3-3A-5 5'-AGATCACCCAGCTGCCCCTG-3' SEQ ID NO: 106 3A-12-sac 5'-CCGAGCTCAGCTCAGATCTAGTTCACGGGGATGAACTCGATCTT-3' SEQ ID NO: 107 C4-3A-10 5'-TGGTGCTGGCGTAGTGGATGCGG-3' SEQ ID NO: 108 C4-3A-9 5'-CCGCATCCACTACGCCAGCACCA-3' SEQ ID NO: 109 C1-1Ab-1 5'-TACGTGCAGGCCGCCAACCTGCACCTG-3' SEQ ID NO: 110 5'8Aa-dm3 5'-AGATCACCCAGCTGCCCCTGGTAAAGGGAGACATGTTATATC-3' SEQ ID NO: 111 3'8Aa-dm3 5'-GAGCTCCTATGTCTCATCTACTGGGATGAA-3' SEQ ID NO: 112 tant-OL-2 5'-GAGGGTGTGGGCCTTCACCAGGGGCAGCTGGGT-3' SEQ ID NO: 113 tant-OL-1 5'-ACCCAGCTGCCCCTGGTGAAGGCCCACACCCTC-3' SEQ ID NO: 114 tant-3'sac 5'-GAGCTCTAGCTTAAGCAGTCCACGAGGTT-3' SEQ ID NO: 115 1Ac-OL-2 5'-TAAAAAGAAAGTTTCCCTTCACCAGGGGCAGCTGGGT-3' SEQ ID NO: 116 1Ac-OL-1 5'-ACCCAGCTGCCCCTGGTGAAGGGAAACTTTCTTTTTA-3' SEQ ID NO: 117 1Ac-3'sac 5'-GAGCTCCTATGTTGCAGTAACTGGAATAAA-3' SEQ ID NO: 118 1Ia-OL-2 5'-AAGACAGATTGAAAGCTTTTACTCAGGGGCAGCTGGGT-3' SEQ ID NO: 119 1Ia-OL-1 5'-ACCCAGCTGCCCCTGAGTAAAAGCTTTCAATCTGTCTT-3' SEQ ID NO: 120 1Ia-3'sac 5'-GAGCTCCTACATGTTACGCTCAATATGGAGT-3' SEQ ID NO: 121 FR-1Ab-2 5'-GATGTTGTTGAACTCGGCGCTCTTGTGGGTCCA-3' SEQ ID NO: 122 FR-1Ab-1 5'-TGGACCCACAAGAGCGCCGAGTTCAACAACATC-3' SEQ ID NO: 123 FR-1Ab-4 5'-GGCTCGTGGGGATGATGTTGTTGAAGTCGACGCTCTTGTGG-3' SEQ ID NO: 124 FR-1Ab-3 5'-CCACAAGAGCGTCGACTTCAACACATCATCCCCAGCAGCC-3' SEQ ID NO: 125 CMS94 5'-GGCGCGCCACCATGGCTAGCATGACTGGTGG-3' SEQ ID NO: 136 CMS95 5'-GCAGGAACAGGTGGGTGTTG-3' SEQ ID NO: 137 CMS96 5'-CCTGAACACCATCTGGCCCA-3' SEQ ID NO: 138 CMS97 5'-CTGGCTGCTGGGGATGATGTTGTTGAAGTCGACGCTCTT-3' SEQ ID NO: 139 CMS98 5'-GAGCTCTTAGGTCACCTCGGC-3' SEQ ID NO: 140 CMS99 5'-AAGAGCGTCGACTTCAACAACATCATCCCCAGCAGCCAG-3' SEQ ID NO: 141 CMS100 5'-GAAGTACCGCGCCCGCATCCGCTACGCCAGCACCACCAAC-3' SEQ ID NO: 142 CMS101 5'-GTTGGTGGTGCTGGCGTAGCGGATGCGGGCGCGGTACTTC-3' SEQ ID NO: 143
TABLE-US-00002 TABLE 2 PCR reaction mixes. Mix 1 Mix 2 Mix 3 50-100 ng template 50-100 ng template 50-100 ng template DNA DNA DNA 0.8 .mu.M primer 1 0.8 .mu.M primer 1 0.8 .mu.M primer 1 0.8 .mu.M primer 2 0.8 .mu.M primer 2 0.8 .mu.M primer 2 1X Pfu buffer 1X Taq buffer 1X cDNA Advantage buffer 0.4 mM dNTPs 0.4 mM dNTPs 0.4 mM dNTPs 2% formamide 2% formamide x units cDNA Advantage 1.25 units Pfu 2.5 units Taq Polymerase (Clontech) Polymerase Polymerase (Qiagen) water to a total (Stratagene) water to a total volume of 50 .mu.l 2.5 units Taq volume of 50 .mu.l Polymerase (Qiagen) water to a total volume of 50 .mu.l Mix 4 Mix 5 50-100 ng template DNA 50-100 ng template DNA 0.4 .mu.M primer 1 0.4 .mu.M primer 1 0.4 .mu.M primer 2 0.4 .mu.M primer 2 1X PCR buffer (Invitrogen) 1X Pfu buffer (Stratagene) 0.4 mM dNTPs 0.2 mM dNTPs 2.5 units HotStart Taq Polymerase 1.25 units Pfu Turbo Polymerase water to a total volume of 50 .mu.l water to a total volume of 50 .mu.l
TABLE-US-00003 TABLE 3 PCR thermocycling profiles. Thermocycle Profile 1 Thermocycle Profile 2 Thermocycle Profile 3 94.degree. C.-5 minutes 94.degree. C.-5 minutes 94.degree. C.-5 minutes 20 cycles: 20 cycles: 20 cycles: 94.degree. C.-30 seconds 94.degree. C.-30 seconds 94.degree. C.-30 seconds 65.degree. C.-30 seconds 55.degree. C.-30 seconds 55.degree. C.-30 seconds 72.degree. C.-30 seconds 72.degree. C.-30 seconds 68.degree. C.-30 seconds 72.degree. C.-7 minutes 72.degree. C.-7 minutes 68.degree. C.-7 minutes hold at 4.degree. C. hold at 4.degree. C. hold at 4.degree. C. Thermocycle Profile 4 Thermocycle Profile 5 Thermocycle Profile 6 94.degree. C.-15 minutes 94.degree. C.-5 minutes 94.degree. C.-5 minutes 20 cycles: 20 cycles: 20 cycles: 94.degree. C.-30 seconds 94.degree. C.-30 seconds 94.degree. C.-30 seconds 50-70.degree. C.-30 seconds 55-75.degree. C.-30 seconds 55-75.degree. C.-30 seconds 72.degree. C.-30 seconds 72.degree. C.-1 minute 72.degree. C.-2 minutes 72.degree. C.-7 minutes 72.degree. C.-15 minutes 72.degree. C.-15 minutes hold at 4.degree. C. hold at 4.degree. C. hold at 4.degree. C.
[0242] Table 4 shows the relationship between the three domains of Cry3A055, Cry1Ab and Cry3A with their respective variable regions and conserved blocks. The amino acids comprised in the domains, conserved blocks and variable regions are shown for each protein.
TABLE-US-00004 TABLE 4 Cry3A055 Cry1Ab Cry3A Cry3A DOMAIN REGION (SEQ ID NO: 70) (SEQ ID NO: 72) (SEQ ID NO: 68) (SEQ ID NO: 131) V1 1-10 1-32 1-10 1-57 I V1 11-142 33-152 11-141 58-188 CB1 143-172 153-182- 142-171 189-218 V2 173-192 183-202 172-191 219-238 CB2 193-244 203-254 192-243 239-290 II 245-259 255-269 244-258 291-305 V3 260-444 270-452 259-443 306-490 CB3 445-454 453-462 444-453 491-500 III 455-492 463-500 454-491 501-538 V4 493-513 501-520 492-512 539-559 CB4 514-523 521-531 513-522 560-569 V5 524-586 532-596 523-585 570-632 CB5 587-598 597-606 586-597 633-644 V6 607-610 Protoxin 611-648
Example 3
Construction of 2OL-8a
[0243] A first nucleic acid fragment encoding an N-terminal portion of a Cry3A055 protein (SEQ ID NO: 70) was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C3-3A-6 (SEQ ID NO: 84) and PCR reaction Mix 1 and thermocycle Profile 1. This PCR reaction introduced a point mutation by deleting nucleotide 28 of SEQ ID NO: 69 (cry3A055), which caused a frame shift in the cry3A055 reading frame, and deleted the BamHI site and Kozak sequence (Kozak, M., 1986. Cell 44:283-92) at the 5` end of the resulting amplicon.
[0244] A second nucleic acid fragment encoding a C-terminal portion of a Cry1Ab protein (SEQ ID NO: 72) was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C3-1Ab-3 (SEQ ID NO: 85) and 1Ab-6-Sac (SEQ ID NO: 86) and PCR reaction Mix 1 and thermocycle Profile 1.
[0245] The first and second nucleic acids described above were connected by uasing them as templates in an overlap PCR reaction (Horton et al., 1989. Gene 77: 61-68) with the primers 5'3A-1-bam (SEQ ID NO: 83) and 1Ab-6-Sac (SEQ ID NO: 86) using PCR reaction Mix 2 and thermocycle Profile 1, except a 45-65.degree. C. gradient was used for the annealing temperature.
[0246] The resulting amplicon was ligated as a blunt ended fragment to a pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) cut with Smal to form plasmid p2OL8a/CR2.1. A BamHI-SacI fragment from p2OL8a/CR2.1 was then ligated to pET21a (EMD Biosciences, Inc., San Diego, Calif.), which was cut with BamHI-SacI, and transformed into E. coli. The BamHI-SacI fragment from p2OL8a/CR2.1 comprised 40 nucleotides derived from the pCR2.1-TOPO vector adjacent to the out of frame amplicon from the first PCR reaction. Ligating this BamHI-SacI fragment to pET21a created an open reading frame starting with the start codon (ATG) of a T7 tag and ending with the Sad site of the inserted DNA. This open reading frame was designated 2OL-8a (SEQ ID NO: 1) and encodes the 2OL-8a chimeric insecticidal protein (SEQ ID NO: 2). Thus, the 2OL-8a chimeric insecticidal protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MASMTGGQQMGRGSTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 126), amino acids 10-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable regions 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6; and 38 amino acids of the Cry1Ab protoxin tail region.
[0247] The nucleotides that encode amino acids 1-14 of the peptidyl fragment are derived from the T7-tag and the BamHI cleavage site of the pET21a vector. The nucleotides that encode amino acids 15-26 of the peptidyl fragment are derived from the pCR2.1-TOPO vector. And the nucleotides that encode amino acids 27-35 of the peptidyl fragment are derived from cry3A055 which are out of frame with the remainder of the cry3A055 coding sequence.
Example 4
Construction of FR8a
[0248] The FR8a coding sequence was constructed by placing a Kozak sequence (ACC) and a start codon (ATG) just downstream of an N-terminal BamHI site in 2OL-8a (See Example 3). In addition, an EcoRI site in 20L-8a was disrupted to aid in future vectoring of FR8a. All of these changes were made using one PCR reaction with 2OL-8a as the template and the primers: 8a-atg-delRJ (SEQ ID NO: 87) and C2-3A-4 (SEQ ID NO: 88) using PCR reaction Mix 2 and thermocycle Profile 2.
[0249] The resulting amplicon was ligated to a pCR2.1-TOPO vector (Invitrogen). A BamHI-PpuMI fragment from the cloned PCR product was then ligated to a PpuMI-NcoI fragment from 2OL8a/pCR2.1 (See Example 3) and a NcoI-BamHI fragment from 2OL8a/pCR2.1 to create FR8a (SEQ ID NO: 3) which encodes the FR8a chimeric insecticidal protein (SEQ ID NO: 4). Thus, the FR8a chimeric insecticidal protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable regions 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6; and 38 amino acids of the Cry1Ab protoxin tail region.
[0250] The FR8a eHIP was very active against western corn rootworm as shown in Table 5. Therefore, elimination of the T7 amino acid sequence from the N-terminal peptidyl fragment from the 2OL-8a eHIP did not have a negative impact on insecticidal activity.
[0251] Adding an additional 34 amino acids to the N-terminus of FR8a created a eHIP, designated FR8a+34 (SEQ ID NO: 160), with an N-terminal peptidyl fragment of 56 amino acids (SEQ ID NO: 131). The 56 amino acid N-terminal prptidyl fragment had no negative effect on activity of FR8a against western corn rootworm (See Table 5).
Example 5
Construction of FRCG
[0252] In order to determine if a cathepsin G protease recognition site was necessary for the insecticidal activity of a hybrid protein comprising an N-terminal fragment of Cry3A055, a construct was made which eliminated the cathepsin G site from the FR8a hybrid protein (Example 4). A first MluI-PpuMI nucleic acid fragment from a plasmid comprising FR8a (SEQ ID NO: 3) and a second PpuMI/MluI nucleic acid fragment from a plasmid comprising mocry3Aa (SEQ ID NO: 67) were ligated using standard molecular biology techniques to create FRCG (also designated FR8a-catg) (SEQ ID NO: 5) which encodes the FRCG hybrid protein (SEQ ID NO: 6). Thus, the FRCG chimeric insecticidal protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-467 of a Cry3A protein (SEQ ID NO: 68), which comprises variable regions 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6; and 38 amino acids of the Cry1Ab protoxin tail region.
[0253] The FRCG protein was as active against western corn rootworm as the FR8a protein (See Table 5) suggesting that a cathepsin G protease site is not required for insecticidal activity of a eHIP.
Example 6
Construction of FR8a-9F
[0254] A first approximately 323 by nucleic acid fragment was PCR amplified from a plasmid comprising FR8a (SEQ ID NO: 3) using primers reverse (SEQ ID NO: 89) and FR8a-OL-1 (SEQ ID NO: 90) and PCR reaction Mix 2 and thermocycle Profile 2. A second approximately 470 by nucleic acid fragment was PCR amplified from a plasmid comprising FR8a using primers FR8a-OL-2 (SEQ ID NO: 91) and C1-3A-2 (SEQ ID NO: 92) and PCR reaction Mix 2 and thermocycle Profile 2. The two resulting amplicons were connected by using them as templates in an overlap PCR reaction with primers 5'FR8a (SEQ ID NO: 93) and C1-3A-2 (SEQ ID NO: 92) using PCR reaction Mix 2 and thermocycle Profile 2 to amplify the 5` end of FR8a-9F. The overlap PCR product was cloned into a pCR2.1-TOPO vector (Invitrogen) designated 5'FR-9F/pCR2.1. A BamHI/PpuMI fragment of 5'FR-9F/pCR2.1 was then ligated to a PpuMI/BamHI fragment of FR8a to create FR8a-9F (SEQ ID NO: 7) which encodes the FR8a-9F chimeric protein (SEQ ID NO: 8). Thus, the FR8a-9F chimeric insecticidal protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRP (SEQ ID NO: 129), amino acids 1-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable regions 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6; and 38 amino acids of the Cry1Ab protoxin tail region.
[0255] The FR8a-9F eHIP was slightly less active against western corn rootworm than the FR8a eHIP (See Table 5) suggesting that the C-terminal 9 amino acids of the peptidyl fragment of SEQ ID NO: 127 play a role in conferring full insecticidal activity to FR8a.
Example 7
Construction of FR-9F-catg
[0256] The FR-9F-catg coding sequence was created to place the out-of-frame cry3A055 derived nucleotides of FR8a back in frame and to eliminate the cathepsin G protease recognition site. A BamHI/PpuMI fragment of 5'FR-9F/pCR2.1 (See Example 6) was ligated with a PpuMI/BamHI fragment of FRCG (See Example 5) to create the FR-9F-catg coding sequence (SEQ ID NO: 9) which encodes the FR-9F-catg chimeric protein (SEQ ID NO: 10). Thus, the FR-9F-catg chimeric protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRP (SEQ ID NO: 129), amino acids 1-467 of a Cry3Aa protein (SEQ ID NO: 68), which comprises variable regions 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6; and 38 amino acids of the Cry1Ab protoxin tail region.
[0257] The FR8a-9F-catg eHIP provided the same level of activity as FR8a against western corn rootworm (See Table 5) confirming that an eHIP can be made from either a modified Cry3A or a native Cry3 sequence.
Example 8
Construction of FR8a-12aa
[0258] The nucleotides encoding amino acids 2-13 of the peptidyl fragment comprised in
[0259] FR8a (SEQ ID NO: 4) were removed using PCR. A fragment was PCR amplified from a plasmid comprising FR8a (SEQ ID NO: 3) using primers 5'FR8a-12aa (SEQ ID NO: 94) and C1-3A-2 (SEQ ID NO: 90) and PCR reaction Mix 1 and thermocycle Profile 1. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen). A BamHI-PpuMI fragment from the pCR2.1-TOPO clone was then ligated with a PpuMI-BamHI fragment from a plasmid comprising FR8a to create FR8a-12aa (SEQ ID NO: 11) which encodes the FR8a-12aa chimeric insecticidal protein (SEQ ID NO: 12). Thus, the FR8a-12aa chimeric insecticidal protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MYDGRQQHRG (SEQ ID NO: 128), amino acids 10-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable regions 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6; and 38 amino acids of the Cry1Ab protoxin tail region.
[0260] The FR8a-12aa eHIP provided the same level of activity as FR8a against western corn rootworm (See Table 5) suggesting that the N-terminal 12 amino acids of the peptidyl fragment of SEQ ID NO: 127 are not necessary for full insecticidal activity of FR8a.
Example 9
Construction of Wr-9mut
[0261] A nucleic acid fragment was PCR amplified from FR8a/pCR2.1 (Example 2) using primers 5'FR8a-12aa (SEQ ID NO: 94) and C1-3A-2 (SEQ ID NO: 92) and PCR reaction Mix 1 and thermocycle Profile 2. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen). A BamHI/PpuMI fragment was then ligated to a PpuMI/BamHI fragment of FR8a (SEQ ID NO: 3) to create Wr-9mut (SEQ ID NO: 13) which encodes the WR-9mut protein (SEQ ID NO: 14), which comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MYDGRQQHRG (SEQ ID NO: 128), and amino acids 10-598 of a Cry3A055 protein (SEQ ID NO: 70). Thus the WR-9mut protein is Cry3A055 with an N-terminal peptidyl fragment of the invention.
[0262] The WR-9mut protein was not active against western corn rootworm. Therefore, the addition of an N-terminal peptidyl fragment to a non-hybrid modified Cry3 a protein destroyed insecticidal activity. This suggests that there may be some interaction between the Cry1Ab C-terminal portion of FR8a and the N-terminal peptidyl fragment that confers full insecticidal activity to FR8a.
Example 10
Construction of FRD3
[0263] The 3' end of this coding sequence was made by PCR amplifying a fragment from a plasmid comprising FR8a (SEQ ID NO: 3) using primers C2-3A-3 (SEQ ID NO: 95) and 3'1Ab-dm3 (SEQ ID NO: 96) and PCR reaction Mix 2 and thermocycle Profile 2. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen). A 364 bp ApaI/SacI fragment of the cloned amplicon, designated 3'FRD3/pCR2.1, was ligated with a SacI/ApaI fragment of FR8a to create FRD3 (SEQ ID NO: 15) which encodes the FRD3 chimeric protein (SEQ ID NO: 16). The FRD3 chimeric protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises complete variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-610 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6. Thus, the FRD3 chimeric insecticidal protein is a variant of an FR8a chimeric insecticidal protein with the 38 amino acid region of the Cry1Ab protoxin tail deleted.
[0264] The FRD3 eHIP provided the same level of activity as FR8a against western corn rootworm (See Table 5) suggesting that the 38 amino acid protoxin tail region of FR8a is not necessary for full insecticidal activity.
Example 11
Construction of FR-12-cg-dm3
[0265] A 3082 bp SacI/PpuMI fragment from a plasmid comprising FR8a-12 (See Example 8), a 721 bp PpuMI/MluI fragment of FRCG (See Example 5) and a 923 bp MluI/SacI fragment of FRD3 (See Example 10) were combined to create the FR-12-cg-dm3 coding sequence (SEQ ID NO: 17) which encodes the FR-12-cg-dm3 chimeric protein (SEQ ID NO: 18). The FR-12-cg-dm3 chimeric protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MYDGRQQHRG (SEQ ID NO: 129), amino acids 10-467 of a Cry3Aa protein (SEQ ID NO: 70), which comprises complete variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-610 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6. Thus, the FR-12-cg-dm3 chimeric protein is a variant of FR8a with 12 N-terminal amino acids of the peptidyl fragment, the cathepsin G protease recognition site, and the 38 amino acid region of the Cry1Ab protoxin tail deleted.
[0266] The FR-12-cg-dm3 eHIP was not as active against western corn rootworm as FR8a (See Table 5) suggesting that some interaction between the C-terminal portion of FR8a and the N-terminal peptidyl fragment is required for full insecticidal activity.
Example 12
Construction of 9F-cg-del6
[0267] The 5' end of this coding sequence was made by PCR amplifying a fragment from a plasmid comprising FR-9F-catg (See Example 7) using primers 5'FR-del6 (SEQ ID NO: 97) and C1-3A-2 (SEQ ID NO: 92) and PCR reaction Mix 3 and thermocycle Profile 3. The resulting amplicon was cloned into pCR2.1-TOPO. A 215 bp BamHI/PpuMI fragment was then ligated with a 4668 bp PpuMI/BamHI fragment of FR-9F-catg to create FR-9F-cg-del6 (SEQ ID NO: 19) which encodes the FR-9F-cg-del6 chimeric protein (SEQ ID NO: 20). The FR-9F-cg-del6 chimeric protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MCAGIRP (SEQ ID NO: 130), amino acids 1-467 of a Cry3A protein (SEQ ID NO: 68), which comprises variable regions 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6; and 38 amino acids of the Cry1Ab protoxin tail region. Thus, the FR-9F-cg-del6 chimeric protein is a variant of FR8a-9F-catg with amino acids 2 to 7 of the peptidyl fragment deleted.
[0268] The FR-9F-cg-del6 was not active against western corn rootworm suggesting that the N-terminal peptidyl fragment needs at least 7 amino acids of the C-terminal 9 amino acids of SEQ ID NO: 127 to be active against western corn rootworm.
Example 13
Construction of FR-cg-dm3
[0269] A 3839 bp MluI/SacI fragment of FRCG (Example 5) and a 923 bp MluI/SacI fragment of FRD3 (Example 10) were ligated to create FR-cg-dm3 (SEQ ID NO: 21) which encodes the FR-cg-dm protein (SEQ ID NO: 22). The FR-cg-dm3 chimeric insecticidal protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-467 of a Cry3A protein (SEQ ID NO: 68), which comprises variable regions 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-610 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6.
[0270] The FRD3 eHIP the same level of activity against western corn rootworm as FR8a (See Table 5) confirming that the cathepsin G site and the protoxin tail region of FR8a were not required for full insecticidal activity against western corn rootworm.
Example 14
Construction of 9F-cg-dm3
[0271] A MluI/SacI fragment from a plasmid comprising FR-9F-cg (See Example 7) was ligated with a 923 bp MluI/SacI fragment from a plasmid comprising FRD3 (See Example 10) to create 9F-cg-dm3 (SEQ ID NO: 23) which encodes the 9F-cg-dm3 chimeric protein (SEQ ID NO: 24). The 9F-cg-dm3 protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRP (SEQ ID NO: 129), amino acids 1-467 of a Cry3A protein (SEQ ID NO: 68), which comprises variable regions 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-610 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6.
[0272] The 9F-cg-dm3 eHIP provided the same level of activity against western corn rootworm (See Table 5) confirming that the C-terminal 9 amino acids of the peptidyl fragment could confer activity when domain I of the eHIP was comprised of either modified Cry3A (Cry3A055) variable regions and conserved blocks or Cry3A variable regions and conserved blocks.
Example 15
Construction of B8a
[0273] A nucleic acid fragment encoding an N-terminal portion of a Cry3A055 protein (SEQ ID NO: 70), was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C3-3A-8 (SEQ ID NO: 99) and PCR reaction Mix 1 and thermocycling Profile 1. A nucleic acid fragment encoding a C-terminal portion of a Cry1Ab protein (SEQ ID NO: 72), comprising variable regions 4-6, was amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C3-3A-7 (SEQ ID NO: 100) and 1Ab-6-sac (SEQ ID NO: 86) and PCR reaction Mix 1 and thermocycling Profile 1. The resulting amplicon was designated 2OL-8b.
[0274] A nucleic acid fragment encoding an N-terminal portion of the Cry3A055 protein (SEQ ID NO: 70), was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C2-3A-4 (SEQ ID NO: 88) and PCR reaction Mix 1 and thermocycling Profile 1.
[0275] A nucleic acid fragment encoding a C-terminal portion of a Cry1Ba protein (SEQ ID NO: 74) was PCR amplified from a plasmid comprising mocry 1Ba (SEQ ID NO: 73) using primers 1B-5 (SEQ ID NO: 101) and 1B-10 (SEQ ID NO: 102) and PCR reaction Mix 1 and thermocycling Profile 1, except a 60.degree. C. annealing temperature was used.
[0276] The two above-described PCR products were then used as the templates in an overlap PCR reaction with primers 5'3A-1-bam (SEQ ID NO: 83) and 1B-10 (SEQ ID NO: 102) using PCR reaction Mix 1 and thermocycling Profile 2. The resulting amplicon was designated B10.
[0277] Next, a nucleic acid fragment of cry3A055 (SEQ ID NO: 69) was PCR amplified using 2OL-8b (see above) as the template and primers 5'A-1-bam (SEQ ID NO: 83) and 3A-22 (SEQ ID NO: 103) with the following PCR conditions: Mix 1, thermocycling profile: 94.degree. C.-45 seconds, 50.degree. C-70.degree. C. gradient-45 seconds, 72.degree. C-90 seconds for 30 cycles. Another nucleic acid fragment was PCR amplified using BIO (see above) as the template and primers 1B-7 (SEQ ID NO: 104) and 1B-10 (SEQ ID NO: 102) using PCR reaction Mix 1 and thermocycling Profile 2, except a 60.degree. C. annealing temperature was used. The two resulting PCR products were then used as templates in an overlap PCR reaction with primers 5'3A-1-bam (SEQ ID NO: 83) and 1B-10 (SEQ ID NO: 102) using the following PCR conditions: Mix 2, thermocycling profile: 94.degree. C.-30 seconds, 40.degree. C.-60.degree. C. gradient-30 seconds, 72.degree. C.-60 seconds for 30 cycles.
[0278] The resulting PCR product was ligated to a pCR2.1-TOPO vector (Invitrogen) and designated B10/pCR2.1. A BamHi-SacI fragment from B8a/pCR2.1 was then ligated to pET21a (Novagen), which was cut with BamHi/SacI, to create the B8a coding sequence (SEQ ID NO: 25), which encodes a B8a hybrid toxin (SEQ ID NO: 26). The B8a hybrid protein comprises, from N-terminus to C-terminus, amino acids 1-468 of a Cry3A055 protein (SEQ ID NO: 70), and amino acids 505-656 of a Cry1Ba protein (SEQ ID NO: 74).
Example 16
Construction of 5*B8a
[0279] A BamHI-XbaI fragment from a plasmid comprising 2OL-8a (See Example 3) and a XbaI-SacI fragment from a plasmid comprising B8a (See Example 15) were ligated to create 5*B8a (SEQ ID NO: 27), which encodes the 5*B8a chimeric protein (SEQ ID NO: 28). The 5*B8a protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-467 of a Cry3A055 protein (SEQ ID NO: 70) and amino acids 505-656 of a Cry1Ba protein (SEQ ID NO: 74). Thus, the 5*B8a chimeric protein is the B8a hybrid protein to which an N-terminal peptidyl fragment has been added.
Example 17
Construction of V3A
[0280] This gene was PCR amplified using 3 fragments together as templates: the first fragment was amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C2-3A-4 (SEQ ID NO: 88) and PCR reaction Mix 1 and thermocycling Profile 1; the second fragment was amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C2-3A-3 (SEQ ID NO: 95) and C3-1Ab-2 (SEQ ID NO: 105) and PCR reaction Mix 1 and thermocycling Profile 1; and the third fragment was amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers C3-3A-5 (SEQ ID NO: 106) and 3A-12-sac (SEQ ID NO: 107) and PCR reaction Mix 1 and thermocycling Profile 1. These 3 PCR products were then used as templates in an overlap PCR reaction with primers 5'3A-1-bam (SEQ ID NO: 83) and 3A-12-sac (SEQ ID NO: 107) using PCR reaction Mix 1 and thermocycling Profile 1, to produce the v3A coding sequence (SEQ ID NO: 29), which encodes the V3A hybrid protein (SEQ ID NO: 30). The V3A hybrid protein comprises, from N-terminus to C-terminus, amino acids 1-226 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable region 1, conserved block 1, variable region 2, and the N-terminal 34 amino acids of conserved block 2, amino acids 237-474 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 33 amino acids of conserved block 2, variable region 3, and the N-terminal 20 amino acids of conserved block 3, and amino acids 467-598 of a Cry3A055 protein (SEQ ID NO: 70), which comprises the C-terminal 28 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, and conserved block 5.
[0281] The V3A eHIP comprises two crossover positions. The first crossover between Cry3A055 and Cry1Ab is located in conserved block 2 and the second crossover between Cry1Ab and Cry3A055 is located in conserved block 3. Therefore, V3A is a variant of Cry3A055 in which all of variable region 3 has been replaced with variable region 3 of a Cry1Ab protein. The V3A eHIP was not as active against western corn rootworm as FR8a, suggesting that having Cry1Ab sequence in conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5 and/or variable region 6 is important for full insecticidal activity of FR8a.
[0282] The v3A coding sequence was ligated to a pCR2.1-TOPO vector and then subcloned into pET21a using a BamHI/SacI fragment. The V3A protein expressed by the pET21a vector has a T7 tag on the N-terminus. This protein was designated T7-V3A.
Example 18
Construction of V4F
[0283] A first nucleic acid fragment encoding variable regions 1-3 of a Cry3A055 was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C3-3A-6 (SEQ ID NO: 84) and PCR reaction Mix 1 and thermocycling Profile 1.
[0284] A second nucleic acid fragment encoding variable region 4 of a Cry1Ab was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C3-1Ab-3 (SEQ ID NO: 85) and C4-3A-10 (SEQ ID NO: 108) and PCR reaction Mix 1 and thermocycling Profile 1.
[0285] A third nucleic acid fragment encoding variable regions 5-6 of Cry3A055 was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers C4-3A-9 (SEQ ID NO: 109) and 3A-12-sac (SEQ ID NO: 107) and PCR reaction Mix 1 and thermocycling Profile 1.
[0286] All three PCR amplicons were combined and used as the template in an overlap PCR reaction with primers 5'3A-1-bam (SEQ ID NO: 83) and 3A-12-sac (SEQ ID NO: 107) using the following PC conditions: Mix 1 and thermocycling profile: 94.degree. C.-30 seconds, 50.degree. C-70.degree. C. gradient-30 seconds, 72.degree. C.-30 seconds for 20 cycles. The resulting amplicon, designated the v4F coding sequence (SEQ ID NO: 31) which encodes the V4F hybrid toxin (SEQ ID NO: 32), was cloned into a pCR2.1-TOPO vector and designated v4F/pCR2.1 . The V4F hybrid protein comprises, from N-terminus to C-terminus, amino acids 1-468 of a Cry3A055 protein (SEQ ID NO: 70), amino acids 477-520, comprising variable region 4, of a Cry1Ab protein (SEQ ID NO: 72), and amino acids 512-598 of a Cry3A055 protein (SEQ ID NO: 70).
[0287] The V4F protein has two crossover positions. The first crossover between Cry3A055 and Cry1Ab is in conserved block 3 and the second crossover between Cry1Ab and Cry3A055 is located in conserved block 4. Therefore, V4F is a variant of Cry3A055 in which all of variable region 4 has been replaced with variable region 4 of a Cry1Ab protein. The V4F hybrid protein was not active against western corn rootworm suggesting that Cry1Ab sequence at the C-terminal portion of FR8a contributes to the insecticidal activity of FR8a.
[0288] A BamHI-SacI fragment of v4F/pCR2.1 was subcloned into pET21. The protein expressed by the resulting plasmid was designated T7-V4F.
Example 19
Construction of 5*V4F
[0289] A BamHI-XbaI fragment from a plasmid comprising FR8a (See Example 4) and a XbaI-SacI fragment from V4F/pCR2.1 (See Example 18) were ligated to pET21 cut with BamHI-SacI to form 5*V4F/pET21. The 5*V4F coding sequence (SEQ ID NO: 33) encodes the 5*V4F chimeric protein (SEQ ID NO: 34). The 5*V4F chimeric insecticidal protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-491 of a Cry3A055 protein (SEQ ID NO: 70), amino acids 501-520, comprising variable region 4, of a Cry1Ab protein (SEQ ID NO: 72), and amino acids 512-598 of a Cry3A055 protein (SEQ ID NO: 70).
[0290] The 5*V4F eHIP is the V4F hybrid protein with an N-terminal peptidyl fragment (SEQ ID NO: 127) added. The 5*V4F eHIP provided insecticidal activity against western corn rootworm although not at the same level as FR8a. Thus, the N-terminal conferred insecticidal activity to V4F confirming that there may be some contributory interaction between the C-terminal portion and the N-terminal peptidyl fragment of FR8a.
[0291] The protein expressed by the 5*V4F/pET21 plasmid was designated T7-5*V4F and has a T7 tag N-terminal to the 5*V4F peptidyl fragment.
Example 20
Construction of 2OL-7
[0292] A nucleic acid fragment encoding variable region 1 of Cry3A055 was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C1-3A-2 (SEQ ID NO: 92) and PCR reaction Mix 1 and thermocycling Profile 1.
[0293] A nucleic acid fragment encoding variable regions 2-6 of Cry1Ab was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C1-1Ab-1 (SEQ ID NO: 110) and 1Ab-6-sac (SEQ ID NO: 86) and PCR reaction Mix 1 and thermocycling Profile 1.
[0294] The resulting two amplicons were used as templates in an overlap PCR reaction with primers 5'3A-1-bam (SEQ ID NO: 83) and 1 Ab-6-sac (SEQ ID NO: 86) using PCR reaction Mix 2 and thermocycling Profile 1, to create the 2OL-7 coding sequence (SEQ ID NO: 35) which encodes the 2OL-7 hybrid protein (SEQ ID NO: 36). The 2OL-7 hybrid protein comprises, from N-terminus to C-terminus, amino acids 1-156 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable region 1 and the N-terminal 14 amino acids of conserved block 1, and amino acids 167-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 15 amino acids of conserved block 1, variable region 2, conserved block 2, variable region 3, conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5 and variable region 6, and 38 amino acids of the Cry1Ab protoxin tail region Thus, 2OL-7 is a variant of a Cry1Ab protein with variable region 1 replaced by variable region 1 from a Cry3A055 protein.
[0295] The 2OL-7 coding sequence was cloned into pCR2.1-TOPO (Invitrogen) and then moved into pET21a using BamHI/SacI which was designated 2OL-7/pET21a. The coding sequence in 2OL-7pET21a was designated T7-2OL-7 (SEQ ID NO: 37). The protein expressed by the 2OL-7/pET21a vector was designated T7-2OL-7 (SEQ ID NO: 38).
Example 21
Construction of 5*2OL-7
[0296] A BamHI/XbaI fragment of FR8a (See Example 4), a PpuMI/SacI fragment of 2OL-7 (See Example 20) and a BamHI/SacI fragment of pET21a were ligated to produce 5*2OL-7/pET21a. The 5*2OL-7 coding sequence (SEQ ID NO: 39) encodes the 5*2OL-7 chimeric protein (SEQ ID NO: 40). The 5*2OL-7 protein comprises, from N-terminus to C-terminus, a peptidyly fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-156 of a Cry3A055 protein (SEQ ID NO: 70), and amino acids 167-643 of a Cry1Ab protein (SEQ ID NO: 72). Thus, the 5*2OL-7 hybrid protein is the 2OL-7 hybrid protein with a N-terminal peptidyl fragment added.
Example 22
Construction of 2OL-10
[0297] A nucleic acid fragment encoding an N-terminal portion of a Cry3A055 protein was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C2-3A-4 (SEQ ID NO: 88) and PCR reaction Mix 1 and thermocycling Profile 1. A nucleic acid fragment encoding a C-terminal portion of a Cry1Ab protein was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C2-3A-3 (SEQ ID NO: 95) and 1Ab-6-sac (SEQ ID NO: 86) and PCR reaction Mix 1 and thermocycling Profile 1. These 2 PCR products were then used as the templates in an overlap PCR reaction with primers 5'3A-1-bam (SEQ ID NO: 83) and 1Ab-6-sac (SEQ ID NO: 86) using the following PCR conditions: Mix 2, thermocycling profile: 94.degree. C.-30 seconds, 45.degree. C-65.degree. C. gradient-30 seconds, 72.degree. C.-30 seconds for 20 cycles, resulting in the 2OL-10 coding sequence (SEQ ID NO: 41) which encodes the 2OL-10 hybrid toxin (SEQ ID NO: 42). The 2OL-10 protein comprises, from N-terminus to C-terminus, amino acids 1-232 of a Cry3A055 protein (SEQ ID NO: 70) and amino acids 243-648 of a Cry1Ab protein (SEQ ID NO: 72). Thus, the 2OL-10 hybrid protein is substantially Domain I of a Cry3A055 protein and Domains II and III of a Cry1Ab protein.
[0298] The 2OL-10 coding sequence was cloned into pCR2.1-TOPO (Invitrogen) then moved to pET21a using BamHI/SacI. The protein expressed by 2OL-10/pET21a was designated T7-2OL-10.
Example 23
Construction of 5*2OL-10
[0299] A BamHI-XbaI fragment from a plasmid comprising FR8a (See Example 4) and a XbaI-SacI fragment from 2OL-10/pCR2.1 (See Example 22) were ligated to pET21 cut with BamHI-SacI to form 5*2OL-10/pET21. The 5*2OL-10 coding sequence (SEQ ID NO: 43) encodes the 5*2OL-10 chimeric protein (SEQ ID NO: 44). The 5*2OL-10 protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-232 of a Cry3A055 protein (SEQ ID NO: 70) and amino acids 243-648 of a Cry1Ab protein (SEQ ID NO: 72). Thus, the 5*2OL-10 chimeric protein is the 2OL-10 hybrid protein with a N-terminal peptidyl fragment added.
Example 24
Construction of 2OL-12A
[0300] A first nucleic acid fragment encoding an N-terminal portion of Cry1Ab was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers 5'1Ab-bam (SEQ ID NO: 98) and C3-1Ab-2 (SEQ ID NO: 105) and PCR reaction Mix 1 and thermocycling Profile 1.
[0301] A second nucleic acid fragment encoding a C-terminal portion of Cry3A055 was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers C3-3A-5 (SEQ ID NO: 106) and 3A-12-sac (SEQ ID NO: 107) and PCR reaction Mix 1 and thermocycling Profile 1.
[0302] The first and second nucleic acid fragment described above were connected by using them as templates in an overlap PCR reaction with primers 5'1Ab-bam (SEQ ID NO: 98) and 3A-12-sac (SEQ ID NO: 107) using Mix 1 and thermocycling Profile 1 to create the 2OL-12A coding sequence (SEQ ID NO: 45) which encodes the 2OL-12A eHIP (SEQ ID NO: 46). The 2OL-12A protein comprises, from N-terminus to C-terminus, amino acids 1-476 of a Cry1Ab protein (SEQ ID NO: 72) and amino acids 469-598 of a Cry3A055 protein (SEQ ID NO: 70).
[0303] The 2OL-12A eHIP was not active against western corn rootworm but was active against European corn borer (See Table 6). This demonstrates that eHIP can be constructed using lepidopteran active and coleopteran active Cry proteins without loss of activity against a lepidopteran insect species.
[0304] The 2OL-12A coding sequence was cloned into pCR2.1-TOPO (Invitrogen) then moved to pET21a with BamHI/SacI. The protein expressed by the 2OL-12A/pET21a vector was designated T7-2OL-12A. The
Example 25
Construction of 2OL-13
[0305] Four nucleic acid fragments were generated as follows: fragment 1 was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C1-3A-2 (SEQ ID NO: 92) and PCR reaction Mix 1 and thermocycling Profile 1; fragment 2 was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C2-3A-3 (SEQ ID NO: 95) and C3-1Ab-2 (SEQ ID NO: 105) and PCR reaction Mix 1 and thermocycling Profile 1; fragment 3 was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C3-1Ab-3 (SEQ ID NO: 85) and C4-3A-10 (SEQ ID NO: 108) and PCR reaction Mix 1 and thermocycling Profile 1; and fragment 4 was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers C4-3A-9 (SEQ ID NO: 109) and 3A-12-sac (SEQ ID NO: 107) and PCR reaction Mix 1 and thermocycling Profile 1.
[0306] All four fragments were then used as templates in an overlap PCR reaction using primers 5'3A-bam (SEQ ID NO: 83) and 3A-12-sac (SEQ ID NO: 107) using PCR reaction Mix 1 and thermocycling Profile 1 to create the 2OL-13 coding sequence (SEQ ID NO: 47) which encodes the 2OL-13 hybrid toxin (SEQ ID NO: 48). The 2OL-13 protein comprises, from N-terminus to C-terminus, amino acids 1-159 of a Cry3A055 protein (SEQ ID NO: 70), amino acids 170-522 of a Cry1Ab protein (SEQ ID NO: 72), and amino acids 515-598 of a Cry3A055 protein (SEQ ID NO: 70). Thus, the 2OL-13 hybrid toxin is comprised of V1 and the N-terminal portion of CB1 from a Cry3A055 protein; the C-terminal portion of CB1, V2, CB2, V3, CB3, and V4 from a Cry1Ab protein; and CB4, V5, and CB5 from a Cry3A055 protein.
[0307] The 2OL-13 coding sequence was cloned into pCR2.1-TOPO (Invitrogen) then moved to pET21a using BamHI/SacI. The protein expressed by the 2OL-13/pET21a vector was designated T7-2OL-13.
Example 26
Construction of 2OL-20
[0308] A BamHI/NspI fragment from a plasmid comprising mocry3A (SEQ ID NO: 67), a NspI/HindIII fragment from a plasmid comprising 2OL-8A (SEQ ID NO: 1), and a HindIII/BamHI fragment from pET21a were ligated to make 2OL-20/pET21a.
Example 27
Construction of V5&6
[0309] A nucleic acid fragment encoding an N-terminal portion of Cry3A055 was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C4-3A-10 (SEQ ID NO: 108) and PCR reaction Mix 1 and thermocycling Profile 1.
[0310] A nucleic acid fragment encoding a C-terminal portion of Cry1Ab was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C4-3A-9 (SEQ ID NO: 109) and 1Ab-6-sac (SEQ ID NO: 86) and PCR reaction Mix 1 and thermocycling Profile 1.
[0311] These two PCR products were then used as the templates in an overlap PCR reaction with primers 5'3A-1-bam (SEQ ID NO: 83) and 1Ab-6-sac (SEQ ID NO: 86) using PCR reaction Mix 1 and thermocycling Profile 2 to create the V5&6 coding sequence (SEQ ID NO: 49), which encodes the V5&6 hybrid toxin (SEQ ID NO: 50). The V5&6 protein comprises, from N-terminus to C-terminus, amino acids 1-524 of a Cry3A055 protein (SEQ ID NO: 70), which comprises V1, CB1, V2, CB2, V3, CB3, V4, and CB4, and amino acids 533-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises V5, CB5 and V6, and 38 amino acids of a Cry1Ab protoxin tail region.
[0312] The V5&6 coding sequence was cloned into pCR2.1-TOPO then moved to pET21 with BamHI/SacI. The protein expressed by V5&6/pET21a was designated T7-V5&6.
Example 28
Construction of 5*V5&6
[0313] A BamHI/XbaI fragment of FR8a (See Example 4), a XbaI/SacI fragment of V5 &6 (See Example 27) and a BamHI/SacI fragment of pET21a were ligated to form 5*V5&6/pET21. The 5*V5&6 coding sequence (SEQ ID NO: 51) encodes the 5*V5&6 chimeric protein (SEQ ID NO: 52). The 5*V5&6 chimeric insecticidal protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-524 of a Cry3A055 protein (SEQ ID NO: 70), which comprises V1, CB1, V2, CB2, V3, CB3, V4, and CB4, and amino acids 533-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises V5, CB5 and V6, and 38 amino acids of a Cry1Ab protoxin tail region. Thus, the 5*V5&6 chimeric insecticidal protein is the V5&6 hybrid protein with an N-terminal peptidyl fragment added.
Example 29
Construction of 88A-dm3
[0314] A nucleic acid fragment encoding a C-terminal portion of a Cry8Aa protein (SEQ ID NO: 78) was PCR amplified from a plasmid comprising cry8Aa (SEQ ID NO: 77) using primers 5'8Aa-dm3 (SEQ ID NO: 111) and 3'8Aa-dm3 (SEQ ID NO: 112) and PCR reaction Mix 2 and thermocycling Profile 2. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen) and designated 88A-dm3/pCR2.1.
[0315] A MluI/SacI fragment from 88A-dm3/pCR2.1 and a SacI/MluI fragment from a plasmid comprising FR8a (See Example 4) were ligated to create the 88A-dm3 coding sequence (SEQ ID NO: 53) which encodes the 88A-dm3 hybrid protein (SEQ ID NO: 54). The 88A-dm3 protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 of a Cry3A055 protein (SEQ ID NO: 70) and amino acids 532-664 of a Cry8Aa protein (SEQ ID NO: 78).
[0316] The 88A-dm3 coding sequence was also transformed into pET21a using a BamHI/SacI restriction digest and ligation. The protein expressed by 88A-dm3/pET21a was designated T7-88A-dm3.
Example 30
Construction of FR(1Fa)
[0317] A nucleic acid fragment encoding an N-terminal portion of FR8a (See Example 3) was PCR amplified from a plasmid comprising FR8a (SEQ ID NO: 1) using primers C2-3A-3 (SEQ ID NO: 95) and tant-OL-2 (SEQ ID NO: 113) and PCR reaction Mix 3 and thermocycling Profile 3.
[0318] A nucleic acid fragment encoding a C-terminal portion of a Cry1Fa protein (SEQ ID NO: 76) was PCR amplified from a plasmid comprising mocry 1Fa (SEQ ID NO: 75) using primers tant-OL-1 (SEQ ID NO: 114) and tant-3'sac (SEQ ID NO: 115) and PCR reaction Mix 3 and thermocycling Profile 3.
[0319] These two PCR products were then used as templates in an overlap PCR reaction with primers C2-3A-3 (SEQ ID NO: 95) and tant-3'sac (SEQ ID NO: 115) using PCR reaction Mix 3 and thermocycling Profile 3. The resulting PCR product was cloned into pCR2.1-TOPO (Invitrogen). A BamHI/MluI fragment from a plasmid comprising FR8a, a Mlul/SacI fragment from the overlap PCR product in pCR2.1 and a BamHI/SacI fragment of pET21a were then ligated to create FR(1Fa)/pET21a. The FR(1Fa) coding sequence (SEQ ID NO: 55) encodes the FR(1Fa) chimeric protein (SEQ ID NO: 56). The FR(1Fa) protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 of a Cry3A055 protein (SEQ ID NO: 70) and amino acids 470-649 of a Cry1Fa protein (SEQ ID NO: 76).
Example 31
Construction of FR(1Ac)
[0320] Domains I & II of FR8a were PCR amplified from a plasmid comprising FR8a (SEQ ID NO: 1) using primers C2-3A-3 (SEQ ID NO: 95) and 1Ac-OL-2 (SEQ ID NO: 116) and PCR reaction Mix 3 and thermocycling Profile 3. Domain III of Cry1Ac (SEQ ID NO: 80) was PCR amplified from a plasmid comprising cry1Ac (SEQ ID NO: 79) using primers 1Ac-OL-1 (SEQ ID NO: 117) and 1Ac-3'sac (SEQ ID NO: 118) and PCR reaction Mix 3 and thermocycling Profile 3.
[0321] These 2 PCR products were used as templates in an overlap PCR reaction with primers C2-3A-3 (SEQ ID NO: 95) and 1Ac-3'sac (SEQ ID NO: 118) and the following conditions: Mix 3 and thermocycling profile: 94.degree. C.-30 seconds, 68.degree. C.-30 seconds, 68.degree. C.-30 seconds for 20 cycles. The overlap PCR product was cloned into pCR2.1-TOPO (Invitrogen). A BamHI/MluI fragment from a plasmid comprising FR8a, the MluI/SacI fragment from the overlap PCR product in pCR2.1 and BamHI/SacI fragment of pET21a were ligated to create FR(lAc)/pET21a. The FR(lAc) protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 of a Cry3A055 protein (SEQ ID NO: 70) and amino acids 477-608 of a Cry1Ac protein (SEQ ID NO: 80).
Example 32
Construction of FR(1I a)
[0322] A nucleotide fragment encoding Domains I and II of FR8a was PCR amplified from a plasmid comprising FR8a (SEQ ID NO: 3) using primers C2-3A-3 (SEQ ID NO: 95) and 1Ia-OL-2 (SEQ ID NO: 119) and PCR reaction Mix 3 and thermocycling Profile 3. A second nucleotide fragment encoding Domain III of a Cry1Ia protein (SEQ ID NO: 82) was PCR amplified from a plasmid comprising cry1Ia (SEQ ID NO: 81) using primers 1Ia-OL-1 (SEQ ID NO: 120) and 1 Ia-3'sac (SEQ ID NO: 121) and PCR reaction Mix 3 and thermocycling Profile 3. These two PCR products were used as templates in an overlap PCR reaction with primers C2-3A-3 (SEQ ID NO: 95) and 1Ia-3` sac (SEQ ID NO: 121) and PCR reaction Mix 3 and thermocycling profile: 94.degree. C.-30 seconds, 68.degree. C.-45 seconds for 20 cycles. The overlap PCR product was cloned into pCR2.1-TOPO (Invitrogen). The BamHI/MluI fragment from a plasmid comprising FR8a, the MluI/SacI fragment from the overlap PCR product in pCR2.1 and BamHI/SacI fragment of pET21a were ligated to create FR(lIa)/pET21a. The FR(lIa) protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 of a Cry3A055 protein (SEQ ID NO: 70) and amino acids 513-719 of a Cry1Ia protein (SEQ ID NO: 82).
Example 33
Construction of Dm2-3A
[0323] Part of the 5' end of this coding sequence was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers C2-3A-3 (SEQ ID NO: 95) and FR-1Ab-2 (SEQ ID NO: 122) and PCR reaction Mix 3 and thermocycling Profile 2. A nucleotide fragment encoding Domain III of Cry1Ab was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers FR1Ab-1 (SEQ ID NO: 123) and 1Ab-6-sac (SEQ ID NO: 86) and PCR reaction Mix 3 and thermocycling Profile 2. These two PCR products were used as the templates in an overlap PCR reaction with primers C2-3A-3 (SEQ ID NO: 95) and 1Ab-6-sac (SEQ ID NO: 86) and PCR reaction Mix 3 and thermocycling Profile 2. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen). FR8a BamHI/MluI, and the above PCR product in pCR2.1-TOPO AflIII, FR8a AflIII/SacI were ligated into pET21a BamHI/SacI. The entire coding sequence (BamHI/SacI) was then moved to 1454. The DM2-3A chimeric insecticidal protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-451 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 7 amino acids of conserved block 3, and amino acids 460-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 41 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, and variable region 6. Thus, the DM2-3A eHIP has a cross-over junction between Cry3A055 and Cry1Ab located in conserved block 3 immediately following Ser451 which is upstream of the domain II domain III junction. DM2-3A has insecticidal activity against western corn rootworm but the activity was less than that of the 8AF and FR8a eHIPs as shown in Table 5.
Example 34
Construction of T7-8AF
[0324] A nucleic acid fragment encoding an N-terminal portion of a Cry3A055 protein (SEQ ID NO: 70) was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO:69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C3-3A-6 (SEQ ID NO: 84) and PCR reaction Mix 1 and thermocycling Profile 1.
[0325] A nucleic acid fragment encoding a C-terminal portion of a Cry1Ab protein (SEQ ID NO: 72) was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C3-1Ab-3 (SEQ ID NO: 85) and 1Ab-6-Sac (SEQ ID NO: 86) and PCR reaction Mix 1 and thermocycle Profile 1.
[0326] The two above-described PCR products were next used as templates in an overlap PCR reaction with the primers 5'3A-1-bam (SEQ ID NO: 83) and 1Ab-6-Sac (SEQ ID NO: 86) using PCR reaction Mix 2 and thermocycling Profile 1.
[0327] The resulting amplicon was ligated as a blunt ended fragment to a pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) cut with Smal to form plasmid p8AF/CR2.1. A BamHI-SacI fragment from p8AF/CR2.1 was then ligated to pET21a (EMD Biosciences, Inc., San Diego, Calif.), which was cut with BamHI-SacI, and transformed into E. coli. The open reading frame was designated T7-8AF (SEQ ID NO: 144) and encodes the T7-8AF hybrid protein (SEQ ID NO: 145). The T7-8AF hybrid protein comprises, from N-terminus to C-terminus, a peptidyl fragment comprising the amino acid sequence MASMTGGQQMGRGS (amino acids 1-14 of SEQ ID NO: 126), amino acids 1-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5 and variable region 6, and a 38 amino acid region of the Cry1Ab protoxin tail. The T7-8AF hybrid protein had little or no insecticidal activity against western corn rootworm.
Example 35
Construction of 8AF
[0328] A BamHI-SacI fragment from plasmid p8AF/CR2.1 (See Example 34) was ligated to a plasmid containing a constitutive Cry1Ac promoter that has been modified from that described by Schnepf et al. (1985. J. Biol. Chem. 260:6264-6272) to correct an internal ATG start codon which exists in the promoter of Schnepf et al. to an ATC codon, which was cut with BamHI-SacI, and transformed into E. coli. The open reading frame was designated 8AF (SEQ ID NO: 63) and encodes the 8AF eHIP (SEQ ID NO: 64). The 8AF eHIP is similar to the FR8a eHIP but does not contain the optional N-terminal peptidyl fragment. The 8AF eHIP comprises, from N-terminus to C-terminus, amino acids 1-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 24 amino acids of conserved block 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5 and variable region 6, and a 38 amino acid region of a Cry1Ab protoxin tail. Thus, the 8AF eHIP has a cross-over junction between Cry3A055 and Cry1Ab located in conserved block 3 immediately following Leu468 of SEQ ID NO: 70 which is downstream of the domain II domain III junction. The 8AF eHIP had high activity against western corn rootworm.
Example 36
Construction of -catG8AF
[0329] A construct was made without the Cathepsin G (Cat G) site to determine whether the Cat G site in domain I of the 8AF eHIP was necessary for rootworm activity. A 1359 by BamHI/SalI fragment from a plasmid comprising moCry3A (SEQ ID NO: 67) and a 3483 by BamHI/SalI fragment from a plasmid comprising 2OL-8a (SEQ ID NO: 1) were ligated to create -catG8AF (SEQ ID NO: 146) which encodes the -catG8AF eHIP (SEQ ID NO: 147).
[0330] The -catG8AF eHIP was very active against western corn rootworm demonstrating that the Cathepsin G protease recognition site in the 8AF eHIP is not required for insecticidal activity.
Example 37
Construction of 8AFdm3
[0331] The 8AF eHIP described in Example 35 has a cross-over point between Cry3A055 and Cry1Ab located in CB3 downstream of the domain II/III junction, resulting in domain III of the 8AF eHIP having a small N-terminal region of domain III of Cry3A055 and the remainder of domain III being Cry1Ab domain III sequence. To determine whether the small N-terminal region of domain III of Cry3A055 was required for insecticidal activity in 8AF, another construct was made having the cross-over between Cry3A055 and Cry1Ab located in CB3 exactly at the domain II-domain III junction.
[0332] A nucleic acid fragment encoding part of domain I and domain II of Cry3A055 was PCR amplified from a plasmid comprising FR8a (SEQ ID NO: 3) using primers CMS96 (SEQ ID NO: 138) and CMS97 (SEQ ID NO: 139) and PCR reaction Mix 5 and thermocycle Profile 5.
[0333] A nucleic acid fragment encoding domain III of moCry1Ab was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers CMS98 (SEQ ID NO: 140) and CMS99 (SEQ ID NO: 141) and PCR reaction Mix 5 and thermocycle Profile 5.
[0334] The resulting two amplicons were used as templates in an overlap PCR reaction with primers CMS96 (SEQ ID NO: 138) and CMS98 (SEQ ID NO: 140) using PCR reaction Mix 5 and thermocycle Profile 6. The resulting amplicon was cloned into pCR4 Blunt (Invitrogen, Carlsbad, Calif.). A 1633 bp StuI/SacI fragment of the cloned amplicon, designated pCR4Blunt-OLWrdm3, and a approximately 3089 by StuI/SacI fragment of a plasmid comprising cry3A055 (SEQ ID NO: 69) were combined to create 8AFdm3 (SEQ ID NO: 148) which encodes the 8AFdm3 hybrid protein (SEQ ID NO: 149).
[0335] The 8AFdm3 hybrid protein comprises, from N-terminus to C-terminus, amino acids 1-454 of a Cry3A055 protein (SEQ ID NO: 70), which comprises domains I and II, which comprise variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 10 amino acids of conserved block 3, and amino acids 463-610 of a Cry1Ab protein (SEQ ID NO: 72), which comprises all of domain III, comprising the C-terminal 38 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5 and variable region 6.
[0336] Thus, the 8AFdm3 protein has a cross-over junction between Cry3A055 and Cry1Ab immediately after Phe454 of SEQ ID NO: 70, which is at the domain II-domain III junction. The 8AFdm3 protein had no activity against western corn rootworm. This suggests that the 24 amino acid N-terminal region of CB3 of Cry3A055 or Cry3A, since they have the same sequence in this region, are necessary for activity of an 8AF eHIP.
Example 38
Construction of 8AFlongdm3
[0337] To determine if the location of the cross-over junction in CB3 between Cry3A or Cry3A005 and Cry1Ab was critical for rootworm activity a construct was made wherein the cross-over junction was placed in CB4 immediately after amino acid 519 of a Cry3A055 protein.
[0338] A nucleic acid fragment encoding part of domain I and all of domain II and part of domain III of Cry3A055 was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) using primers CMS96 (SEQ ID NO: 138) and CMS101 (SEQ ID NO: 143) and PCR reaction Mix 5 and thermocycle Profile 5.
[0339] A nucleic acid fragment encoding part of domain III of Cry1Ab was PCR amplified froma plasmid comprising mocry1Ab(SEQ ID NO: 71) using primers CMS98 (SEQ ID NO: 140) and CMS100 (SEQ ID NO: 142) and PCR reaction Mix 5 and thermocycle Profile 5.
[0340] The resulting two amplicons were used as templates in an overlap PCR reaction with primers CMS96 (SEQ ID NO: 138) and CMS98 (SEQ ID NO: 140) using PCR reaction Mix 5 and thermocycle Profile 6. The resulting amplicon was cloned into pCR4 Blunt (Invitrogen, Carlsbad, Calif.). A approximately 460 bp SalI/SacI fragment of the cloned amplicon, designated pCR4Blunt-OL8AFlongdm3, and a approximately 4265 bp SalI/SacI fragment of a plasmid comprising 8AFdm3 (SEQ ID NO: 147) were combined to create 8AFlongdm3 (SEQ ID NO: 150) which encodes the 8AFlongdm3 hybrid protein (SEQ ID NO: 151).
[0341] The 8AFlongdm3 hybrid protein comprises, from N-terminus to C-terminus, amino acids 1-519 of a Cry3A055 protein (SEQ ID NO: 70), which comprises domains I and II, which comprise variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, conserved block 3, variable region 4, and the N-terminal 6 amino acids of conserved block 4, and amino acids 528-610 of a Cry1Ab protein (SEQ ID NO: 72), which comprises a C-terminal region of domain III, comprising the C-terminal 4 amino acids of conserved block 4, variable region 5, conserved block 5, and variable region 6.
[0342] Thus, the 8AFlongdm3 protein has a cross-over junction between Cry3A055 and Cry1Ab in conserved block 4 immediately after Ile519 of SEQ ID NO: 70. The 8AFlongdm3 hybrid Cry protein had no activity against western corn rootworm. This suggests that a critical region for corn rootworm activity of a Cry3A-Cry1A eHIP lies in a region between amino acids corresponding to amino acid 6 of CB3 to amino acid 7 of CB4.
Example 39
Construction of cap8AFdm3
[0343] A approximately 1363 bp BamHI/SalI fragment from a plasmid comprising 8AFdm3 (SEQ ID NO: 148) and a approximately 3362 bp BamHI/SalI fragment from a plasmid comprising FR8a (SEQ ID NO: 3) were ligated to create cap8AFdm3 (SEQ ID NO: 152) which encodes the cap8AFdm3 eHIP (SEQ ID NO: 153).
[0344] The cap8AFdm3 protein had some activity against western corn rootworm as indicated in Table 5. The only difference between the 8AFdm3 hybrid protein, which was not insecticidal, and the cap8AFdm3 eHIP is the presence of an N-terminal peptidyl fragment (SEQ ID NO: 127). Thus, adding a peptidyl fragment to a non-active hybrid Cry protein created a rootworm active engineered hybrid insecticidal protein.
Example 40
Construction of 8AFdm3T
[0345] A approximately 4654 bp Pm1I/SacI fragment from a plasmid comprising 8AFdm3 (SEQ ID NO: 148) and a approximately 190 bp Pm1I/SacI fragment from a plasmid comprising FR8a (SEQ ID NO: 3) were ligated to create 8AFdm3T (SEQ ID NO: 154) which encodes the 8AFdm3T eHIP (SEQ ID NO: 155). The 8AFdm3T eHIP comprises from N-terminus to C-terminus, amino acids 1-454 of a Cry3A055 protein (SEQ ID NO: 70), which comprises domains I and II, which comprise variable region 1, conserved block 1, variable region 2, conserved block 2, variable region 3, and the N-terminal 10 amino acids of conserved block 3, and amino acids 463-610 of a Cry1Ab protein (SEQ ID NO: 72), which comprises all of domain III, comprising the C-terminal 38 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, conserved block 5, variable region 6, and a 38 amino acid region of a Cry1Ab protoxin tail.
[0346] The only difference between the 8AFdm3 hybrid protein and the 8AFdm3T eHIP is the addition of the 38 amino acid Cry1Ab protoxin tail region indicating that addition of a protoxin tail region can change a non-active hybrid Cry protein into an active eHIP.
Example 41
Construction of 8AFlongdm3T
[0347] A approximately 4693 bp Pm1I/SacI fragment from a plasmid comprising 8AFlongdm3 (SEQ ID NO: 150) and a approximately 190 bp Pm1I/SacI fragment from a plasmid comprising FR8a (SEQ ID NO: 3) were ligated to create 8AFlongdm3T (SEQ ID NO: 156) which encodes the 8AFlongdmT hybrid Cry protein (SEQ ID NO: 157).
[0348] The only difference between the 8AFlongdm3 hybrid Cry protein and the 8AFlongdm3T hybrid Cry protein, which was not active against western corn rootworm, is the addition of a 38 amino acid Cry1Ab protoxin tail region indicating that the protoxin region was not itself sufficient to confer insecticidal activity to the 8AFlongdm3 hybrid Cry protein. This indicates that a combination of variable regions and conserved blocks in addition to a protoxin tail region and/or an N-terminal peptidyl fragment may be necessary to create some eHIPs.
Example 42
Construction of cap8AFdm3 T
[0349] A approximately 4693 bp Pm1I/SacI fragment from a plasmid comprising cap8AFdm3 (SEQ ID NO: 152) and a approximately 190 bp Pm1I/SacI fragment from a plasmid comprising FR8A (SEQ ID NO: 3) were ligated to create cap8AFdm3T (SEQ ID NO: 158) which encodes the cap8AFdm3T eHIP (SEQ ID NO: 159).
[0350] The cap8AFdm3T protein had increased activity against western corn rootworm over the cap8AFdm3 eHIP as indicated in Table 5. The only difference between the cap8AFdm3 eHIP, which had some insecticidal activity against corn rootworm, and the cap8AFdm3T eHIP is the presence of a 38 amino acid protoxin tail region from Cry1Ab. Thus, some hybrid Cry proteins can be made active by adding an N-terminal peptidyl fragment and a protoxin tail region.
Example 43
Testing hybrid proteins for insecticidal activity
Western Corn Rootworm
[0351] Hybrid proteins generated in the above described Examples were tested for insecticidal activity against western corn rootworm in laboratory bioassays. Bioassays were performed using a diet incorporation method. E. coli clones that express one of the proteins were grown overnight. 500 .mu.l of an overnight culture was sonicated and the amount of protein to be tested was determined. The protein solution was then mixed with 500 .mu.l of molten artificial diet similar to that described in Marrone et al. (1985, J. of Economic Entomology 78:290-293). After the diet solidified, it was dispensed in a petri-dish and 20 neonate corn rootworm were placed on the diet. The petri-dishes were held at approximately 30.degree. C. Mortality was recorded after 6 days.
[0352] Results of the bioassays are shown in Table 5. Column 1 indicates the names of the hybrid Cry proteins, engineered hybrid insecticidal proteins and chimeric insecticidal proteins. Column 2 indicates relative levels of western corn rootworm activity ("-"=<40% mortality; "+"=40-49% mortality; "++"=50-59% mortality; "+++"=60-80% mortality; and "++++"=>80% mortality). Column 3 indicates relative levels of the appropriate protein detected by Western blot. Column 4 indicates presence of a peptidyl fragment ("-"=No peptidyl fragment; #1=SEQ ID NO: 126; #2=SEQ ID NO: 127; #3=SEQ ID NO: 128; #4=SEQ ID NO: 129; #5=SEQ ID NO: 130; #6=SEQ ID NO: 131; #7=SEQ ID NO: 132). Columns 5-7 show the combinations and arrangement of the variable regions (V1-V6), conserved blocks (C1-C5) and associated domains (Domain I-III) from a first Bt Cry protein or modified Cry protein and a second Bt Cry protein different from the first Cry protein or modified Cry protein that make up a core hybrid protein, which are not active against western corn rootworm and eHIPs, which have activity against western corn rootworm. Column 8 indicates the number of amino acids in a protoxin tail region if present and the Cry protein from which the tail region is derived ("1Ab-38"=38 amino acids from a Cry1Ab protoxin tail; "1Ba-18"=18 amino acids from a Cry1Ba protoxin tail).
TABLE-US-00005 TABLE 5 Results of western corn rootworm bioassays. Domain Proteins CRW Protein Peptidyl Domain I II Domain III Protoxin Tested Activity Expressed Fragment V1 C1 V2 C2 V3 C3 V4 C4 V5 C5 V6 Region 8AF ++++ ++ -- 3A055 3A055 3A055 1Ab 1Ab-38 T7-8AF - + #7 3A055 3A055 3A055 1Ab 1Ab-38 -CatG8F ++++ ++ -- 3A 3A 3A 1Ab 1Ab-38 8APdm3 - + -- 3A055 3A055 1Ab -- 8APdm3T +++ ++ -- 3A055 3A055 1Ab 1Ab-38 8APlongdm3 - + -- 3A055 3A055 3A055 1Ab -- 8APlongdm3T - + -- 3A055 3A055 3A055 1Ab 1Ab-38 Cap8AFdm3 + + #2 3A055 3A055 1Ab -- Cap8AFdm3T ++ ++ #2 3A055 3A055 1Ab 1Ab-38 2OL-8a ++++ ++ #1 3A055 3A055 3A055 1Ab 1Ab-38 FR8a +34 ++++ ++ #6 3A055 3A055 3A055 1Ab 1Ab-38 PR8a ++++ ++ #2 3A055 3A055 3A055 1Ab 1Ab-38 FRCG ++++ ++ #2 3A 3A 3A 1Ab 1Ab-38 FR8a-9F +++ ++ #5 3A055 3A055 3A055 1Ab 1Ab-38 FR8a-9F-catg ++++ ++ #5 3A 3A 3A 1Ab 1Ab-38 FR8a-12aa ++++ ++ #3 3A055 3A055 3A055 1Ab 1Ab-38 Cry3A055 ++++ ++ -- 3A055 3A055 3A055 -- 5*Cry3A055 - ++ #2 3A055 3A055 3A055 -- Wr-9mut - ++ #3 3A055 3A055 3A055 -- FRD3 ++++ ++ #2 3A055 3A055 3A055 1Ab -- FR-12-cg-dm3 ++ ++ #3 3A055 3A055 3A055 1Ab -- 9F-cg-del6 - ++ #5 3A 3A 3A 1Ab 1Ab-38 FR-cg-dm3 ++++ ++ #2 3A 3A 3A 1Ab -- 9F-cg-dm3 ++++ ++ #5 3A 3A 3A 1Ab -- B8a - + -- 3A055 3A055 3A055 1Ba 1Ba-18 5*B8a - + #2 3A055 3A055 3A055 1Ba 1Ba-18 V3A ++ + -- 3A055 1Ab 3A055 -- V4F - ++ -- 3A055 3A055 1Ab 3A055 -- 5*V4F ++ + #2 3A055 3A055 1Ab 3A055 -- 2OL-7 - ++ -- 3A055 1Ab 1Ab 1Ab 1Ab-38 5*2OL-7 - + #2 3A055 1Ab 1Ab 1Ab 1Ab-38 2OL-10 - + -- 3A055 1Ab 1Ab 1Ab-38 5*2OL-10 +/- +/- #2 3A055 1Ab 1Ab 1Ab-38 2OL-12A - ++ -- 1Ab 1Ab 3A -- 2OL-13 - - -- 3A055 1Ab 1Ab 3A055 -- 2OL-20 - + -- 3A 3A 3A 1Ab 1Ab-38 V5&6 - ++ -- 3A055 3A055 3A055 1Ab 1Ab-38 5*V5&6 - ++ #2 3A055 3A055 3A055 1Ab 1Ab-38 88A-dm3 - ++ #2 3A055 3A055 3A055 8Aa -- PR(1Fa) - ++ #2 3A055 3A055 3A055 1Fa -- FR(1Ac) - + #2 3A055 3A055 3A055 1Ac -- FR(1Ia) - - #2 3A055 3A055 3A055 1Ia -- DM23A + + #2 3A055 3A055 3A055 1Ab 1Ab-38
[0353] The chimeric insecticidal proteins, 2OL-8a and FR8a, and the 2OL-12A eHIP, were tested against several insect species to determine spectrum of activity. The insects tested included western corn rootworm (WCR), northern corn rootworm (NCR), southern corn rootworm (SCR), Colorado potato beetle (CPB), and European corn borer (ECB). Results of the assays are shown in Table 6. A "+" indicates insecticidal activity. A "-" indicates no activity. The 2OL-8a and FR8a CIPs were active against WCR, NCR and CPB. The 2OL-12A eHIP was surprisingly active against ECB.
TABLE-US-00006 TABLE 6 Activity spectrum of CIPs. Activity Spectrum Protein WCR NCR SCR CPB ECB 2OL-8a + + - + - FR8a + + - + - 2OL-12A - nt nt nt + Cry3A055 + + - + - Cry3A - - - + - Cry1Ab - - - - +
Example 44
Insertion of Genes Encoding eHIPs into Plants
[0354] Three genes encoding the chimeric insecticidal proteins FR8a, FRCG and FRD3 were chosen for transformation into maize plants. An expression cassette comprising the FR8a or FRCG or FRD3 coding sequence was transferred to a suitable vector for Agrobacterium-mediated maize transformation. For this example, the following vectors were used in the transformation experiments: 12207 (FIG. 3), 12161 (FIG. 4), 12208 (FIG. 5), 12274 (FIG. 6), 12473 (FIG. 7) and 12474 (FIG. 8).
[0355] Transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19: 798-803. For this example, all media constituents were essentially as described in Negrotto et al., supra. However, various media constituents known in the art may be substituted.
[0356] The genes used for transformation were cloned into a vector suitable for maize transformation. Vectors used in this example contain the phosphomannose isomerase (PMI) gene for selection of transgenic lines (Negrotto et al., supra).
[0357] Briefly, Agrobacterium strain LBA4404 (pSB 1) containing a plant transformation plasmid was grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl (5 g/L), 15 g/l agar, pH 6.8) solid medium for 2-4 days at 28.degree. C. Approximately 0.8.times.10.sup.9 Agrobacterium were suspended in LS-inf media supplemented with 100 .mu.M As (Negrotto et al., supra). Bacteria were pre-induced in this medium for 30-60 minutes.
[0358] Immature embryos from A188 or other suitable genotype are excised from 8-12 day old ears into liquid LS-inf +100 .mu.M As. Embryos are rinsed once with fresh infection medium. Agrobacterium solution is then added and embryos are vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos are then transferred scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri plate are transferred to LSDc medium supplemented with cefotaxime (250 mg/l) and silver nitrate (1.6 mg/l) and cultured in the dark for 28.degree. C. for 10 days.
[0359] Immature embryos, producing embryogenic callus were transferred to LSD1M0.5S medium. The cultures were selected on this medium for about 6 weeks with a subculture step at about 3 weeks. Surviving calli were transferred to Regl medium supplemented with mannose. Following culturing in the light (16 hour light/8 hour dark regiment), green tissues were then transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Plantlets were transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium and grown in the light. After about 2-3 weeks, plants were tested for the presence of the pmi gene and the FR8a or FRCG genes by PCR. Positive plants from the PCR assay were transferred to the greenhouse and tested for resistance to corn rootworm.
Example 45
Analysis of Transgenic Maize Plants for Corn Rootworm Efficacy: Root Excision Bioassay
[0360] Typically, corn plants are sampled as they are being transplanted from Magenta GA-7 boxes into soil. This allows the roots to be sampled from a reasonably sterile environment relative to soil conditions. Sampling consists of cutting a small piece of root (ca. 2-4 cm long) and placing it onto enriched phytagar (phytagar, 12 g., sucrose, 9 g., MS salts, 3 ml., MS vitamins, 3 ml., Nystatin(25 mg/ml), 3 ml., Cefotaxime (50 mg/ml), 7 ml., Aureomycin (50 mg/ml), 7 ml., Streptomycin (50 mg/ml), 7 ml., dH.sub.2O, 600 ml) in a small petri-dish. Negative controls are either transgenic plants that are PCR negative for the FR8a or FRCG gene from the same transformation experiment, or from non-transgenic plants (of a similar size to test plants) that were being grown in the phytotron.
[0361] Roots are also sampled after plants have been growing in soil. If sampling roots from soil, the root pieces are washed with water to remove soil residue, dipped in Nystatin solution (5mg/m1), removed from the dip, blotted dry with paper toweling, and placed into a phytagar dish as above.
[0362] Root samples are inoculated with western corn rootworms by placing about 10 first instar larvae onto the inside surface of the lid of each phytagar dish and the lids then tightly resealed over the exposed root piece. Larvae are handled using a fine tip paintbrush. After all dishes were inoculated, the tray of dishes was placed in the dark at room temperature until data collection.
[0363] At about 2-4 days after root inoculation, data were collected. The percent mortality of the larvae was calculated along with a visual damage rating of the root. Feeding damage was scored by observing the number of feeding holes (FH) in the root piece caused by the rootworm larvae and was rated as high, moderate, low, or absent and given a numerical value of category 3, 2, or 1, respectively (with Category 1 including damage ratings of absent and/or low). Category 1 plants typically have 0-FH to 2-FH; Category 2 plants have 3 to 4-FH; and Category 3 plants have >5-FH. Root samples having a damage rating in Category 1 were considered excellent performers, category 2: average performers and category 3: poor performers. Category 1 plants were selected for further testing in the greenhouse and field.
[0364] Results in Table 7 show that plants expressing a the FR8a and FRCG eHIPs protected roots from feeding damage caused by western corn rootworm. A majority of events expressing the chimeric insecticidal protein were considered category 1 plants, whereas control plants not expressing a chimeric insecticidal protein were in category 3. Plants expressing the FRD3 eHIP provided comparable levels of control of western corn rootworm.
TABLE-US-00007 TABLE 7 Efficacy of transgenic plants expressing FR8a and FRCG against WCR. Damage Damage Rating Rating (No. (No. Catego- Vector Event FH) Category Vector Event FH) ry 12161 1 1 1 12207 1 0 1 2 1 1 2 2 1 3 0 1 3 1 1 4 3 2 4 2 1 5 2 1 5 1 1 6 0 1 6 2 1 7 0 1 7 4 2 8 0 1 8 3 2 9 1 1 9 4 2 10 1 1 11 4 2 Control 1 7 3 12 0 1 2 9 3 3 8 3 Control 1 6 3 4 11 3 2 6 3 5 7 3 3 6 3 6 12 3 4 18 3 12208 1 0 1 12274 1 3 2 2 0 1 2 0 1 3 3 2 3 3 2 4 4 2 4 3 2 5 1 1 5 0 1 6 4 2 6 3 2 7 0 1 7 3 2 8 4 2 8 0 1 9 4 2 9 3 2 10 1 1 10 3 2 11 1 1 11 0 1 12 1 1 13 0 1 Control 1 10 3 2 10 3 Control 1 10 3 3 10 3 2 5 3 4 7 3 3 7 3 5 8 3 4 7 3 6 6 3 5 8 3 6 8 3
Example 46
Analysis of Transgenic Maize Plants for Corn Rootworm Efficacy in the Field
[0365] Some positive plants identified using the root excision bioassay described above were evaluated in the field. Eighteen plants from each event were removed from field plots and evaluated for damage to the roots. Root damage was rated using the Iowa State 0 to 3 linear root damage scale (Oleson, J. D. et al., 2005. J. Econ Entomol. 98(1): 1-8), where 0.00=no feeding damage (lowest rating that can be given); 1.00=one node (circle of roots), or the equivalent of an entire node, eaten back to within approximately 11/2 inches of the stalk (soil line on the 7th node); 2.00=two complete nodes eaten; 3.00=three or more nodes eaten (highest rating that can be given); and damage in between complete nodes eaten is noted as the percentage of the node missing, i.e. 1.50=11/2 nodes eaten, 0.25=1/4 of one node eaten, etc.
[0366] Results of the field trials against western and northern corn rootworm are shown in Table 8 and against Mexican corn rootworm in Table 9. All transgenic corn expressing the FR8a chimeric insecticidal protein performed better than a standard commercial chemical insecticide against western, northern and Mexican corn rootworm.
TABLE-US-00008 TABLE 8 Results of western and northern corn rootworm field trials. Event Plasmid Root Rating 1 12161 (ubi:FR8a) 0.08 2 12161 0.05 3 12161 0.09 4 12161 0.04 5 12274 (cmp:FR8a) 0.04 6 12274 0.08 7 12274 0.05 Chemical 0.15 Neg Check 0.87
TABLE-US-00009 TABLE 9 Results of Mexican corn rootworm field trials. Event Plasmid Root Rating 1 12161 (ubi:FR8a) 0.04 5 12274 (cmp:FR8a) 0.22 6 12274 0.05 Chemical 0.15 Neg Check 1.04
Sequence CWU
1
1
16012001DNAArtificial Sequence2OL-8a coding sequence 1atggctagca
tgactggtgg acagcaaatg ggtcgcggat ccactagtaa cggccgccag 60tgtgctggaa
ttcgccctta tgacggccga caacaacacc gaggcctgga cagcagcacc 120accaaggacg
tgatccagaa gggcatcagc gtggtgggcg acctgctggg cgtggtgggc 180ttccccttcg
gcggcgccct ggtgagcttc tacaccaact tcctgaacac catctggccc 240agcgaggacc
cctggaaggc cttcatggag caggtggagg ccctgatgga ccagaagatc 300gccgactacg
ccaagaacaa ggcactggcc gagctacagg gcctccagaa caacgtggag 360gactatgtga
gcgccctgag cagctggcag aagaaccccg ctgcaccgtt ccgcaacccc 420cacagccagg
gccgcatccg cgagctgttc agccaggccg agagccactt ccgcaacagc 480atgcccagct
tcgccatcag cggctacgag gtgctgttcc tgaccaccta cgcccaggcc 540gccaacaccc
acctgttcct gctgaaggac gcccaaatct acggagagga gtggggctac 600gagaaggagg
acatcgccga gttctacaag cgccagctga agctgaccca ggagtacacc 660gaccactgcg
tgaagtggta caacgtgggt ctagacaagc tccgcggcag cagctacgag 720agctgggtga
acttcaaccg ctaccgccgc gagatgaccc tgaccgtgct ggacctgatc 780gccctgttcc
ccctgtacga cgtgcgcctg taccccaagg aggtgaagac cgagctgacc 840cgcgacgtgc
tgaccgaccc catcgtgggc gtgaacaacc tgcgcggcta cggcaccacc 900ttcagcaaca
tcgagaacta catccgcaag ccccacctgt tcgactacct gcaccgcatc 960cagttccaca
cgcgtttcca gcccggctac tacggcaacg acagcttcaa ctactggagc 1020ggcaactacg
tgagcacccg ccccagcatc ggcagcaacg acatcatcac cagccccttc 1080tacggcaaca
agagcagcga gcccgtgcag aaccttgagt tcaacggcga gaaggtgtac 1140cgcgccgtgg
ctaacaccaa cctggccgtg tggccctctg cagtgtacag cggcgtgacc 1200aaggtggagt
tcagccagta caacgaccag accgacgagg ccagcaccca gacctacgac 1260agcaagcgca
acgtgggcgc cgtgagctgg gacagcatcg accagctgcc ccccgagacc 1320accgacgagc
ccctggagaa gggctacagc caccagctga actacgtgat gtgcttcctg 1380atgcagggca
gccgcggcac catccccgtg ctgacctgga cccacaagag cgtcgacttc 1440ttcaacatga
tcgacagcaa gaagatcacc cagctgcccc tgaccaagag caccaacctg 1500ggcagcggca
ccagcgtggt gaagggcccc ggcttcaccg gcggcgacat cctgcgccgc 1560accagccccg
gccagatcag caccctgcgc gtgaacatca ccgcccccct gagccagcgc 1620taccgcgtcc
gcatccgcta cgccagcacc accaacctgc agttccacac cagcatcgac 1680ggccgcccca
tcaaccaggg caacttcagc gccaccatga gcagcggcag caacctgcag 1740agcggcagct
tccgcaccgt gggcttcacc acccccttca acttcagcaa cggcagcagc 1800gtgttcaccc
tgagcgccca cgtgttcaac agcggcaacg aggtgtacat cgaccgcatc 1860gagttcgtgc
ccgccgaggt gaccttcgag gccgagtacg acctggagag ggctcagaag 1920gccgtgaacg
agctgttcac cagcagcaac cagatcggcc tgaagaccga cgtgaccgac 1980taccacatcg
atcaggtgta g
20012668PRTArtificial Sequence2OL-8a protein 2Met Ala Ser Met Thr Gly Gly
Gln Gln Met Gly Arg Gly Ser Gly Ser 1 5
10 15 Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg
Pro Tyr Asp Gly Arg 20 25
30 Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp Val Ile
Gln 35 40 45 Lys
Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly Phe Pro 50
55 60 Phe Gly Gly Ala Leu Val
Ser Phe Tyr Thr Asn Phe Leu Asn Thr Ile 65 70
75 80 Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met
Glu Gln Val Glu Ala 85 90
95 Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu Ala
100 105 110 Glu Leu
Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala Leu 115
120 125 Ser Ser Trp Gln Lys Asn Pro
Ala Ala Pro Phe Arg Asn Pro His Ser 130 135
140 Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
Ser His Phe Arg 145 150 155
160 Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe Leu
165 170 175 Thr Thr Tyr
Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys Asp 180
185 190 Ala Gln Ile Tyr Gly Glu Glu Trp
Gly Tyr Glu Lys Glu Asp Ile Ala 195 200
205 Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr
Thr Asp His 210 215 220
Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser Ser 225
230 235 240 Tyr Glu Ser Trp
Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu 245
250 255 Thr Val Leu Asp Leu Ile Ala Leu Phe
Pro Leu Tyr Asp Val Arg Leu 260 265
270 Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu
Thr Asp 275 280 285
Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe Ser 290
295 300 Asn Ile Glu Asn Tyr
Ile Arg Lys Pro His Leu Phe Asp Tyr Leu His 305 310
315 320 Arg Ile Gln Phe His Thr Arg Phe Gln Pro
Gly Tyr Tyr Gly Asn Asp 325 330
335 Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser
Ile 340 345 350 Gly
Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser Ser 355
360 365 Glu Pro Val Gln Asn Leu
Glu Phe Asn Gly Glu Lys Val Tyr Arg Ala 370 375
380 Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser
Ala Val Tyr Ser Gly 385 390 395
400 Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala
405 410 415 Ser Thr
Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser Trp 420
425 430 Asp Ser Ile Asp Gln Leu Pro
Pro Glu Thr Thr Asp Glu Pro Leu Glu 435 440
445 Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys
Phe Leu Met Gln 450 455 460
Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser Val 465
470 475 480 Asp Phe Phe
Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu 485
490 495 Thr Lys Ser Thr Asn Leu Gly Ser
Gly Thr Ser Val Val Lys Gly Pro 500 505
510 Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro
Gly Gln Ile 515 520 525
Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg 530
535 540 Val Arg Ile Arg
Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser 545 550
555 560 Ile Asp Gly Arg Pro Ile Asn Gln Gly
Asn Phe Ser Ala Thr Met Ser 565 570
575 Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly
Phe Thr 580 585 590
Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala
595 600 605 His Val Phe Asn
Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe 610
615 620 Val Pro Ala Glu Val Thr Phe Glu
Ala Glu Tyr Asp Leu Glu Arg Ala 625 630
635 640 Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn
Gln Ile Gly Leu 645 650
655 Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val 660
665 31962DNAArtificial SequenceFR8a coding
sequence 3atgactagta acggccgcca gtgtgctggt attcgccctt atgacggccg
acaacaacac 60cgaggcctgg acagcagcac caccaaggac gtgatccaga agggcatcag
cgtggtgggc 120gacctgctgg gcgtggtggg cttccccttc ggcggcgccc tggtgagctt
ctacaccaac 180ttcctgaaca ccatctggcc cagcgaggac ccctggaagg ccttcatgga
gcaggtggag 240gccctgatgg accagaagat cgccgactac gccaagaaca aggcactggc
cgagctacag 300ggcctccaga acaacgtgga ggactatgtg agcgccctga gcagctggca
gaagaacccc 360gctgcaccgt tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt
cagccaggcc 420gagagccact tccgcaacag catgcccagc ttcgccatca gcggctacga
ggtgctgttc 480ctgaccacct acgcccaggc cgccaacacc cacctgttcc tgctgaagga
cgcccaaatc 540tacggagagg agtggggcta cgagaaggag gacatcgccg agttctacaa
gcgccagctg 600aagctgaccc aggagtacac cgaccactgc gtgaagtggt acaacgtggg
tctagacaag 660ctccgcggca gcagctacga gagctgggtg aacttcaacc gctaccgccg
cgagatgacc 720ctgaccgtgc tggacctgat cgccctgttc cccctgtacg acgtgcgcct
gtaccccaag 780gaggtgaaga ccgagctgac ccgcgacgtg ctgaccgacc ccatcgtggg
cgtgaacaac 840ctgcgcggct acggcaccac cttcagcaac atcgagaact acatccgcaa
gccccacctg 900ttcgactacc tgcaccgcat ccagttccac acgcgtttcc agcccggcta
ctacggcaac 960gacagcttca actactggag cggcaactac gtgagcaccc gccccagcat
cggcagcaac 1020gacatcatca ccagcccctt ctacggcaac aagagcagcg agcccgtgca
gaaccttgag 1080ttcaacggcg agaaggtgta ccgcgccgtg gctaacacca acctggccgt
gtggccctct 1140gcagtgtaca gcggcgtgac caaggtggag ttcagccagt acaacgacca
gaccgacgag 1200gccagcaccc agacctacga cagcaagcgc aacgtgggcg ccgtgagctg
ggacagcatc 1260gaccagctgc cccccgagac caccgacgag cccctggaga agggctacag
ccaccagctg 1320aactacgtga tgtgcttcct gatgcagggc agccgcggca ccatccccgt
gctgacctgg 1380acccacaaga gcgtcgactt cttcaacatg atcgacagca agaagatcac
ccagctgccc 1440ctgaccaaga gcaccaacct gggcagcggc accagcgtgg tgaagggccc
cggcttcacc 1500ggcggcgaca tcctgcgccg caccagcccc ggccagatca gcaccctgcg
cgtgaacatc 1560accgcccccc tgagccagcg ctaccgcgtc cgcatccgct acgccagcac
caccaacctg 1620cagttccaca ccagcatcga cggccgcccc atcaaccagg gcaacttcag
cgccaccatg 1680agcagcggca gcaacctgca gagcggcagc ttccgcaccg tgggcttcac
cacccccttc 1740aacttcagca acggcagcag cgtgttcacc ctgagcgccc acgtgttcaa
cagcggcaac 1800gaggtgtaca tcgaccgcat cgagttcgtg cccgccgagg tgaccttcga
ggccgagtac 1860gacctggaga gggctcagaa ggccgtgaac gagctgttca ccagcagcaa
ccagatcggc 1920ctgaagaccg acgtgaccga ctaccacatc gatcaggtgt ag
19624653PRTArtificial SequenceFR8a protein 4Met Thr Ser Asn
Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp Ser
Ser Thr Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val
Gly Phe 35 40 45
Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu
Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr
Ala Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser
Ala 100 105 110 Leu
Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His 115
120 125 Ser Gln Gly Arg Ile Arg
Glu Leu Phe Ser Gln Ala Glu Ser His Phe 130 135
140 Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly
Tyr Glu Val Leu Phe 145 150 155
160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys
165 170 175 Asp Ala
Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile 180
185 190 Ala Glu Phe Tyr Lys Arg Gln
Leu Lys Leu Thr Gln Glu Tyr Thr Asp 195 200
205 His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys
Leu Arg Gly Ser 210 215 220
Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225
230 235 240 Leu Thr Val
Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg 245
250 255 Leu Tyr Pro Lys Glu Val Lys Thr
Glu Leu Thr Arg Asp Val Leu Thr 260 265
270 Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly
Thr Thr Phe 275 280 285
Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu 290
295 300 His Arg Ile Gln
Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn 305 310
315 320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn
Tyr Val Ser Thr Arg Pro Ser 325 330
335 Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn
Lys Ser 340 345 350
Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg
355 360 365 Ala Val Ala Asn
Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser 370
375 380 Gly Val Thr Lys Val Glu Phe Ser
Gln Tyr Asn Asp Gln Thr Asp Glu 385 390
395 400 Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val
Gly Ala Val Ser 405 410
415 Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu
420 425 430 Glu Lys Gly
Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu Met 435
440 445 Gln Gly Ser Arg Gly Thr Ile Pro
Val Leu Thr Trp Thr His Lys Ser 450 455
460 Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr
Gln Leu Pro 465 470 475
480 Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly
485 490 495 Pro Gly Phe Thr
Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln 500
505 510 Ile Ser Thr Leu Arg Val Asn Ile Thr
Ala Pro Leu Ser Gln Arg Tyr 515 520
525 Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe
His Thr 530 535 540
Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met 545
550 555 560 Ser Ser Gly Ser Asn
Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe 565
570 575 Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser
Ser Val Phe Thr Leu Ser 580 585
590 Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile
Glu 595 600 605 Phe
Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg 610
615 620 Ala Gln Lys Ala Val Asn
Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly 625 630
635 640 Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp
Gln Val 645 650
51959DNAArtificial SequenceFRCG coding sequence 5atgactagta acggccgcca
gtgtgctggt attcgccctt atgacggccg acaacaacac 60cgaggcctgg acagcagcac
caccaaggac gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg gcgtggtggg
cttccccttc ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca ccatctggcc
cagcgaggac ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg accagaagat
cgccgactac gccaagaaca aggcactggc cgagctacag 300ggcctccaga acaacgtgga
ggactatgtg agcgccctga gcagctggca gaagaacccc 360gtctcgagcc gcaaccccca
cagccagggc cgcatccgcg agctgttcag ccaggccgag 420agccacttcc gcaacagcat
gcccagcttc gccatcagcg gctacgaggt gctgttcctg 480accacctacg cccaggccgc
caacacccac ctgttcctgc tgaaggacgc ccaaatctac 540ggagaggagt ggggctacga
gaaggaggac atcgccgagt tctacaagcg ccagctgaag 600ctgacccagg agtacaccga
ccactgcgtg aagtggtaca acgtgggtct agacaagctc 660cgcggcagca gctacgagag
ctgggtgaac ttcaaccgct accgccgcga gatgaccctg 720accgtgctgg acctgatcgc
cctgttcccc ctgtacgacg tgcgcctgta ccccaaggag 780gtgaagaccg agctgacccg
cgacgtgctg accgacccca tcgtgggcgt gaacaacctg 840cgcggctacg gcaccacctt
cagcaacatc gagaactaca tccgcaagcc ccacctgttc 900gactacctgc accgcatcca
gttccacacg cgtttccagc ccggctacta cggcaacgac 960agcttcaact actggagcgg
caactacgtg agcacccgcc ccagcatcgg cagcaacgac 1020atcatcacca gccccttcta
cggcaacaag agcagcgagc ccgtgcagaa ccttgagttc 1080aacggcgaga aggtgtaccg
cgccgtggct aacaccaacc tggccgtgtg gccctctgca 1140gtgtacagcg gcgtgaccaa
ggtggagttc agccagtaca acgaccagac cgacgaggcc 1200agcacccaga cctacgacag
caagcgcaac gtgggcgccg tgagctggga cagcatcgac 1260cagctgcccc ccgagaccac
cgacgagccc ctggagaagg gctacagcca ccagctgaac 1320tacgtgatgt gcttcctgat
gcagggcagc cgcggcacca tccccgtgct gacctggacc 1380cacaagagcg tcgacttctt
caacatgatc gacagcaaga agatcaccca gctgcccctg 1440accaagagca ccaacctggg
cagcggcacc agcgtggtga agggccccgg cttcaccggc 1500ggcgacatcc tgcgccgcac
cagccccggc cagatcagca ccctgcgcgt gaacatcacc 1560gcccccctga gccagcgcta
ccgcgtccgc atccgctacg ccagcaccac caacctgcag 1620ttccacacca gcatcgacgg
ccgccccatc aaccagggca acttcagcgc caccatgagc 1680agcggcagca acctgcagag
cggcagcttc cgcaccgtgg gcttcaccac ccccttcaac 1740ttcagcaacg gcagcagcgt
gttcaccctg agcgcccacg tgttcaacag cggcaacgag 1800gtgtacatcg accgcatcga
gttcgtgccc gccgaggtga ccttcgaggc cgagtacgac 1860ctggagaggg ctcagaaggc
cgtgaacgag ctgttcacca gcagcaacca gatcggcctg 1920aagaccgacg tgaccgacta
ccacatcgat caggtgtag 19596652PRTArtificial
SequenceFRCG protein 6Met Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro
Tyr Asp Gly 1 5 10 15
Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp Val Ile
20 25 30 Gln Lys Gly Ile
Ser Val Val Gly Asp Leu Leu Gly Val Val Gly Phe 35
40 45 Pro Phe Gly Gly Ala Leu Val Ser Phe
Tyr Thr Asn Phe Leu Asn Thr 50 55
60 Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
Gln Val Glu 65 70 75
80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu
85 90 95 Ala Glu Leu Gln
Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala 100
105 110 Leu Ser Ser Trp Gln Lys Asn Pro Val
Ser Ser Arg Asn Pro His Ser 115 120
125 Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
Phe Arg 130 135 140
Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe Leu 145
150 155 160 Thr Thr Tyr Ala Gln
Ala Ala Asn Thr His Leu Phe Leu Leu Lys Asp 165
170 175 Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr
Glu Lys Glu Asp Ile Ala 180 185
190 Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp
His 195 200 205 Cys
Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser Ser 210
215 220 Tyr Glu Ser Trp Val Asn
Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu 225 230
235 240 Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu
Tyr Asp Val Arg Leu 245 250
255 Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr Asp
260 265 270 Pro Ile
Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe Ser 275
280 285 Asn Ile Glu Asn Tyr Ile Arg
Lys Pro His Leu Phe Asp Tyr Leu His 290 295
300 Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
Tyr Gly Asn Asp 305 310 315
320 Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser Ile
325 330 335 Gly Ser Asn
Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser Ser 340
345 350 Glu Pro Val Gln Asn Leu Glu Phe
Asn Gly Glu Lys Val Tyr Arg Ala 355 360
365 Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
Tyr Ser Gly 370 375 380
Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala 385
390 395 400 Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser Trp 405
410 415 Asp Ser Ile Asp Gln Leu Pro Pro Glu
Thr Thr Asp Glu Pro Leu Glu 420 425
430 Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
Met Gln 435 440 445
Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser Val 450
455 460 Asp Phe Phe Asn Met
Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu 465 470
475 480 Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr
Ser Val Val Lys Gly Pro 485 490
495 Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln
Ile 500 505 510 Ser
Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg 515
520 525 Val Arg Ile Arg Tyr Ala
Ser Thr Thr Asn Leu Gln Phe His Thr Ser 530 535
540 Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe
Ser Ala Thr Met Ser 545 550 555
560 Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr
565 570 575 Thr Pro
Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala 580
585 590 His Val Phe Asn Ser Gly Asn
Glu Val Tyr Ile Asp Arg Ile Glu Phe 595 600
605 Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp
Leu Glu Arg Ala 610 615 620
Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu 625
630 635 640 Lys Thr Asp
Val Thr Asp Tyr His Ile Asp Gln Val 645
650 71962DNAArtificial SequenceFR8a-9F coding sequence
7atgactagta acggccgcca gtgtgctggt attcgcccta tgacggccga caacaacacc
60gaggccctgg acagcagcac caccaaggac gtgatccaga agggcatcag cgtggtgggc
120gacctgctgg gcgtggtggg cttccccttc ggcggcgccc tggtgagctt ctacaccaac
180ttcctgaaca ccatctggcc cagcgaggac ccctggaagg ccttcatgga gcaggtggag
240gccctgatgg accagaagat cgccgactac gccaagaaca aggcactggc cgagctacag
300ggcctccaga acaacgtgga ggactatgtg agcgccctga gcagctggca gaagaacccc
360gctgcaccgt tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt cagccaggcc
420gagagccact tccgcaacag catgcccagc ttcgccatca gcggctacga ggtgctgttc
480ctgaccacct acgcccaggc cgccaacacc cacctgttcc tgctgaagga cgcccaaatc
540tacggagagg agtggggcta cgagaaggag gacatcgccg agttctacaa gcgccagctg
600aagctgaccc aggagtacac cgaccactgc gtgaagtggt acaacgtggg tctagacaag
660ctccgcggca gcagctacga gagctgggtg aacttcaacc gctaccgccg cgagatgacc
720ctgaccgtgc tggacctgat cgccctgttc cccctgtacg acgtgcgcct gtaccccaag
780gaggtgaaga ccgagctgac ccgcgacgtg ctgaccgacc ccatcgtggg cgtgaacaac
840ctgcgcggct acggcaccac cttcagcaac atcgagaact acatccgcaa gccccacctg
900ttcgactacc tgcaccgcat ccagttccac acgcgtttcc agcccggcta ctacggcaac
960gacagcttca actactggag cggcaactac gtgagcaccc gccccagcat cggcagcaac
1020gacatcatca ccagcccctt ctacggcaac aagagcagcg agcccgtgca gaaccttgag
1080ttcaacggcg agaaggtgta ccgcgccgtg gctaacacca acctggccgt gtggccctct
1140gcagtgtaca gcggcgtgac caaggtggag ttcagccagt acaacgacca gaccgacgag
1200gccagcaccc agacctacga cagcaagcgc aacgtgggcg ccgtgagctg ggacagcatc
1260gaccagctgc cccccgagac caccgacgag cccctggaga agggctacag ccaccagctg
1320aactacgtga tgtgcttcct gatgcagggc agccgcggca ccatccccgt gctgacctgg
1380acccacaaga gcgtcgactt cttcaacatg atcgacagca agaagatcac ccagctgccc
1440ctgaccaaga gcaccaacct gggcagcggc accagcgtgg tgaagggccc cggcttcacc
1500ggcggcgaca tcctgcgccg caccagcccc ggccagatca gcaccctgcg cgtgaacatc
1560accgcccccc tgagccagcg ctaccgcgtc cgcatccgct acgccagcac caccaacctg
1620cagttccaca ccagcatcga cggccgcccc atcaaccagg gcaacttcag cgccaccatg
1680agcagcggca gcaacctgca gagcggcagc ttccgcaccg tgggcttcac cacccccttc
1740aacttcagca acggcagcag cgtgttcacc ctgagcgccc acgtgttcaa cagcggcaac
1800gaggtgtaca tcgaccgcat cgagttcgtg cccgccgagg tgaccttcga ggccgagtac
1860gacctggaga gggctcagaa ggccgtgaac gagctgttca ccagcagcaa ccagatcggc
1920ctgaagaccg acgtgaccga ctaccacatc gatcaggtgt ag
19628653PRTArtificial SequenceFR8a-9F protein 8Met Thr Ser Asn Gly Arg
Gln Cys Ala Gly Ile Arg Pro Met Thr Ala 1 5
10 15 Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr
Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly
Phe 35 40 45 Pro
Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110 Leu Ser
Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His 115
120 125 Ser Gln Gly Arg Ile Arg Glu
Leu Phe Ser Gln Ala Glu Ser His Phe 130 135
140 Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr
Glu Val Leu Phe 145 150 155
160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys
165 170 175 Asp Ala Gln
Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile 180
185 190 Ala Glu Phe Tyr Lys Arg Gln Leu
Lys Leu Thr Gln Glu Tyr Thr Asp 195 200
205 His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
Arg Gly Ser 210 215 220
Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225
230 235 240 Leu Thr Val Leu
Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg 245
250 255 Leu Tyr Pro Lys Glu Val Lys Thr Glu
Leu Thr Arg Asp Val Leu Thr 260 265
270 Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
Thr Phe 275 280 285
Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu 290
295 300 His Arg Ile Gln Phe
His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn 305 310
315 320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr
Val Ser Thr Arg Pro Ser 325 330
335 Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
Ser 340 345 350 Ser
Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg 355
360 365 Ala Val Ala Asn Thr Asn
Leu Ala Val Trp Pro Ser Ala Val Tyr Ser 370 375
380 Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn
Asp Gln Thr Asp Glu 385 390 395
400 Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser
405 410 415 Trp Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu 420
425 430 Glu Lys Gly Tyr Ser His Gln
Leu Asn Tyr Val Met Cys Phe Leu Met 435 440
445 Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp
Thr His Lys Ser 450 455 460
Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro 465
470 475 480 Leu Thr Lys
Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly 485
490 495 Pro Gly Phe Thr Gly Gly Asp Ile
Leu Arg Arg Thr Ser Pro Gly Gln 500 505
510 Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser
Gln Arg Tyr 515 520 525
Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr 530
535 540 Ser Ile Asp Gly
Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met 545 550
555 560 Ser Ser Gly Ser Asn Leu Gln Ser Gly
Ser Phe Arg Thr Val Gly Phe 565 570
575 Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr
Leu Ser 580 585 590
Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu
595 600 605 Phe Val Pro Ala
Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg 610
615 620 Ala Gln Lys Ala Val Asn Glu Leu
Phe Thr Ser Ser Asn Gln Ile Gly 625 630
635 640 Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln
Val 645 650
91959DNAArtificial SequenceFR-9F-catg coding sequence 9atgactagta
acggccgcca gtgtgctggt attcgcccta tgacggccga caacaacacc 60gaggccctgg
acagcagcac caccaaggac gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg
gcgtggtggg cttccccttc ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca
ccatctggcc cagcgaggac ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg
accagaagat cgccgactac gccaagaaca aggcactggc cgagctacag 300ggcctccaga
acaacgtgga ggactatgtg agcgccctga gcagctggca gaagaacccc 360gtctcgagcc
gcaaccccca cagccagggc cgcatccgcg agctgttcag ccaggccgag 420agccacttcc
gcaacagcat gcccagcttc gccatcagcg gctacgaggt gctgttcctg 480accacctacg
cccaggccgc caacacccac ctgttcctgc tgaaggacgc ccaaatctac 540ggagaggagt
ggggctacga gaaggaggac atcgccgagt tctacaagcg ccagctgaag 600ctgacccagg
agtacaccga ccactgcgtg aagtggtaca acgtgggtct agacaagctc 660cgcggcagca
gctacgagag ctgggtgaac ttcaaccgct accgccgcga gatgaccctg 720accgtgctgg
acctgatcgc cctgttcccc ctgtacgacg tgcgcctgta ccccaaggag 780gtgaagaccg
agctgacccg cgacgtgctg accgacccca tcgtgggcgt gaacaacctg 840cgcggctacg
gcaccacctt cagcaacatc gagaactaca tccgcaagcc ccacctgttc 900gactacctgc
accgcatcca gttccacacg cgtttccagc ccggctacta cggcaacgac 960agcttcaact
actggagcgg caactacgtg agcacccgcc ccagcatcgg cagcaacgac 1020atcatcacca
gccccttcta cggcaacaag agcagcgagc ccgtgcagaa ccttgagttc 1080aacggcgaga
aggtgtaccg cgccgtggct aacaccaacc tggccgtgtg gccctctgca 1140gtgtacagcg
gcgtgaccaa ggtggagttc agccagtaca acgaccagac cgacgaggcc 1200agcacccaga
cctacgacag caagcgcaac gtgggcgccg tgagctggga cagcatcgac 1260cagctgcccc
ccgagaccac cgacgagccc ctggagaagg gctacagcca ccagctgaac 1320tacgtgatgt
gcttcctgat gcagggcagc cgcggcacca tccccgtgct gacctggacc 1380cacaagagcg
tcgacttctt caacatgatc gacagcaaga agatcaccca gctgcccctg 1440accaagagca
ccaacctggg cagcggcacc agcgtggtga agggccccgg cttcaccggc 1500ggcgacatcc
tgcgccgcac cagccccggc cagatcagca ccctgcgcgt gaacatcacc 1560gcccccctga
gccagcgcta ccgcgtccgc atccgctacg ccagcaccac caacctgcag 1620ttccacacca
gcatcgacgg ccgccccatc aaccagggca acttcagcgc caccatgagc 1680agcggcagca
acctgcagag cggcagcttc cgcaccgtgg gcttcaccac ccccttcaac 1740ttcagcaacg
gcagcagcgt gttcaccctg agcgcccacg tgttcaacag cggcaacgag 1800gtgtacatcg
accgcatcga gttcgtgccc gccgaggtga ccttcgaggc cgagtacgac 1860ctggagaggg
ctcagaaggc cgtgaacgag ctgttcacca gcagcaacca gatcggcctg 1920aagaccgacg
tgaccgacta ccacatcgat caggtgtag
195910652PRTArtificial SequenceFR-9F-catg protein 10Met Thr Ser Asn Gly
Arg Gln Cys Ala Gly Ile Arg Pro Met Thr Ala 1 5
10 15 Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser
Thr Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly
Phe 35 40 45 Pro
Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110 Leu Ser
Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn Pro His Ser 115
120 125 Gln Gly Arg Ile Arg Glu Leu
Phe Ser Gln Ala Glu Ser His Phe Arg 130 135
140 Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu
Val Leu Phe Leu 145 150 155
160 Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys Asp
165 170 175 Ala Gln Ile
Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala 180
185 190 Glu Phe Tyr Lys Arg Gln Leu Lys
Leu Thr Gln Glu Tyr Thr Asp His 195 200
205 Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg
Gly Ser Ser 210 215 220
Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu 225
230 235 240 Thr Val Leu Asp
Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg Leu 245
250 255 Tyr Pro Lys Glu Val Lys Thr Glu Leu
Thr Arg Asp Val Leu Thr Asp 260 265
270 Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
Phe Ser 275 280 285
Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu His 290
295 300 Arg Ile Gln Phe His
Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn Asp 305 310
315 320 Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val
Ser Thr Arg Pro Ser Ile 325 330
335 Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser
Ser 340 345 350 Glu
Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg Ala 355
360 365 Val Ala Asn Thr Asn Leu
Ala Val Trp Pro Ser Ala Val Tyr Ser Gly 370 375
380 Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp
Gln Thr Asp Glu Ala 385 390 395
400 Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser Trp
405 410 415 Asp Ser
Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu 420
425 430 Lys Gly Tyr Ser His Gln Leu
Asn Tyr Val Met Cys Phe Leu Met Gln 435 440
445 Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr
His Lys Ser Val 450 455 460
Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu 465
470 475 480 Thr Lys Ser
Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro 485
490 495 Gly Phe Thr Gly Gly Asp Ile Leu
Arg Arg Thr Ser Pro Gly Gln Ile 500 505
510 Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln
Arg Tyr Arg 515 520 525
Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser 530
535 540 Ile Asp Gly Arg
Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser 545 550
555 560 Ser Gly Ser Asn Leu Gln Ser Gly Ser
Phe Arg Thr Val Gly Phe Thr 565 570
575 Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu
Ser Ala 580 585 590
His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe
595 600 605 Val Pro Ala Glu
Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala 610
615 620 Gln Lys Ala Val Asn Glu Leu Phe
Thr Ser Ser Asn Gln Ile Gly Leu 625 630
635 640 Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val
645 650 111926DNAArtificial
SequenceFR8a-12AA coding sequence 11atgtatgacg gccgacaaca acaccgaggc
ctggacagca gcaccaccaa ggacgtgatc 60cagaagggca tcagcgtggt gggcgacctg
ctgggcgtgg tgggcttccc cttcggcggc 120gccctggtga gcttctacac caacttcctg
aacaccatct ggcccagcga ggacccctgg 180aaggccttca tggagcaggt ggaggccctg
atggaccaga agatcgccga ctacgccaag 240aacaaggcac tggccgagct acagggcctc
cagaacaacg tggaggacta tgtgagcgcc 300ctgagcagct ggcagaagaa ccccgctgca
ccgttccgca acccccacag ccagggccgc 360atccgcgagc tgttcagcca ggccgagagc
cacttccgca acagcatgcc cagcttcgcc 420atcagcggct acgaggtgct gttcctgacc
acctacgccc aggccgccaa cacccacctg 480ttcctgctga aggacgccca aatctacgga
gaggagtggg gctacgagaa ggaggacatc 540gccgagttct acaagcgcca gctgaagctg
acccaggagt acaccgacca ctgcgtgaag 600tggtacaacg tgggtctaga caagctccgc
ggcagcagct acgagagctg ggtgaacttc 660aaccgctacc gccgcgagat gaccctgacc
gtgctggacc tgatcgccct gttccccctg 720tacgacgtgc gcctgtaccc caaggaggtg
aagaccgagc tgacccgcga cgtgctgacc 780gaccccatcg tgggcgtgaa caacctgcgc
ggctacggca ccaccttcag caacatcgag 840aactacatcc gcaagcccca cctgttcgac
tacctgcacc gcatccagtt ccacacgcgt 900ttccagcccg gctactacgg caacgacagc
ttcaactact ggagcggcaa ctacgtgagc 960acccgcccca gcatcggcag caacgacatc
atcaccagcc ccttctacgg caacaagagc 1020agcgagcccg tgcagaacct tgagttcaac
ggcgagaagg tgtaccgcgc cgtggctaac 1080accaacctgg ccgtgtggcc ctctgcagtg
tacagcggcg tgaccaaggt ggagttcagc 1140cagtacaacg accagaccga cgaggccagc
acccagacct acgacagcaa gcgcaacgtg 1200ggcgccgtga gctgggacag catcgaccag
ctgccccccg agaccaccga cgagcccctg 1260gagaagggct acagccacca gctgaactac
gtgatgtgct tcctgatgca gggcagccgc 1320ggcaccatcc ccgtgctgac ctggacccac
aagagcgtcg acttcttcaa catgatcgac 1380agcaagaaga tcacccagct gcccctgacc
aagagcacca acctgggcag cggcaccagc 1440gtggtgaagg gccccggctt caccggcggc
gacatcctgc gccgcaccag ccccggccag 1500atcagcaccc tgcgcgtgaa catcaccgcc
cccctgagcc agcgctaccg cgtccgcatc 1560cgctacgcca gcaccaccaa cctgcagttc
cacaccagca tcgacggccg ccccatcaac 1620cagggcaact tcagcgccac catgagcagc
ggcagcaacc tgcagagcgg cagcttccgc 1680accgtgggct tcaccacccc cttcaacttc
agcaacggca gcagcgtgtt caccctgagc 1740gcccacgtgt tcaacagcgg caacgaggtg
tacatcgacc gcatcgagtt cgtgcccgcc 1800gaggtgacct tcgaggccga gtacgacctg
gagagggctc agaaggccgt gaacgagctg 1860ttcaccagca gcaaccagat cggcctgaag
accgacgtga ccgactacca catcgatcag 1920gtgtag
192612641PRTArtificial SequenceFR8a-12AA
protein 12Met Tyr Asp Gly Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr
1 5 10 15 Lys Asp
Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly 20
25 30 Val Val Gly Phe Pro Phe Gly
Gly Ala Leu Val Ser Phe Tyr Thr Asn 35 40
45 Phe Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp
Lys Ala Phe Met 50 55 60
Glu Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys 65
70 75 80 Asn Lys Ala
Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp 85
90 95 Tyr Val Ser Ala Leu Ser Ser Trp
Gln Lys Asn Pro Ala Ala Pro Phe 100 105
110 Arg Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe
Ser Gln Ala 115 120 125
Glu Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr 130
135 140 Glu Val Leu Phe
Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu 145 150
155 160 Phe Leu Leu Lys Asp Ala Gln Ile Tyr
Gly Glu Glu Trp Gly Tyr Glu 165 170
175 Lys Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu
Thr Gln 180 185 190
Glu Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys
195 200 205 Leu Arg Gly Ser
Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg 210
215 220 Arg Glu Met Thr Leu Thr Val Leu
Asp Leu Ile Ala Leu Phe Pro Leu 225 230
235 240 Tyr Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr
Glu Leu Thr Arg 245 250
255 Asp Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr
260 265 270 Gly Thr Thr
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu 275
280 285 Phe Asp Tyr Leu His Arg Ile Gln
Phe His Thr Arg Phe Gln Pro Gly 290 295
300 Tyr Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn
Tyr Val Ser 305 310 315
320 Thr Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr
325 330 335 Gly Asn Lys Ser
Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu 340
345 350 Lys Val Tyr Arg Ala Val Ala Asn Thr
Asn Leu Ala Val Trp Pro Ser 355 360
365 Ala Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr
Asn Asp 370 375 380
Gln Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val 385
390 395 400 Gly Ala Val Ser Trp
Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 405
410 415 Asp Glu Pro Leu Glu Lys Gly Tyr Ser His
Gln Leu Asn Tyr Val Met 420 425
430 Cys Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr
Trp 435 440 445 Thr
His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile 450
455 460 Thr Gln Leu Pro Leu Thr
Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser 465 470
475 480 Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp
Ile Leu Arg Arg Thr 485 490
495 Ser Pro Gly Gln Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu
500 505 510 Ser Gln
Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515
520 525 Gln Phe His Thr Ser Ile Asp
Gly Arg Pro Ile Asn Gln Gly Asn Phe 530 535
540 Ser Ala Thr Met Ser Ser Gly Ser Asn Leu Gln Ser
Gly Ser Phe Arg 545 550 555
560 Thr Val Gly Phe Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val
565 570 575 Phe Thr Leu
Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile 580
585 590 Asp Arg Ile Glu Phe Val Pro Ala
Glu Val Thr Phe Glu Ala Glu Tyr 595 600
605 Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Glu Leu Phe
Thr Ser Ser 610 615 620
Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln 625
630 635 640 Val
131800DNAArtificial SequenceWR-9mut coding sequence 13atgtatgacg
gccgacaaca acaccgaggc ctggacagca gcaccaccaa ggacgtgatc 60cagaagggca
tcagcgtggt gggcgacctg ctgggcgtgg tgggcttccc cttcggcggc 120gccctggtga
gcttctacac caacttcctg aacaccatct ggcccagcga ggacccctgg 180aaggccttca
tggagcaggt ggaggccctg atggaccaga agatcgccga ctacgccaag 240aacaaggcac
tggccgagct acagggcctc cagaacaacg tggaggacta tgtgagcgcc 300ctgagcagct
ggcagaagaa ccccgctgca ccgttccgca acccccacag ccagggccgc 360atccgcgagc
tgttcagcca ggccgagagc cacttccgca acagcatgcc cagcttcgcc 420atcagcggct
acgaggtgct gttcctgacc acctacgccc aggccgccaa cacccacctg 480ttcctgctga
aggacgccca aatctacgga gaggagtggg gctacgagaa ggaggacatc 540gccgagttct
acaagcgcca gctgaagctg acccaggagt acaccgacca ctgcgtgaag 600tggtacaacg
tgggtctaga caagctccgc ggcagcagct acgagagctg ggtgaacttc 660aaccgctacc
gccgcgagat gaccctgacc gtgctggacc tgatcgccct gttccccctg 720tacgacgtgc
gcctgtaccc caaggaggtg aagaccgagc tgacccgcga cgtgctgacc 780gaccccatcg
tgggcgtgaa caacctgcgc ggctacggca ccaccttcag caacatcgag 840aactacatcc
gcaagcccca cctgttcgac tacctgcacc gcatccagtt ccacacgcgt 900ttccagcccg
gctactacgg caacgacagc ttcaactact ggagcggcaa ctacgtgagc 960acccgcccca
gcatcggcag caacgacatc atcaccagcc ccttctacgg caacaagagc 1020agcgagcccg
tgcagaacct tgagttcaac ggcgagaagg tgtaccgcgc cgtggctaac 1080accaacctgg
ccgtgtggcc ctctgcagtg tacagcggcg tgaccaaggt ggagttcagc 1140cagtacaacg
accagaccga cgaggccagc acccagacct acgacagcaa gcgcaacgtg 1200ggcgccgtga
gctgggacag catcgaccag ctgccccccg agaccaccga cgagcccctg 1260gagaagggct
acagccacca gctgaactac gtgatgtgct tcctgatgca gggcagccgc 1320ggcaccatcc
ccgtgctgac ctggacccac aagagcgtcg acttcttcaa catgatcgac 1380agcaagaaga
tcacccagct gcccctggtg aaggcctaca agctccagag cggcgccagc 1440gtggtggcag
gcccccgctt caccggcggc gacatcatcc agtgcaccga gaacggcagc 1500gccgccacca
tctacgtgac ccccgacgtg agctacagcc agaagtaccg cgcccgcatc 1560cactacgcca
gcaccagcca gatcaccttc accctgagcc tggacggggc ccccttcaac 1620caatactact
tcgacaagac catcaacaag ggcgacaccc tgacctacaa cagcttcaac 1680ctggccagct
tcagcacccc tttcgagctg agcggcaaca acctccagat cggcgtgacc 1740ggcctgagcg
ccggcgacaa ggtgtacatc gacaagatcg agttcatccc cgtgaactag
180014599PRTArtificial SequenceWR-9mut protein 14Met Tyr Asp Gly Arg Gln
Gln His Arg Gly Leu Asp Ser Ser Thr Thr 1 5
10 15 Lys Asp Val Ile Gln Lys Gly Ile Ser Val Val
Gly Asp Leu Leu Gly 20 25
30 Val Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr
Asn 35 40 45 Phe
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met 50
55 60 Glu Gln Val Glu Ala Leu
Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys 65 70
75 80 Asn Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln
Asn Asn Val Glu Asp 85 90
95 Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe
100 105 110 Arg Asn
Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala 115
120 125 Glu Ser His Phe Arg Asn Ser
Met Pro Ser Phe Ala Ile Ser Gly Tyr 130 135
140 Glu Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala
Asn Thr His Leu 145 150 155
160 Phe Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu
165 170 175 Lys Glu Asp
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln 180
185 190 Glu Tyr Thr Asp His Cys Val Lys
Trp Tyr Asn Val Gly Leu Asp Lys 195 200
205 Leu Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn
Arg Tyr Arg 210 215 220
Arg Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu 225
230 235 240 Tyr Asp Val Arg
Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg 245
250 255 Asp Val Leu Thr Asp Pro Ile Val Gly
Val Asn Asn Leu Arg Gly Tyr 260 265
270 Gly Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro
His Leu 275 280 285
Phe Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly 290
295 300 Tyr Tyr Gly Asn Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser 305 310
315 320 Thr Arg Pro Ser Ile Gly Ser Asn Asp Ile
Ile Thr Ser Pro Phe Tyr 325 330
335 Gly Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly
Glu 340 345 350 Lys
Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser 355
360 365 Ala Val Tyr Ser Gly Val
Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp 370 375
380 Gln Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp
Ser Lys Arg Asn Val 385 390 395
400 Gly Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr
405 410 415 Asp Glu
Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met 420
425 430 Cys Phe Leu Met Gln Gly Ser
Arg Gly Thr Ile Pro Val Leu Thr Trp 435 440
445 Thr His Lys Ser Val Asp Phe Phe Asn Met Ile Asp
Ser Lys Lys Ile 450 455 460
Thr Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser 465
470 475 480 Val Val Ala
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr 485
490 495 Glu Asn Gly Ser Ala Ala Thr Ile
Tyr Val Thr Pro Asp Val Ser Tyr 500 505
510 Ser Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr
Ser Gln Ile 515 520 525
Thr Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe 530
535 540 Asp Lys Thr Ile
Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn 545 550
555 560 Leu Ala Ser Phe Ser Thr Pro Phe Glu
Leu Ser Gly Asn Asn Leu Gln 565 570
575 Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile
Asp Lys 580 585 590
Ile Glu Phe Ile Pro Val Asn 595 15
1848DNAArtificial SequenceFRD3 coding sequence 15atgactagta acggccgcca
gtgtgctggt attcgccctt atgacggccg acaacaacac 60cgaggcctgg acagcagcac
caccaaggac gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg gcgtggtggg
cttccccttc ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca ccatctggcc
cagcgaggac ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg accagaagat
cgccgactac gccaagaaca aggcactggc cgagctacag 300ggcctccaga acaacgtgga
ggactatgtg agcgccctga gcagctggca gaagaacccc 360gctgcaccgt tccgcaaccc
ccacagccag ggccgcatcc gcgagctgtt cagccaggcc 420gagagccact tccgcaacag
catgcccagc ttcgccatca gcggctacga ggtgctgttc 480ctgaccacct acgcccaggc
cgccaacacc cacctgttcc tgctgaagga cgcccaaatc 540tacggagagg agtggggcta
cgagaaggag gacatcgccg agttctacaa gcgccagctg 600aagctgaccc aggagtacac
cgaccactgc gtgaagtggt acaacgtggg tctagacaag 660ctccgcggca gcagctacga
gagctgggtg aacttcaacc gctaccgccg cgagatgacc 720ctgaccgtgc tggacctgat
cgccctgttc cccctgtacg acgtgcgcct gtaccccaag 780gaggtgaaga ccgagctgac
ccgcgacgtg ctgaccgacc ccatcgtggg cgtgaacaac 840ctgcgcggct acggcaccac
cttcagcaac atcgagaact acatccgcaa gccccacctg 900ttcgactacc tgcaccgcat
ccagttccac acgcgtttcc agcccggcta ctacggcaac 960gacagcttca actactggag
cggcaactac gtgagcaccc gccccagcat cggcagcaac 1020gacatcatca ccagcccctt
ctacggcaac aagagcagcg agcccgtgca gaaccttgag 1080ttcaacggcg agaaggtgta
ccgcgccgtg gctaacacca acctggccgt gtggccctct 1140gcagtgtaca gcggcgtgac
caaggtggag ttcagccagt acaacgacca gaccgacgag 1200gccagcaccc agacctacga
cagcaagcgc aacgtgggcg ccgtgagctg ggacagcatc 1260gaccagctgc cccccgagac
caccgacgag cccctggaga agggctacag ccaccagctg 1320aactacgtga tgtgcttcct
gatgcagggc agccgcggca ccatccccgt gctgacctgg 1380acccacaaga gcgtcgactt
cttcaacatg atcgacagca agaagatcac ccagctgccc 1440ctgaccaaga gcaccaacct
gggcagcggc accagcgtgg tgaagggccc cggcttcacc 1500ggcggcgaca tcctgcgccg
caccagcccc ggccagatca gcaccctgcg cgtgaacatc 1560accgcccccc tgagccagcg
ctaccgcgtc cgcatccgct acgccagcac caccaacctg 1620cagttccaca ccagcatcga
cggccgcccc atcaaccagg gcaacttcag cgccaccatg 1680agcagcggca gcaacctgca
gagcggcagc ttccgcaccg tgggcttcac cacccccttc 1740aacttcagca acggcagcag
cgtgttcacc ctgagcgccc acgtgttcaa cagcggcaac 1800gaggtgtaca tcgaccgcat
cgagttcgtg cccgccgagg tgacctag 184816615PRTArtificial
SequenceFRD3 protein 16Met Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg
Pro Tyr Asp Gly 1 5 10
15 Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp Val Ile
20 25 30 Gln Lys Gly
Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly Phe 35
40 45 Pro Phe Gly Gly Ala Leu Val Ser
Phe Tyr Thr Asn Phe Leu Asn Thr 50 55
60 Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
Gln Val Glu 65 70 75
80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu
85 90 95 Ala Glu Leu Gln
Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala 100
105 110 Leu Ser Ser Trp Gln Lys Asn Pro Ala
Ala Pro Phe Arg Asn Pro His 115 120
125 Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
His Phe 130 135 140
Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe 145
150 155 160 Leu Thr Thr Tyr Ala
Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys 165
170 175 Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly
Tyr Glu Lys Glu Asp Ile 180 185
190 Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
Asp 195 200 205 His
Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser 210
215 220 Ser Tyr Glu Ser Trp Val
Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225 230
235 240 Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro
Leu Tyr Asp Val Arg 245 250
255 Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr
260 265 270 Asp Pro
Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe 275
280 285 Ser Asn Ile Glu Asn Tyr Ile
Arg Lys Pro His Leu Phe Asp Tyr Leu 290 295
300 His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly
Tyr Tyr Gly Asn 305 310 315
320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser
325 330 335 Ile Gly Ser
Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser 340
345 350 Ser Glu Pro Val Gln Asn Leu Glu
Phe Asn Gly Glu Lys Val Tyr Arg 355 360
365 Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
Val Tyr Ser 370 375 380
Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu 385
390 395 400 Ala Ser Thr Gln
Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser 405
410 415 Trp Asp Ser Ile Asp Gln Leu Pro Pro
Glu Thr Thr Asp Glu Pro Leu 420 425
430 Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
Leu Met 435 440 445
Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser 450
455 460 Val Asp Phe Phe Asn
Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro 465 470
475 480 Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly
Thr Ser Val Val Lys Gly 485 490
495 Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly
Gln 500 505 510 Ile
Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr 515
520 525 Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr Asn Leu Gln Phe His Thr 530 535
540 Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn
Phe Ser Ala Thr Met 545 550 555
560 Ser Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe
565 570 575 Thr Thr
Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser 580
585 590 Ala His Val Phe Asn Ser Gly
Asn Glu Val Tyr Ile Asp Arg Ile Glu 595 600
605 Phe Val Pro Ala Glu Val Thr 610
615 171809DNAArtificial SequenceFR-12-cg-dm3 coding sequence
17atgtatgacg gccgacaaca acaccgaggc ctggacagca gcaccaccaa ggacgtgatc
60cagaagggca tcagcgtggt gggcgacctg ctgggcgtgg tgggcttccc cttcggcggc
120gccctggtga gcttctacac caacttcctg aacaccatct ggcccagcga ggacccctgg
180aaggccttca tggagcaggt ggaggccctg atggaccaga agatcgccga ctacgccaag
240aacaaggcac tggccgagct acagggcctc cagaacaacg tggaggacta tgtgagcgcc
300ctgagcagct ggcagaagaa ccccgtctcg agccgcaacc cccacagcca gggccgcatc
360cgcgagctgt tcagccaggc cgagagccac ttccgcaaca gcatgcccag cttcgccatc
420agcggctacg aggtgctgtt cctgaccacc tacgcccagg ccgccaacac ccacctgttc
480ctgctgaagg acgcccaaat ctacggagag gagtggggct acgagaagga ggacatcgcc
540gagttctaca agcgccagct gaagctgacc caggagtaca ccgaccactg cgtgaagtgg
600tacaacgtgg gtctagacaa gctccgcggc agcagctacg agagctgggt gaacttcaac
660cgctaccgcc gcgagatgac cctgaccgtg ctggacctga tcgccctgtt ccccctgtac
720gacgtgcgcc tgtaccccaa ggaggtgaag accgagctga cccgcgacgt gctgaccgac
780cccatcgtgg gcgtgaacaa cctgcgcggc tacggcacca ccttcagcaa catcgagaac
840tacatccgca agccccacct gttcgactac ctgcaccgca tccagttcca cacgcgtttc
900cagcccggct actacggcaa cgacagcttc aactactgga gcggcaacta cgtgagcacc
960cgccccagca tcggcagcaa cgacatcatc accagcccct tctacggcaa caagagcagc
1020gagcccgtgc agaaccttga gttcaacggc gagaaggtgt accgcgccgt ggctaacacc
1080aacctggccg tgtggccctc tgcagtgtac agcggcgtga ccaaggtgga gttcagccag
1140tacaacgacc agaccgacga ggccagcacc cagacctacg acagcaagcg caacgtgggc
1200gccgtgagct gggacagcat cgaccagctg ccccccgaga ccaccgacga gcccctggag
1260aagggctaca gccaccagct gaactacgtg atgtgcttcc tgatgcaggg cagccgcggc
1320accatccccg tgctgacctg gacccacaag agcgtcgact tcttcaacat gatcgacagc
1380aagaagatca cccagctgcc cctgaccaag agcaccaacc tgggcagcgg caccagcgtg
1440gtgaagggcc ccggcttcac cggcggcgac atcctgcgcc gcaccagccc cggccagatc
1500agcaccctgc gcgtgaacat caccgccccc ctgagccagc gctaccgcgt ccgcatccgc
1560tacgccagca ccaccaacct gcagttccac accagcatcg acggccgccc catcaaccag
1620ggcaacttca gcgccaccat gagcagcggc agcaacctgc agagcggcag cttccgcacc
1680gtgggcttca ccaccccctt caacttcagc aacggcagca gcgtgttcac cctgagcgcc
1740cacgtgttca acagcggcaa cgaggtgtac atcgaccgca tcgagttcgt gcccgccgag
1800gtgacctag
180918602PRTArtificial SequenceFR-12-cg-dm3 protein 18Met Tyr Asp Gly Arg
Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr 1 5
10 15 Lys Asp Val Ile Gln Lys Gly Ile Ser Val
Val Gly Asp Leu Leu Gly 20 25
30 Val Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr
Asn 35 40 45 Phe
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met 50
55 60 Glu Gln Val Glu Ala Leu
Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys 65 70
75 80 Asn Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln
Asn Asn Val Glu Asp 85 90
95 Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg
100 105 110 Asn Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 115
120 125 Ser His Phe Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130 135
140 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe 145 150 155
160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175 Glu Asp Ile
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu 180
185 190 Tyr Thr Asp His Cys Val Lys Trp
Tyr Asn Val Gly Leu Asp Lys Leu 195 200
205 Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg
Tyr Arg Arg 210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr 225
230 235 240 Asp Val Arg Leu
Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp 245
250 255 Val Leu Thr Asp Pro Ile Val Gly Val
Asn Asn Leu Arg Gly Tyr Gly 260 265
270 Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His
Leu Phe 275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr 290
295 300 Tyr Gly Asn Asp Ser
Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 305 310
315 320 Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile
Thr Ser Pro Phe Tyr Gly 325 330
335 Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu
Lys 340 345 350 Val
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala 355
360 365 Val Tyr Ser Gly Val Thr
Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 370 375
380 Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Val Gly 385 390 395
400 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415 Glu Pro
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys 420
425 430 Phe Leu Met Gln Gly Ser Arg
Gly Thr Ile Pro Val Leu Thr Trp Thr 435 440
445 His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser
Lys Lys Ile Thr 450 455 460
Gln Leu Pro Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val 465
470 475 480 Val Lys Gly
Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser 485
490 495 Pro Gly Gln Ile Ser Thr Leu Arg
Val Asn Ile Thr Ala Pro Leu Ser 500 505
510 Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr
Asn Leu Gln 515 520 525
Phe His Thr Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser 530
535 540 Ala Thr Met Ser
Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr 545 550
555 560 Val Gly Phe Thr Thr Pro Phe Asn Phe
Ser Asn Gly Ser Ser Val Phe 565 570
575 Thr Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr
Ile Asp 580 585 590
Arg Ile Glu Phe Val Pro Ala Glu Val Thr 595 600
19 1941DNAArtificial Sequence9F-cg-del6 coding sequence
19atgtgtgctg gtattcgccc tatgacggcc gacaacaaca ccgaggccct ggacagcagc
60accaccaagg acgtgatcca gaagggcatc agcgtggtgg gcgacctgct gggcgtggtg
120ggcttcccct tcggcggcgc cctggtgagc ttctacacca acttcctgaa caccatctgg
180cccagcgagg acccctggaa ggccttcatg gagcaggtgg aggccctgat ggaccagaag
240atcgccgact acgccaagaa caaggcactg gccgagctac agggcctcca gaacaacgtg
300gaggactatg tgagcgccct gagcagctgg cagaagaacc ccgtctcgag ccgcaacccc
360cacagccagg gccgcatccg cgagctgttc agccaggccg agagccactt ccgcaacagc
420atgcccagct tcgccatcag cggctacgag gtgctgttcc tgaccaccta cgcccaggcc
480gccaacaccc acctgttcct gctgaaggac gcccaaatct acggagagga gtggggctac
540gagaaggagg acatcgccga gttctacaag cgccagctga agctgaccca ggagtacacc
600gaccactgcg tgaagtggta caacgtgggt ctagacaagc tccgcggcag cagctacgag
660agctgggtga acttcaaccg ctaccgccgc gagatgaccc tgaccgtgct ggacctgatc
720gccctgttcc ccctgtacga cgtgcgcctg taccccaagg aggtgaagac cgagctgacc
780cgcgacgtgc tgaccgaccc catcgtgggc gtgaacaacc tgcgcggcta cggcaccacc
840ttcagcaaca tcgagaacta catccgcaag ccccacctgt tcgactacct gcaccgcatc
900cagttccaca cgcgtttcca gcccggctac tacggcaacg acagcttcaa ctactggagc
960ggcaactacg tgagcacccg ccccagcatc ggcagcaacg acatcatcac cagccccttc
1020tacggcaaca agagcagcga gcccgtgcag aaccttgagt tcaacggcga gaaggtgtac
1080cgcgccgtgg ctaacaccaa cctggccgtg tggccctctg cagtgtacag cggcgtgacc
1140aaggtggagt tcagccagta caacgaccag accgacgagg ccagcaccca gacctacgac
1200agcaagcgca acgtgggcgc cgtgagctgg gacagcatcg accagctgcc ccccgagacc
1260accgacgagc ccctggagaa gggctacagc caccagctga actacgtgat gtgcttcctg
1320atgcagggca gccgcggcac catccccgtg ctgacctgga cccacaagag cgtcgacttc
1380ttcaacatga tcgacagcaa gaagatcacc cagctgcccc tgaccaagag caccaacctg
1440ggcagcggca ccagcgtggt gaagggcccc ggcttcaccg gcggcgacat cctgcgccgc
1500accagccccg gccagatcag caccctgcgc gtgaacatca ccgcccccct gagccagcgc
1560taccgcgtcc gcatccgcta cgccagcacc accaacctgc agttccacac cagcatcgac
1620ggccgcccca tcaaccaggg caacttcagc gccaccatga gcagcggcag caacctgcag
1680agcggcagct tccgcaccgt gggcttcacc acccccttca acttcagcaa cggcagcagc
1740gtgttcaccc tgagcgccca cgtgttcaac agcggcaacg aggtgtacat cgaccgcatc
1800gagttcgtgc ccgccgaggt gaccttcgag gccgagtacg acctggagag ggctcagaag
1860gccgtgaacg agctgttcac cagcagcaac cagatcggcc tgaagaccga cgtgaccgac
1920taccacatcg atcaggtgta g
194120646PRTArtificial Sequence9F-cg-del6 protein 20Met Cys Ala Gly Ile
Arg Pro Met Thr Ala Asp Asn Asn Thr Glu Ala 1 5
10 15 Leu Asp Ser Ser Thr Thr Lys Asp Val Ile
Gln Lys Gly Ile Ser Val 20 25
30 Val Gly Asp Leu Leu Gly Val Val Gly Phe Pro Phe Gly Gly Ala
Leu 35 40 45 Val
Ser Phe Tyr Thr Asn Phe Leu Asn Thr Ile Trp Pro Ser Glu Asp 50
55 60 Pro Trp Lys Ala Phe Met
Glu Gln Val Glu Ala Leu Met Asp Gln Lys 65 70
75 80 Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu Ala
Glu Leu Gln Gly Leu 85 90
95 Gln Asn Asn Val Glu Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys
100 105 110 Asn Pro
Val Ser Ser Arg Asn Pro His Ser Gln Gly Arg Ile Arg Glu 115
120 125 Leu Phe Ser Gln Ala Glu Ser
His Phe Arg Asn Ser Met Pro Ser Phe 130 135
140 Ala Ile Ser Gly Tyr Glu Val Leu Phe Leu Thr Thr
Tyr Ala Gln Ala 145 150 155
160 Ala Asn Thr His Leu Phe Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu
165 170 175 Glu Trp Gly
Tyr Glu Lys Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln 180
185 190 Leu Lys Leu Thr Gln Glu Tyr Thr
Asp His Cys Val Lys Trp Tyr Asn 195 200
205 Val Gly Leu Asp Lys Leu Arg Gly Ser Ser Tyr Glu Ser
Trp Val Asn 210 215 220
Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu Thr Val Leu Asp Leu Ile 225
230 235 240 Ala Leu Phe Pro
Leu Tyr Asp Val Arg Leu Tyr Pro Lys Glu Val Lys 245
250 255 Thr Glu Leu Thr Arg Asp Val Leu Thr
Asp Pro Ile Val Gly Val Asn 260 265
270 Asn Leu Arg Gly Tyr Gly Thr Thr Phe Ser Asn Ile Glu Asn
Tyr Ile 275 280 285
Arg Lys Pro His Leu Phe Asp Tyr Leu His Arg Ile Gln Phe His Thr 290
295 300 Arg Phe Gln Pro Gly
Tyr Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser 305 310
315 320 Gly Asn Tyr Val Ser Thr Arg Pro Ser Ile
Gly Ser Asn Asp Ile Ile 325 330
335 Thr Ser Pro Phe Tyr Gly Asn Lys Ser Ser Glu Pro Val Gln Asn
Leu 340 345 350 Glu
Phe Asn Gly Glu Lys Val Tyr Arg Ala Val Ala Asn Thr Asn Leu 355
360 365 Ala Val Trp Pro Ser Ala
Val Tyr Ser Gly Val Thr Lys Val Glu Phe 370 375
380 Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala Ser
Thr Gln Thr Tyr Asp 385 390 395
400 Ser Lys Arg Asn Val Gly Ala Val Ser Trp Asp Ser Ile Asp Gln Leu
405 410 415 Pro Pro
Glu Thr Thr Asp Glu Pro Leu Glu Lys Gly Tyr Ser His Gln 420
425 430 Leu Asn Tyr Val Met Cys Phe
Leu Met Gln Gly Ser Arg Gly Thr Ile 435 440
445 Pro Val Leu Thr Trp Thr His Lys Ser Val Asp Phe
Phe Asn Met Ile 450 455 460
Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu Thr Lys Ser Thr Asn Leu 465
470 475 480 Gly Ser Gly
Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp 485
490 495 Ile Leu Arg Arg Thr Ser Pro Gly
Gln Ile Ser Thr Leu Arg Val Asn 500 505
510 Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile
Arg Tyr Ala 515 520 525
Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg Pro Ile 530
535 540 Asn Gln Gly Asn
Phe Ser Ala Thr Met Ser Ser Gly Ser Asn Leu Gln 545 550
555 560 Ser Gly Ser Phe Arg Thr Val Gly Phe
Thr Thr Pro Phe Asn Phe Ser 565 570
575 Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn
Ser Gly 580 585 590
Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu Val Thr
595 600 605 Phe Glu Ala Glu
Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Glu 610
615 620 Leu Phe Thr Ser Ser Asn Gln Ile
Gly Leu Lys Thr Asp Val Thr Asp 625 630
635 640 Tyr His Ile Asp Gln Val 645
211845DNAArtificial SequenceFR-cg-dm3 coding sequence 21atgactagta
acggccgcca gtgtgctggt attcgccctt atgacggccg acaacaacac 60cgaggcctgg
acagcagcac caccaaggac gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg
gcgtggtggg cttccccttc ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca
ccatctggcc cagcgaggac ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg
accagaagat cgccgactac gccaagaaca aggcactggc cgagctacag 300ggcctccaga
acaacgtgga ggactatgtg agcgccctga gcagctggca gaagaacccc 360gtctcgagcc
gcaaccccca cagccagggc cgcatccgcg agctgttcag ccaggccgag 420agccacttcc
gcaacagcat gcccagcttc gccatcagcg gctacgaggt gctgttcctg 480accacctacg
cccaggccgc caacacccac ctgttcctgc tgaaggacgc ccaaatctac 540ggagaggagt
ggggctacga gaaggaggac atcgccgagt tctacaagcg ccagctgaag 600ctgacccagg
agtacaccga ccactgcgtg aagtggtaca acgtgggtct agacaagctc 660cgcggcagca
gctacgagag ctgggtgaac ttcaaccgct accgccgcga gatgaccctg 720accgtgctgg
acctgatcgc cctgttcccc ctgtacgacg tgcgcctgta ccccaaggag 780gtgaagaccg
agctgacccg cgacgtgctg accgacccca tcgtgggcgt gaacaacctg 840cgcggctacg
gcaccacctt cagcaacatc gagaactaca tccgcaagcc ccacctgttc 900gactacctgc
accgcatcca gttccacacg cgtttccagc ccggctacta cggcaacgac 960agcttcaact
actggagcgg caactacgtg agcacccgcc ccagcatcgg cagcaacgac 1020atcatcacca
gccccttcta cggcaacaag agcagcgagc ccgtgcagaa ccttgagttc 1080aacggcgaga
aggtgtaccg cgccgtggct aacaccaacc tggccgtgtg gccctctgca 1140gtgtacagcg
gcgtgaccaa ggtggagttc agccagtaca acgaccagac cgacgaggcc 1200agcacccaga
cctacgacag caagcgcaac gtgggcgccg tgagctggga cagcatcgac 1260cagctgcccc
ccgagaccac cgacgagccc ctggagaagg gctacagcca ccagctgaac 1320tacgtgatgt
gcttcctgat gcagggcagc cgcggcacca tccccgtgct gacctggacc 1380cacaagagcg
tcgacttctt caacatgatc gacagcaaga agatcaccca gctgcccctg 1440accaagagca
ccaacctggg cagcggcacc agcgtggtga agggccccgg cttcaccggc 1500ggcgacatcc
tgcgccgcac cagccccggc cagatcagca ccctgcgcgt gaacatcacc 1560gcccccctga
gccagcgcta ccgcgtccgc atccgctacg ccagcaccac caacctgcag 1620ttccacacca
gcatcgacgg ccgccccatc aaccagggca acttcagcgc caccatgagc 1680agcggcagca
acctgcagag cggcagcttc cgcaccgtgg gcttcaccac ccccttcaac 1740ttcagcaacg
gcagcagcgt gttcaccctg agcgcccacg tgttcaacag cggcaacgag 1800gtgtacatcg
accgcatcga gttcgtgccc gccgaggtga cctag
184522614PRTArtificial SequenceFR-cg-dm3 protein 22Met Thr Ser Asn Gly
Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp Ser Ser
Thr Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly
Phe 35 40 45 Pro
Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110 Leu Ser
Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn Pro His Ser 115
120 125 Gln Gly Arg Ile Arg Glu Leu
Phe Ser Gln Ala Glu Ser His Phe Arg 130 135
140 Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu
Val Leu Phe Leu 145 150 155
160 Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys Asp
165 170 175 Ala Gln Ile
Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala 180
185 190 Glu Phe Tyr Lys Arg Gln Leu Lys
Leu Thr Gln Glu Tyr Thr Asp His 195 200
205 Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg
Gly Ser Ser 210 215 220
Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu 225
230 235 240 Thr Val Leu Asp
Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg Leu 245
250 255 Tyr Pro Lys Glu Val Lys Thr Glu Leu
Thr Arg Asp Val Leu Thr Asp 260 265
270 Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
Phe Ser 275 280 285
Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu His 290
295 300 Arg Ile Gln Phe His
Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn Asp 305 310
315 320 Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val
Ser Thr Arg Pro Ser Ile 325 330
335 Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser
Ser 340 345 350 Glu
Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg Ala 355
360 365 Val Ala Asn Thr Asn Leu
Ala Val Trp Pro Ser Ala Val Tyr Ser Gly 370 375
380 Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp
Gln Thr Asp Glu Ala 385 390 395
400 Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser Trp
405 410 415 Asp Ser
Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu 420
425 430 Lys Gly Tyr Ser His Gln Leu
Asn Tyr Val Met Cys Phe Leu Met Gln 435 440
445 Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr
His Lys Ser Val 450 455 460
Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu 465
470 475 480 Thr Lys Ser
Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro 485
490 495 Gly Phe Thr Gly Gly Asp Ile Leu
Arg Arg Thr Ser Pro Gly Gln Ile 500 505
510 Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln
Arg Tyr Arg 515 520 525
Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser 530
535 540 Ile Asp Gly Arg
Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser 545 550
555 560 Ser Gly Ser Asn Leu Gln Ser Gly Ser
Phe Arg Thr Val Gly Phe Thr 565 570
575 Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu
Ser Ala 580 585 590
His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe
595 600 605 Val Pro Ala Glu
Val Thr 610 231845DNAArtificial Sequence9F-cg-dm3
coding sequence 23atgactagta acggccgcca gtgtgctggt attcgcccta tgacggccga
caacaacacc 60gaggccctgg acagcagcac caccaaggac gtgatccaga agggcatcag
cgtggtgggc 120gacctgctgg gcgtggtggg cttccccttc ggcggcgccc tggtgagctt
ctacaccaac 180ttcctgaaca ccatctggcc cagcgaggac ccctggaagg ccttcatgga
gcaggtggag 240gccctgatgg accagaagat cgccgactac gccaagaaca aggcactggc
cgagctacag 300ggcctccaga acaacgtgga ggactatgtg agcgccctga gcagctggca
gaagaacccc 360gtctcgagcc gcaaccccca cagccagggc cgcatccgcg agctgttcag
ccaggccgag 420agccacttcc gcaacagcat gcccagcttc gccatcagcg gctacgaggt
gctgttcctg 480accacctacg cccaggccgc caacacccac ctgttcctgc tgaaggacgc
ccaaatctac 540ggagaggagt ggggctacga gaaggaggac atcgccgagt tctacaagcg
ccagctgaag 600ctgacccagg agtacaccga ccactgcgtg aagtggtaca acgtgggtct
agacaagctc 660cgcggcagca gctacgagag ctgggtgaac ttcaaccgct accgccgcga
gatgaccctg 720accgtgctgg acctgatcgc cctgttcccc ctgtacgacg tgcgcctgta
ccccaaggag 780gtgaagaccg agctgacccg cgacgtgctg accgacccca tcgtgggcgt
gaacaacctg 840cgcggctacg gcaccacctt cagcaacatc gagaactaca tccgcaagcc
ccacctgttc 900gactacctgc accgcatcca gttccacacg cgtttccagc ccggctacta
cggcaacgac 960agcttcaact actggagcgg caactacgtg agcacccgcc ccagcatcgg
cagcaacgac 1020atcatcacca gccccttcta cggcaacaag agcagcgagc ccgtgcagaa
ccttgagttc 1080aacggcgaga aggtgtaccg cgccgtggct aacaccaacc tggccgtgtg
gccctctgca 1140gtgtacagcg gcgtgaccaa ggtggagttc agccagtaca acgaccagac
cgacgaggcc 1200agcacccaga cctacgacag caagcgcaac gtgggcgccg tgagctggga
cagcatcgac 1260cagctgcccc ccgagaccac cgacgagccc ctggagaagg gctacagcca
ccagctgaac 1320tacgtgatgt gcttcctgat gcagggcagc cgcggcacca tccccgtgct
gacctggacc 1380cacaagagcg tcgacttctt caacatgatc gacagcaaga agatcaccca
gctgcccctg 1440accaagagca ccaacctggg cagcggcacc agcgtggtga agggccccgg
cttcaccggc 1500ggcgacatcc tgcgccgcac cagccccggc cagatcagca ccctgcgcgt
gaacatcacc 1560gcccccctga gccagcgcta ccgcgtccgc atccgctacg ccagcaccac
caacctgcag 1620ttccacacca gcatcgacgg ccgccccatc aaccagggca acttcagcgc
caccatgagc 1680agcggcagca acctgcagag cggcagcttc cgcaccgtgg gcttcaccac
ccccttcaac 1740ttcagcaacg gcagcagcgt gttcaccctg agcgcccacg tgttcaacag
cggcaacgag 1800gtgtacatcg accgcatcga gttcgtgccc gccgaggtga cctag
184524614PRTArtificial Sequence9F-cg-dm3 protein 24Met Thr Ser
Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Met Thr Ala 1 5
10 15 Asp Asn Asn Thr Glu Ala Leu Asp
Ser Ser Thr Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
Val Gly Phe 35 40 45
Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser
Glu Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp
Tyr Ala Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val
Ser Ala 100 105 110
Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn Pro His Ser
115 120 125 Gln Gly Arg Ile
Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg 130
135 140 Asn Ser Met Pro Ser Phe Ala Ile
Ser Gly Tyr Glu Val Leu Phe Leu 145 150
155 160 Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe
Leu Leu Lys Asp 165 170
175 Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala
180 185 190 Glu Phe Tyr
Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp His 195
200 205 Cys Val Lys Trp Tyr Asn Val Gly
Leu Asp Lys Leu Arg Gly Ser Ser 210 215
220 Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu
Met Thr Leu 225 230 235
240 Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg Leu
245 250 255 Tyr Pro Lys Glu
Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr Asp 260
265 270 Pro Ile Val Gly Val Asn Asn Leu Arg
Gly Tyr Gly Thr Thr Phe Ser 275 280
285 Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr
Leu His 290 295 300
Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn Asp 305
310 315 320 Ser Phe Asn Tyr Trp
Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser Ile 325
330 335 Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe
Tyr Gly Asn Lys Ser Ser 340 345
350 Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg
Ala 355 360 365 Val
Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser Gly 370
375 380 Val Thr Lys Val Glu Phe
Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala 385 390
395 400 Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val
Gly Ala Val Ser Trp 405 410
415 Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu
420 425 430 Lys Gly
Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu Met Gln 435
440 445 Gly Ser Arg Gly Thr Ile Pro
Val Leu Thr Trp Thr His Lys Ser Val 450 455
460 Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr
Gln Leu Pro Leu 465 470 475
480 Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro
485 490 495 Gly Phe Thr
Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile 500
505 510 Ser Thr Leu Arg Val Asn Ile Thr
Ala Pro Leu Ser Gln Arg Tyr Arg 515 520
525 Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe
His Thr Ser 530 535 540
Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser 545
550 555 560 Ser Gly Ser Asn
Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr 565
570 575 Thr Pro Phe Asn Phe Ser Asn Gly Ser
Ser Val Phe Thr Leu Ser Ala 580 585
590 His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile
Glu Phe 595 600 605
Val Pro Ala Glu Val Thr 610 251863DNAArtificial
SequenceB8a coding sequence 25atgacggccg acaacaacac cgaggccctg gacagcagca
ccaccaagga cgtgatccag 60aagggcatca gcgtggtggg cgacctgctg ggcgtggtgg
gcttcccctt cggcggcgcc 120ctggtgagct tctacaccaa cttcctgaac accatctggc
ccagcgagga cccctggaag 180gccttcatgg agcaggtgga ggccctgatg gaccagaaga
tcgccgacta cgccaagaac 240aaggcactgg ccgagctaca gggcctccag aacaacgtgg
aggactatgt gagcgccctg 300agcagctggc agaagaaccc cgctgcaccg ttccgcaacc
cccacagcca gggccgcatc 360cgcgagctgt tcagccaggc cgagagccac ttccgcaaca
gcatgcccag cttcgccatc 420agcggctacg aggtgctgtt cctgaccacc tacgcccagg
ccgccaacac ccacctgttc 480ctgctgaagg acgcccaaat ctacggagag gagtggggct
acgagaagga ggacatcgcc 540gagttctaca agcgccagct gaagctgacc caggagtaca
ccgaccactg cgtgaagtgg 600tacaacgtgg gtctagacaa gctccgcggc agcagctacg
agagctgggt gaacttcaac 660cgctaccgcc gcgagatgac cctgaccgtg ctggacctga
tcgccctgtt ccccctgtac 720gacgtgcgcc tgtaccccaa ggaggtgaag accgagctga
cccgcgacgt gctgaccgac 780cccatcgtgg gcgtgaacaa cctgcgcggc tacggcacca
ccttcagcaa catcgagaac 840tacatccgca agccccacct gttcgactac ctgcaccgca
tccagttcca cacgcgtttc 900cagcccggct actacggcaa cgacagcttc aactactgga
gcggcaacta cgtgagcacc 960cgccccagca tcggcagcaa cgacatcatc accagcccct
tctacggcaa caagagcagc 1020gagcccgtgc agaaccttga gttcaacggc gagaaggtgt
accgcgccgt ggctaacacc 1080aacctggccg tgtggccctc tgcagtgtac agcggcgtga
ccaaggtgga gttcagccag 1140tacaacgacc agaccgacga ggccagcacc cagacctacg
acagcaagcg caacgtgggc 1200gccgtgagct gggacagcat cgaccagctg ccccccgaga
ccaccgacga gcccctggag 1260aagggctaca gccaccagct gaactacgtg atgtgcttcc
tgatgcaggg cagccgcggc 1320accatccccg tgctgacctg gacccacaag agcgtcgact
tcttcaacat gatcgacagc 1380aagaagatca cccagctgcc cctggtgaag gccagcgagc
tgccccaggg caccaccgtg 1440gttcgcggcc ccggcttcac cggaggcgac atcctgcgac
gcaccaacac cggcggcttc 1500ggccccatcc gcgtgaccgt gaacggcccc ctgacccagc
gctaccgcat cggcttccgc 1560tacgccagca ccgtggactt cgacttcttc gtgagccgcg
gcggcaccac cgtgaacaac 1620ttccgcttcc tgcgcaccat gaacagcggc gacgagctga
agtacggcaa cttcgtgcgc 1680cgcgccttca ccaccccctt caccttcacc cagatccagg
acatcatccg caccagcatc 1740cagggcctga gcggcaacgg cgaggtgtac atcgacaaga
tcgagatcat ccccgtgacc 1800gccaccttcg aggccgagta cgacctagag cgcgcccagg
aggccgtgaa cgccctgttc 1860tag
186326620PRTArtificial SequenceB8a protein 26Met
Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1
5 10 15 Asp Val Ile Gln Lys Gly
Ile Ser Val Val Gly Asp Leu Leu Gly Val 20
25 30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val
Ser Phe Tyr Thr Asn Phe 35 40
45 Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe
Met Glu 50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65
70 75 80 Lys Ala Leu Ala Glu
Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr 85
90 95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn
Pro Ala Ala Pro Phe Arg 100 105
110 Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala
Glu 115 120 125 Ser
His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130
135 140 Val Leu Phe Leu Thr Thr
Tyr Ala Gln Ala Ala Asn Thr His Leu Phe 145 150
155 160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu
Trp Gly Tyr Glu Lys 165 170
175 Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu
180 185 190 Tyr Thr
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu 195
200 205 Arg Gly Ser Ser Tyr Glu Ser
Trp Val Asn Phe Asn Arg Tyr Arg Arg 210 215
220 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu
Phe Pro Leu Tyr 225 230 235
240 Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp
245 250 255 Val Leu Thr
Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly 260
265 270 Thr Thr Phe Ser Asn Ile Glu Asn
Tyr Ile Arg Lys Pro His Leu Phe 275 280
285 Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln
Pro Gly Tyr 290 295 300
Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 305
310 315 320 Arg Pro Ser Ile
Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly 325
330 335 Asn Lys Ser Ser Glu Pro Val Gln Asn
Leu Glu Phe Asn Gly Glu Lys 340 345
350 Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro
Ser Ala 355 360 365
Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 370
375 380 Thr Asp Glu Ala Ser
Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly 385 390
395 400 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu
Pro Pro Glu Thr Thr Asp 405 410
415 Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met
Cys 420 425 430 Phe
Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr 435
440 445 His Lys Ser Val Asp Phe
Phe Asn Met Ile Asp Ser Lys Lys Ile Thr 450 455
460 Gln Leu Pro Leu Val Lys Ala Ser Glu Leu Pro
Gln Gly Thr Thr Val 465 470 475
480 Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn
485 490 495 Thr Gly
Gly Phe Gly Pro Ile Arg Val Thr Val Asn Gly Pro Leu Thr 500
505 510 Gln Arg Tyr Arg Ile Gly Phe
Arg Tyr Ala Ser Thr Val Asp Phe Asp 515 520
525 Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn
Phe Arg Phe Leu 530 535 540
Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly Asn Phe Val Arg 545
550 555 560 Arg Ala Phe
Thr Thr Pro Phe Thr Phe Thr Gln Ile Gln Asp Ile Ile 565
570 575 Arg Thr Ser Ile Gln Gly Leu Ser
Gly Asn Gly Glu Val Tyr Ile Asp 580 585
590 Lys Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Glu Ala
Glu Tyr Asp 595 600 605
Leu Glu Arg Ala Gln Glu Ala Val Asn Ala Leu Phe 610
615 620 271902DNAArtificial Sequence5*B8a coding sequence
27atgactagta acggccgcca gtgtgctggt attcgccctt atgacggccg acaacaacac
60cgaggcctgg acagcagcac caccaaggac gtgatccaga agggcatcag cgtggtgggc
120gacctgctgg gcgtggtggg cttccccttc ggcggcgccc tggtgagctt ctacaccaac
180ttcctgaaca ccatctggcc cagcgaggac ccctggaagg ccttcatgga gcaggtggag
240gccctgatgg accagaagat cgccgactac gccaagaaca aggcactggc cgagctacag
300ggcctccaga acaacgtgga ggactatgtg agcgccctga gcagctggca gaagaacccc
360gctgcaccgt tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt cagccaggcc
420gagagccact tccgcaacag catgcccagc ttcgccatca gcggctacga ggtgctgttc
480ctgaccacct acgcccaggc cgccaacacc cacctgttcc tgctgaagga cgcccaaatc
540tacggagagg agtggggcta cgagaaggag gacatcgccg agttctacaa gcgccagctg
600aagctgaccc aggagtacac cgaccactgc gtgaagtggt acaacgtggg tctagacaag
660ctccgcggca gcagctacga gagctgggtg aacttcaacc gctaccgccg cgagatgacc
720ctgaccgtgc tggacctgat cgccctgttc cccctgtacg acgtgcgcct gtaccccaag
780gaggtgaaga ccgagctgac ccgcgacgtg ctgaccgacc ccatcgtggg cgtgaacaac
840ctgcgcggct acggcaccac cttcagcaac atcgagaact acatccgcaa gccccacctg
900ttcgactacc tgcaccgcat ccagttccac acgcgtttcc agcccggcta ctacggcaac
960gacagcttca actactggag cggcaactac gtgagcaccc gccccagcat cggcagcaac
1020gacatcatca ccagcccctt ctacggcaac aagagcagcg agcccgtgca gaaccttgag
1080ttcaacggcg agaaggtgta ccgcgccgtg gctaacacca acctggccgt gtggccctct
1140gcagtgtaca gcggcgtgac caaggtggag ttcagccagt acaacgacca gaccgacgag
1200gccagcaccc agacctacga cagcaagcgc aacgtgggcg ccgtgagctg ggacagcatc
1260gaccagctgc cccccgagac caccgacgag cccctggaga agggctacag ccaccagctg
1320aactacgtga tgtgcttcct gatgcagggc agccgcggca ccatccccgt gctgacctgg
1380acccacaaga gcgtcgactt cttcaacatg atcgacagca agaagatcac ccagctgccc
1440ctggtgaagg ccagcgagct gccccagggc accaccgtgg ttcgcggccc cggcttcacc
1500ggaggcgaca tcctgcgacg caccaacacc ggcggcttcg gccccatccg cgtgaccgtg
1560aacggccccc tgacccagcg ctaccgcatc ggcttccgct acgccagcac cgtggacttc
1620gacttcttcg tgagccgcgg cggcaccacc gtgaacaact tccgcttcct gcgcaccatg
1680aacagcggcg acgagctgaa gtacggcaac ttcgtgcgcc gcgccttcac cacccccttc
1740accttcaccc agatccagga catcatccgc accagcatcc agggcctgag cggcaacggc
1800gaggtgtaca tcgacaagat cgagatcatc cccgtgaccg ccaccttcga ggccgagtac
1860gacctagagc gcgcccagga ggccgtgaac gccctgttct ag
190228633PRTArtificial Sequence5*B8a Protein 28Met Thr Ser Asn Gly Arg
Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr
Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly
Phe 35 40 45 Pro
Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110 Leu Ser
Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His 115
120 125 Ser Gln Gly Arg Ile Arg Glu
Leu Phe Ser Gln Ala Glu Ser His Phe 130 135
140 Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr
Glu Val Leu Phe 145 150 155
160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys
165 170 175 Asp Ala Gln
Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile 180
185 190 Ala Glu Phe Tyr Lys Arg Gln Leu
Lys Leu Thr Gln Glu Tyr Thr Asp 195 200
205 His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
Arg Gly Ser 210 215 220
Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225
230 235 240 Leu Thr Val Leu
Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg 245
250 255 Leu Tyr Pro Lys Glu Val Lys Thr Glu
Leu Thr Arg Asp Val Leu Thr 260 265
270 Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
Thr Phe 275 280 285
Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu 290
295 300 His Arg Ile Gln Phe
His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn 305 310
315 320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr
Val Ser Thr Arg Pro Ser 325 330
335 Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
Ser 340 345 350 Ser
Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg 355
360 365 Ala Val Ala Asn Thr Asn
Leu Ala Val Trp Pro Ser Ala Val Tyr Ser 370 375
380 Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn
Asp Gln Thr Asp Glu 385 390 395
400 Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser
405 410 415 Trp Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu 420
425 430 Glu Lys Gly Tyr Ser His Gln
Leu Asn Tyr Val Met Cys Phe Leu Met 435 440
445 Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp
Thr His Lys Ser 450 455 460
Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro 465
470 475 480 Leu Val Lys
Ala Ser Glu Leu Pro Gln Gly Thr Thr Val Val Arg Gly 485
490 495 Pro Gly Phe Thr Gly Gly Asp Ile
Leu Arg Arg Thr Asn Thr Gly Gly 500 505
510 Phe Gly Pro Ile Arg Val Thr Val Asn Gly Pro Leu Thr
Gln Arg Tyr 515 520 525
Arg Ile Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe Phe Val 530
535 540 Ser Arg Gly Gly
Thr Thr Val Asn Asn Phe Arg Phe Leu Arg Thr Met 545 550
555 560 Asn Ser Gly Asp Glu Leu Lys Tyr Gly
Asn Phe Val Arg Arg Ala Phe 565 570
575 Thr Thr Pro Phe Thr Phe Thr Gln Ile Gln Asp Ile Ile Arg
Thr Ser 580 585 590
Ile Gln Gly Leu Ser Gly Asn Gly Glu Val Tyr Ile Asp Lys Ile Glu
595 600 605 Ile Ile Pro Val
Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg 610
615 620 Ala Gln Glu Ala Val Asn Ala Leu
Phe 625 630 291791DNAArtificial SequenceV3A
coding sequence 29atgacggccg acaacaacac cgaggccctg gacagcagca ccaccaagga
cgtgatccag 60aagggcatca gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt
cggcggcgcc 120ctggtgagct tctacaccaa cttcctgaac accatctggc ccagcgagga
cccctggaag 180gccttcatgg agcaggtgga ggccctgatg gaccagaaga tcgccgacta
cgccaagaac 240aaggcactgg ccgagctaca gggcctccag aacaacgtgg aggactatgt
gagcgccctg 300agcagctggc agaagaaccc cgctgcaccg ttccgcaacc cccacagcca
gggccgcatc 360cgcgagctgt tcagccaggc cgagagccac ttccgcaaca gcatgcccag
cttcgccatc 420agcggctacg aggtgctgtt cctgaccacc tacgcccagg ccgccaacac
ccacctgttc 480ctgctgaagg acgcccaaat ctacggagag gagtggggct acgagaagga
ggacatcgcc 540gagttctaca agcgccagct gaagctgacc caggagtaca ccgaccactg
cgtgaagtgg 600tacaacgtgg gtctagacaa gctccgcggc agcagctacg agagctgggt
gaacttcaac 660cgctaccgcc gcgagatgac cctgaccgtg ctggacatcg tgagcctgtt
ccccaactac 720gacagccgca cctaccccat ccgcaccgtg agccagctga cccgcgagat
ttacaccaac 780cccgtgctgg agaacttcga cggcagcttc cgcggcagcg cccagggcat
cgagggcagc 840atccgcagcc cccacctgat ggacatcctg aacagcatca ccatctacac
cgacgcccac 900cgcggcgagt actactggag cggccaccag atcatggcca gccccgtcgg
cttcagcggc 960cccgagttca ccttccccct gtacggcacc atgggcaacg ctgcacctca
gcagcgcatc 1020gtggcacagc tgggccaggg agtgtaccgc accctgagca gcaccctgta
ccgtcgacct 1080ttcaacatcg gcatcaacaa ccagcagctg agcgtgctgg acggcaccga
gttcgcctac 1140ggcaccagca gcaacctgcc cagcgccgtg taccgcaaga gcggcaccgt
ggacagcctg 1200gacgagatcc cccctcagaa caacaacgtg ccacctcgac agggcttcag
ccaccgtctg 1260agccacgtga gcatgttccg cagtggcttc agcaacagca gcgtgagcat
catccgtgca 1320cctatgttca gctggattca ccgcagtgcc gagttcaaca acatcatccc
cagcagccag 1380atcacccaga tccccctggt gaaggcctac aagctccaga gcggcgccag
cgtggtggca 1440ggcccccgct tcaccggcgg cgacatcatc cagtgcaccg agaacggcag
cgccgccacc 1500atctacgtga cccccgacgt gagctacagc cagaagtacc gcgcccgcat
ccactacgcc 1560agcaccagcc agatcacctt caccctgagc ctggacgggg cccccttcaa
ccaatactac 1620ttcgacaaga ccatcaacaa gggcgacacc ctgacctaca acagcttcaa
cctggccagc 1680ttcagcaccc ctttcgagct gagcggcaac aacctccaga tcggcgtgac
cggcctgagc 1740gccggcgaca aggtgtacat cgacaagatc gagttcatcc ccgtgaacta g
179130596PRTArtificial SequenceV3A protein 30Met Thr Ala Asp
Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val
Val Gly Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr
Asn Phe 35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu
Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln
Asn Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe
Arg 100 105 110 Asn
Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 115
120 125 Ser His Phe Arg Asn Ser
Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130 135
140 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala
Asn Thr His Leu Phe 145 150 155
160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175 Glu Asp
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu 180
185 190 Tyr Thr Asp His Cys Val Lys
Trp Tyr Asn Val Gly Leu Asp Lys Leu 195 200
205 Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn
Arg Tyr Arg Arg 210 215 220
Glu Met Thr Leu Thr Val Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr 225
230 235 240 Asp Ser Arg
Thr Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu 245
250 255 Ile Tyr Thr Asn Pro Val Leu Glu
Asn Phe Asp Gly Ser Phe Arg Gly 260 265
270 Ser Ala Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His
Leu Met Asp 275 280 285
Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr 290
295 300 Tyr Trp Ser Gly
His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly 305 310
315 320 Pro Glu Phe Thr Phe Pro Leu Tyr Gly
Thr Met Gly Asn Ala Ala Pro 325 330
335 Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg
Thr Leu 340 345 350
Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln
355 360 365 Gln Leu Ser Val
Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser 370
375 380 Asn Leu Pro Ser Ala Val Tyr Arg
Lys Ser Gly Thr Val Asp Ser Leu 385 390
395 400 Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro
Arg Gln Gly Phe 405 410
415 Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn
420 425 430 Ser Ser Val
Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg 435
440 445 Ser Ala Glu Phe Asn Asn Ile Ile
Pro Ser Ser Gln Ile Thr Gln Ile 450 455
460 Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser
Val Val Ala 465 470 475
480 Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly
485 490 495 Ser Ala Ala Thr
Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys 500
505 510 Tyr Arg Ala Arg Ile His Tyr Ala Ser
Thr Ser Gln Ile Thr Phe Thr 515 520
525 Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp
Lys Thr 530 535 540
Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser 545
550 555 560 Phe Ser Thr Pro Phe
Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val 565
570 575 Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr
Ile Asp Lys Ile Glu Phe 580 585
590 Ile Pro Val Asn 595 31 1797DNAArtificial
SequenceV4F coding sequence 31atgacggccg acaacaacac cgaggccctg gacagcagca
ccaccaagga cgtgatccag 60aagggcatca gcgtggtggg cgacctgctg ggcgtggtgg
gcttcccctt cggcggcgcc 120ctggtgagct tctacaccaa cttcctgaac accatctggc
ccagcgagga cccctggaag 180gccttcatgg agcaggtgga ggccctgatg gaccagaaga
tcgccgacta cgccaagaac 240aaggcactgg ccgagctaca gggcctccag aacaacgtgg
aggactatgt gagcgccctg 300agcagctggc agaagaaccc cgctgcaccg ttccgcaacc
cccacagcca gggccgcatc 360cgcgagctgt tcagccaggc cgagagccac ttccgcaaca
gcatgcccag cttcgccatc 420agcggctacg aggtgctgtt cctgaccacc tacgcccagg
ccgccaacac ccacctgttc 480ctgctgaagg acgcccaaat ctacggagag gagtggggct
acgagaagga ggacatcgcc 540gagttctaca agcgccagct gaagctgacc caggagtaca
ccgaccactg cgtgaagtgg 600tacaacgtgg gtctagacaa gctccgcggc agcagctacg
agagctgggt gaacttcaac 660cgctaccgcc gcgagatgac cctgaccgtg ctggacctga
tcgccctgtt ccccctgtac 720gacgtgcgcc tgtaccccaa ggaggtgaag accgagctga
cccgcgacgt gctgaccgac 780cccatcgtgg gcgtgaacaa cctgcgcggc tacggcacca
ccttcagcaa catcgagaac 840tacatccgca agccccacct gttcgactac ctgcaccgca
tccagttcca cacgcgtttc 900cagcccggct actacggcaa cgacagcttc aactactgga
gcggcaacta cgtgagcacc 960cgccccagca tcggcagcaa cgacatcatc accagcccct
tctacggcaa caagagcagc 1020gagcccgtgc agaaccttga gttcaacggc gagaaggtgt
accgcgccgt ggctaacacc 1080aacctggccg tgtggccctc tgcagtgtac agcggcgtga
ccaaggtgga gttcagccag 1140tacaacgacc agaccgacga ggccagcacc cagacctacg
acagcaagcg caacgtgggc 1200gccgtgagct gggacagcat cgaccagctg ccccccgaga
ccaccgacga gcccctggag 1260aagggctaca gccaccagct gaactacgtg atgtgcttcc
tgatgcaggg cagccgcggc 1320accatccccg tgctgacctg gacccacaag agcgtcgact
tcttcaacat gatcgacagc 1380aagaagatca cccagctcgc cctgaccaag agcaccaacc
tgggcagcgg caccagcgtg 1440gtgaagggcc ccggcttcac cggcggcgac atcctgcgcc
gcaccagccc cggccagatc 1500agcaccctgc gcgtgaacat caccgccccc ctgagccagc
gctaccgcgt ccgcatccac 1560tacgccagca ccagccagat caccttcacc ctgagcctgg
acggggcccc cttcaaccaa 1620tactacttcg acaagaccat caacaagggc gacaccctga
cctacaacag cttcaacctg 1680gccagcttca gcaccccttt cgagctgagc ggcaacaacc
tccagatcgg cgtgaccggc 1740ctgagcgccg gcgacaaggt gtacatcgac aagatcgagt
tcatccccgt gaactag 179732598PRTArtificial SequenceV4F protein 32Met
Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1
5 10 15 Asp Val Ile Gln Lys Gly
Ile Ser Val Val Gly Asp Leu Leu Gly Val 20
25 30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val
Ser Phe Tyr Thr Asn Phe 35 40
45 Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe
Met Glu 50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65
70 75 80 Lys Ala Leu Ala Glu
Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr 85
90 95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn
Pro Ala Ala Pro Phe Arg 100 105
110 Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala
Glu 115 120 125 Ser
His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130
135 140 Val Leu Phe Leu Thr Thr
Tyr Ala Gln Ala Ala Asn Thr His Leu Phe 145 150
155 160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu
Trp Gly Tyr Glu Lys 165 170
175 Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu
180 185 190 Tyr Thr
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu 195
200 205 Arg Gly Ser Ser Tyr Glu Ser
Trp Val Asn Phe Asn Arg Tyr Arg Arg 210 215
220 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu
Phe Pro Leu Tyr 225 230 235
240 Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp
245 250 255 Val Leu Thr
Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly 260
265 270 Thr Thr Phe Ser Asn Ile Glu Asn
Tyr Ile Arg Lys Pro His Leu Phe 275 280
285 Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln
Pro Gly Tyr 290 295 300
Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 305
310 315 320 Arg Pro Ser Ile
Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly 325
330 335 Asn Lys Ser Ser Glu Pro Val Gln Asn
Leu Glu Phe Asn Gly Glu Lys 340 345
350 Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro
Ser Ala 355 360 365
Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 370
375 380 Thr Asp Glu Ala Ser
Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly 385 390
395 400 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu
Pro Pro Glu Thr Thr Asp 405 410
415 Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met
Cys 420 425 430 Phe
Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr 435
440 445 His Lys Ser Val Asp Phe
Phe Asn Met Ile Asp Ser Lys Lys Ile Thr 450 455
460 Gln Leu Ala Leu Thr Lys Ser Thr Asn Leu Gly
Ser Gly Thr Ser Val 465 470 475
480 Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser
485 490 495 Pro Gly
Gln Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser 500
505 510 Gln Arg Tyr Arg Val Arg Ile
His Tyr Ala Ser Thr Ser Gln Ile Thr 515 520
525 Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln
Tyr Tyr Phe Asp 530 535 540
Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu 545
550 555 560 Ala Ser Phe
Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile 565
570 575 Gly Val Thr Gly Leu Ser Ala Gly
Asp Lys Val Tyr Ile Asp Lys Ile 580 585
590 Glu Phe Ile Pro Val Asn 595 33
1836DNAArtificial Sequence5*V4F coding sequence 33atgactagta acggccgcca
gtgtgctggt attcgccctt atgacggccg acaacaacac 60cgaggcctgg acagcagcac
caccaaggac gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg gcgtggtggg
cttccccttc ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca ccatctggcc
cagcgaggac ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg accagaagat
cgccgactac gccaagaaca aggcactggc cgagctacag 300ggcctccaga acaacgtgga
ggactatgtg agcgccctga gcagctggca gaagaacccc 360gctgcaccgt tccgcaaccc
ccacagccag ggccgcatcc gcgagctgtt cagccaggcc 420gagagccact tccgcaacag
catgcccagc ttcgccatca gcggctacga ggtgctgttc 480ctgaccacct acgcccaggc
cgccaacacc cacctgttcc tgctgaagga cgcccaaatc 540tacggagagg agtggggcta
cgagaaggag gacatcgccg agttctacaa gcgccagctg 600aagctgaccc aggagtacac
cgaccactgc gtgaagtggt acaacgtggg tctagacaag 660ctccgcggca gcagctacga
gagctgggtg aacttcaacc gctaccgccg cgagatgacc 720ctgaccgtgc tggacctgat
cgccctgttc cccctgtacg acgtgcgcct gtaccccaag 780gaggtgaaga ccgagctgac
ccgcgacgtg ctgaccgacc ccatcgtggg cgtgaacaac 840ctgcgcggct acggcaccac
cttcagcaac atcgagaact acatccgcaa gccccacctg 900ttcgactacc tgcaccgcat
ccagttccac acgcgtttcc agcccggcta ctacggcaac 960gacagcttca actactggag
cggcaactac gtgagcaccc gccccagcat cggcagcaac 1020gacatcatca ccagcccctt
ctacggcaac aagagcagcg agcccgtgca gaaccttgag 1080ttcaacggcg agaaggtgta
ccgcgccgtg gctaacacca acctggccgt gtggccctct 1140gcagtgtaca gcggcgtgac
caaggtggag ttcagccagt acaacgacca gaccgacgag 1200gccagcaccc agacctacga
cagcaagcgc aacgtgggcg ccgtgagctg ggacagcatc 1260gaccagctgc cccccgagac
caccgacgag cccctggaga agggctacag ccaccagctg 1320aactacgtga tgtgcttcct
gatgcagggc agccgcggca ccatccccgt gctgacctgg 1380acccacaaga gcgtcgactt
cttcaacatg atcgacagca agaagatcac ccagctcgcc 1440ctgaccaaga gcaccaacct
gggcagcggc accagcgtgg tgaagggccc cggcttcacc 1500ggcggcgaca tcctgcgccg
caccagcccc ggccagatca gcaccctgcg cgtgaacatc 1560accgcccccc tgagccagcg
ctaccgcgtc cgcatccact acgccagcac cagccagatc 1620accttcaccc tgagcctgga
cggggccccc ttcaaccaat actacttcga caagaccatc 1680aacaagggcg acaccctgac
ctacaacagc ttcaacctgg ccagcttcag cacccctttc 1740gagctgagcg gcaacaacct
ccagatcggc gtgaccggcc tgagcgccgg cgacaaggtg 1800tacatcgaca agatcgagtt
catccccgtg aactag 183634611PRTArtificial
Sequence5*V4F Protein 34Met Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg
Pro Tyr Asp Gly 1 5 10
15 Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp Val Ile
20 25 30 Gln Lys Gly
Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly Phe 35
40 45 Pro Phe Gly Gly Ala Leu Val Ser
Phe Tyr Thr Asn Phe Leu Asn Thr 50 55
60 Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
Gln Val Glu 65 70 75
80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu
85 90 95 Ala Glu Leu Gln
Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala 100
105 110 Leu Ser Ser Trp Gln Lys Asn Pro Ala
Ala Pro Phe Arg Asn Pro His 115 120
125 Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
His Phe 130 135 140
Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe 145
150 155 160 Leu Thr Thr Tyr Ala
Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys 165
170 175 Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly
Tyr Glu Lys Glu Asp Ile 180 185
190 Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
Asp 195 200 205 His
Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser 210
215 220 Ser Tyr Glu Ser Trp Val
Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225 230
235 240 Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro
Leu Tyr Asp Val Arg 245 250
255 Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr
260 265 270 Asp Pro
Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe 275
280 285 Ser Asn Ile Glu Asn Tyr Ile
Arg Lys Pro His Leu Phe Asp Tyr Leu 290 295
300 His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly
Tyr Tyr Gly Asn 305 310 315
320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser
325 330 335 Ile Gly Ser
Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser 340
345 350 Ser Glu Pro Val Gln Asn Leu Glu
Phe Asn Gly Glu Lys Val Tyr Arg 355 360
365 Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
Val Tyr Ser 370 375 380
Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu 385
390 395 400 Ala Ser Thr Gln
Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser 405
410 415 Trp Asp Ser Ile Asp Gln Leu Pro Pro
Glu Thr Thr Asp Glu Pro Leu 420 425
430 Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
Leu Met 435 440 445
Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser 450
455 460 Val Asp Phe Phe Asn
Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Ala 465 470
475 480 Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly
Thr Ser Val Val Lys Gly 485 490
495 Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly
Gln 500 505 510 Ile
Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr 515
520 525 Arg Val Arg Ile His Tyr
Ala Ser Thr Ser Gln Ile Thr Phe Thr Leu 530 535
540 Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr
Phe Asp Lys Thr Ile 545 550 555
560 Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser Phe
565 570 575 Ser Thr
Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val Thr 580
585 590 Gly Leu Ser Ala Gly Asp Lys
Val Tyr Ile Asp Lys Ile Glu Phe Ile 595 600
605 Pro Val Asn 610 351901DNAArtificial
Sequence2OL-7 coding sequence 35atgacggccg acaacaacac cgaggccctg
gacagcagca ccaccaagga cgtgatccag 60aagggcatca gcgtggtggg cgacctgctg
ggcgtggtgg gcttcccctt cggcggcgcc 120ctggtgagct tctacaccaa cttcctgaac
accatctggc ccagcgagga cccctggaag 180gccttcatgg agcaggtgga ggccctgatg
gaccagaaga tcgccgacta cgccaagaac 240aaggcactgg ccgagctaca gggcctccag
aacaacgtgg aggactatgt gagcgccctg 300agcagctggc agaagaaccc cgctgcaccg
ttccgcaacc cccacagcca gggccgcatc 360cgcgagctgt tcagccaggc cgagagccac
ttccgcaaca gcatgcccag cttcgccatc 420agcggctacg aggtgctgtt cctgaccacc
tacgtgcagg ccgccaacct gcacctgagc 480gtgctgcgcg acgtcagcgt gttcggccag
cgctggggct tcgacgccgc caccatcaac 540agccgctaca acgacctgac ccgcctgatc
ggcaactaca ccgaccacgc cgtgcgctgg 600tacaacaccg gcctggagcg cgtgtggggt
cccgacagcc gcgactggat caggtacaac 660cagttccgcc gcgagctgac cctgaccgtg
ctggacatcg tgagcctgtt ccccaactac 720gacagccgca cctaccccat ccgcaccgtg
agccagctga cccgcgagat ttacaccaac 780cccgtgctgg agaacttcga cggcagcttc
cgcggcagcg cccagggcat cgagggcagc 840atccgcagcc cccacctgat ggacatcctg
aacagcatca ccatctacac cgacgcccac 900cgcggcgagt actactggag cggccaccag
atcatggcca gccccgtcgg cttcagcggc 960cccgagttca ccttccccct gtacggcacc
atgggcaacg ctgcacctca gcagcgcatc 1020gtggcacagc tgggccaggg agtgtaccgc
accctgagca gcaccctgta ccgtcgacct 1080ttcaacatcg gcatcaacaa ccagcagctg
agcgtgctgg acggcaccga gttcgcctac 1140ggcaccagca gcaacctgcc cagcgccgtg
taccgcaaga gcggcaccgt ggacagcctg 1200gacgagatcc cccctcagaa caacaacgtg
ccacctcgac agggcttcag ccaccgtctg 1260agccacgtga gcatgttccg cagtggcttc
agcaacagca gcgtgagcat catccgtgca 1320cctatgttca gctggattca ccgcagtgcc
gagttcaaca acatcatccc cagcagccag 1380atcacccaga tccccctgac caagagcacc
aacctgggca gcggcaccag cgtggtgaag 1440ggccccggct tcaccggcgg cgacatcctg
cgccgcacca gccccggcca gatcagcacc 1500ctgcgcgtga acatcaccgc ccccctgagc
cagcgctacc gcgtccgcat ccgctacgcc 1560agcaccacca acctgcagtt ccacaccagc
atcgacggcc gccccatcaa ccagggcaac 1620ttcagcgcca ccatgagcag cggcagcaac
ctgcagagcg gcagcttccg caccgtgggc 1680ttcaccaccc ccttcaactt cagcaacggc
agcagcgtgt tcaccctgag cgcccacgtg 1740ttcaacagcg gcaacgaggt gtacatcgac
cgcatcgagt tcgtgcccgc cgaggtgacc 1800ttcgaggccg agtacgacct ggagagggct
cagaaggccg tgaacgagct gttcaccagc 1860agcaaccaga tcggcctgaa gaccgacgtg
accgactacc a 190136633PRTArtificial Sequence2OL-7
protein 36Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15 Asp Val
Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val 20
25 30 Val Gly Phe Pro Phe Gly Gly
Ala Leu Val Ser Phe Tyr Thr Asn Phe 35 40
45 Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys
Ala Phe Met Glu 50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65
70 75 80 Lys Ala Leu
Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr 85
90 95 Val Ser Ala Leu Ser Ser Trp Gln
Lys Asn Pro Ala Ala Pro Phe Arg 100 105
110 Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser
Gln Ala Glu 115 120 125
Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130
135 140 Val Leu Phe Leu
Thr Thr Tyr Val Gln Ala Ala Asn Leu His Leu Ser 145 150
155 160 Val Leu Arg Asp Val Ser Val Phe Gly
Gln Arg Trp Gly Phe Asp Ala 165 170
175 Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile
Gly Asn 180 185 190
Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val
195 200 205 Trp Gly Pro Asp
Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg 210
215 220 Glu Leu Thr Leu Thr Val Leu Asp
Ile Val Ser Leu Phe Pro Asn Tyr 225 230
235 240 Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val Ser Gln
Leu Thr Arg Glu 245 250
255 Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly
260 265 270 Ser Ala Gln
Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp 275
280 285 Ile Leu Asn Ser Ile Thr Ile Tyr
Thr Asp Ala His Arg Gly Glu Tyr 290 295
300 Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly
Phe Ser Gly 305 310 315
320 Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro
325 330 335 Gln Gln Arg Ile
Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu 340
345 350 Ser Ser Thr Leu Tyr Arg Arg Pro Phe
Asn Ile Gly Ile Asn Asn Gln 355 360
365 Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr
Ser Ser 370 375 380
Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu 385
390 395 400 Asp Glu Ile Pro Pro
Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe 405
410 415 Ser His Arg Leu Ser His Val Ser Met Phe
Arg Ser Gly Phe Ser Asn 420 425
430 Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His
Arg 435 440 445 Ser
Ala Glu Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile 450
455 460 Pro Leu Thr Lys Ser Thr
Asn Leu Gly Ser Gly Thr Ser Val Val Lys 465 470
475 480 Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg
Arg Thr Ser Pro Gly 485 490
495 Gln Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg
500 505 510 Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His 515
520 525 Thr Ser Ile Asp Gly Arg Pro
Ile Asn Gln Gly Asn Phe Ser Ala Thr 530 535
540 Met Ser Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe
Arg Thr Val Gly 545 550 555
560 Phe Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu
565 570 575 Ser Ala His
Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile 580
585 590 Glu Phe Val Pro Ala Glu Val Thr
Phe Glu Ala Glu Tyr Asp Leu Glu 595 600
605 Arg Ala Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser
Asn Gln Ile 610 615 620
Gly Leu Lys Thr Asp Val Thr Asp Tyr 625 630
371943DNAArtificial SequenceT7-2OL-7 coding sequence 37atggctagca
tgactggtgg acagcaaatg ggtcgcggat ccatgacggc cgacaacaac 60accgaggccc
tggacagcag caccaccaag gacgtgatcc agaagggcat cagcgtggtg 120ggcgacctgc
tgggcgtggt gggcttcccc ttcggcggcg ccctggtgag cttctacacc 180aacttcctga
acaccatctg gcccagcgag gacccctgga aggccttcat ggagcaggtg 240gaggccctga
tggaccagaa gatcgccgac tacgccaaga acaaggcact ggccgagcta 300cagggcctcc
agaacaacgt ggaggactat gtgagcgccc tgagcagctg gcagaagaac 360cccgctgcac
cgttccgcaa cccccacagc cagggccgca tccgcgagct gttcagccag 420gccgagagcc
acttccgcaa cagcatgccc agcttcgcca tcagcggcta cgaggtgctg 480ttcctgacca
cctacgtgca ggccgccaac ctgcacctga gcgtgctgcg cgacgtcagc 540gtgttcggcc
agcgctgggg cttcgacgcc gccaccatca acagccgcta caacgacctg 600acccgcctga
tcggcaacta caccgaccac gccgtgcgct ggtacaacac cggcctggag 660cgcgtgtggg
gtcccgacag ccgcgactgg atcaggtaca accagttccg ccgcgagctg 720accctgaccg
tgctggacat cgtgagcctg ttccccaact acgacagccg cacctacccc 780atccgcaccg
tgagccagct gacccgcgag atttacacca accccgtgct ggagaacttc 840gacggcagct
tccgcggcag cgcccagggc atcgagggca gcatccgcag cccccacctg 900atggacatcc
tgaacagcat caccatctac accgacgccc accgcggcga gtactactgg 960agcggccacc
agatcatggc cagccccgtc ggcttcagcg gccccgagtt caccttcccc 1020ctgtacggca
ccatgggcaa cgctgcacct cagcagcgca tcgtggcaca gctgggccag 1080ggagtgtacc
gcaccctgag cagcaccctg taccgtcgac ctttcaacat cggcatcaac 1140aaccagcagc
tgagcgtgct ggacggcacc gagttcgcct acggcaccag cagcaacctg 1200cccagcgccg
tgtaccgcaa gagcggcacc gtggacagcc tggacgagat cccccctcag 1260aacaacaacg
tgccacctcg acagggcttc agccaccgtc tgagccacgt gagcatgttc 1320cgcagtggct
tcagcaacag cagcgtgagc atcatccgtg cacctatgtt cagctggatt 1380caccgcagtg
ccgagttcaa caacatcatc cccagcagcc agatcaccca gatccccctg 1440accaagagca
ccaacctggg cagcggcacc agcgtggtga agggccccgg cttcaccggc 1500ggcgacatcc
tgcgccgcac cagccccggc cagatcagca ccctgcgcgt gaacatcacc 1560gcccccctga
gccagcgcta ccgcgtccgc atccgctacg ccagcaccac caacctgcag 1620ttccacacca
gcatcgacgg ccgccccatc aaccagggca acttcagcgc caccatgagc 1680agcggcagca
acctgcagag cggcagcttc cgcaccgtgg gcttcaccac ccccttcaac 1740ttcagcaacg
gcagcagcgt gttcaccctg agcgcccacg tgttcaacag cggcaacgag 1800gtgtacatcg
accgcatcga gttcgtgccc gccgaggtga ccttcgaggc cgagtacgac 1860ctggagaggg
ctcagaaggc cgtgaacgag ctgttcacca gcagcaacca gatcggcctg 1920aagaccgacg
tgaccgacta cca
194338647PRTArtificial SequenceT7-2OL-7 protein 38Met Ala Ser Met Thr Gly
Gly Gln Gln Met Gly Arg Gly Ser Met Thr 1 5
10 15 Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser
Thr Thr Lys Asp Val 20 25
30 Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val
Gly 35 40 45 Phe
Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn 50
55 60 Thr Ile Trp Pro Ser Glu
Asp Pro Trp Lys Ala Phe Met Glu Gln Val 65 70
75 80 Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr
Ala Lys Asn Lys Ala 85 90
95 Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser
100 105 110 Ala Leu
Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro 115
120 125 His Ser Gln Gly Arg Ile Arg
Glu Leu Phe Ser Gln Ala Glu Ser His 130 135
140 Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly
Tyr Glu Val Leu 145 150 155
160 Phe Leu Thr Thr Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu
165 170 175 Arg Asp Val
Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr 180
185 190 Ile Asn Ser Arg Tyr Asn Asp Leu
Thr Arg Leu Ile Gly Asn Tyr Thr 195 200
205 Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg
Val Trp Gly 210 215 220
Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu 225
230 235 240 Thr Leu Thr Val
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser 245
250 255 Arg Thr Tyr Pro Ile Arg Thr Val Ser
Gln Leu Thr Arg Glu Ile Tyr 260 265
270 Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly
Ser Ala 275 280 285
Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu 290
295 300 Asn Ser Ile Thr Ile
Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp 305 310
315 320 Ser Gly His Gln Ile Met Ala Ser Pro Val
Gly Phe Ser Gly Pro Glu 325 330
335 Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln
Gln 340 345 350 Arg
Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser 355
360 365 Thr Leu Tyr Arg Arg Pro
Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu 370 375
380 Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly
Thr Ser Ser Asn Leu 385 390 395
400 Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu
405 410 415 Ile Pro
Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His 420
425 430 Arg Leu Ser His Val Ser Met
Phe Arg Ser Gly Phe Ser Asn Ser Ser 435 440
445 Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile
His Arg Ser Ala 450 455 460
Glu Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu 465
470 475 480 Thr Lys Ser
Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro 485
490 495 Gly Phe Thr Gly Gly Asp Ile Leu
Arg Arg Thr Ser Pro Gly Gln Ile 500 505
510 Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln
Arg Tyr Arg 515 520 525
Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser 530
535 540 Ile Asp Gly Arg
Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser 545 550
555 560 Ser Gly Ser Asn Leu Gln Ser Gly Ser
Phe Arg Thr Val Gly Phe Thr 565 570
575 Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu
Ser Ala 580 585 590
His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe
595 600 605 Val Pro Ala Glu
Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala 610
615 620 Gln Lys Ala Val Asn Glu Leu Phe
Thr Ser Ser Asn Gln Ile Gly Leu 625 630
635 640 Lys Thr Asp Val Thr Asp Tyr 645
391940DNAArtificial Sequence5*2OL-7 coding sequence 39atgactagta
acggccgcca gtgtgctggt attcgccctt atgacggccg acaacaacac 60cgaggcctgg
acagcagcac caccaaggac gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg
gcgtggtggg cttccccttc ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca
ccatctggcc cagcgaggac ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg
accagaagat cgccgactac gccaagaaca aggcactggc cgagctacag 300ggcctccaga
acaacgtgga ggactatgtg agcgccctga gcagctggca gaagaacccc 360gctgcaccgt
tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt cagccaggcc 420gagagccact
tccgcaacag catgcccagc ttcgccatca gcggctacga ggtgctgttc 480ctgaccacct
acgtgcaggc cgccaacctg cacctgagcg tgctgcgcga cgtcagcgtg 540ttcggccagc
gctggggctt cgacgccgcc accatcaaca gccgctacaa cgacctgacc 600cgcctgatcg
gcaactacac cgaccacgcc gtgcgctggt acaacaccgg cctggagcgc 660gtgtggggtc
ccgacagccg cgactggatc aggtacaacc agttccgccg cgagctgacc 720ctgaccgtgc
tggacatcgt gagcctgttc cccaactacg acagccgcac ctaccccatc 780cgcaccgtga
gccagctgac ccgcgagatt tacaccaacc ccgtgctgga gaacttcgac 840ggcagcttcc
gcggcagcgc ccagggcatc gagggcagca tccgcagccc ccacctgatg 900gacatcctga
acagcatcac catctacacc gacgcccacc gcggcgagta ctactggagc 960ggccaccaga
tcatggccag ccccgtcggc ttcagcggcc ccgagttcac cttccccctg 1020tacggcacca
tgggcaacgc tgcacctcag cagcgcatcg tggcacagct gggccaggga 1080gtgtaccgca
ccctgagcag caccctgtac cgtcgacctt tcaacatcgg catcaacaac 1140cagcagctga
gcgtgctgga cggcaccgag ttcgcctacg gcaccagcag caacctgccc 1200agcgccgtgt
accgcaagag cggcaccgtg gacagcctgg acgagatccc ccctcagaac 1260aacaacgtgc
cacctcgaca gggcttcagc caccgtctga gccacgtgag catgttccgc 1320agtggcttca
gcaacagcag cgtgagcatc atccgtgcac ctatgttcag ctggattcac 1380cgcagtgccg
agttcaacaa catcatcccc agcagccaga tcacccagat ccccctgacc 1440aagagcacca
acctgggcag cggcaccagc gtggtgaagg gccccggctt caccggcggc 1500gacatcctgc
gccgcaccag ccccggccag atcagcaccc tgcgcgtgaa catcaccgcc 1560cccctgagcc
agcgctaccg cgtccgcatc cgctacgcca gcaccaccaa cctgcagttc 1620cacaccagca
tcgacggccg ccccatcaac cagggcaact tcagcgccac catgagcagc 1680ggcagcaacc
tgcagagcgg cagcttccgc accgtgggct tcaccacccc cttcaacttc 1740agcaacggca
gcagcgtgtt caccctgagc gcccacgtgt tcaacagcgg caacgaggtg 1800tacatcgacc
gcatcgagtt cgtgcccgcc gaggtgacct tcgaggccga gtacgacctg 1860gagagggctc
agaaggccgt gaacgagctg ttcaccagca gcaaccagat cggcctgaag 1920accgacgtga
ccgactacca
194040646PRTArtificial Sequence5*2OL-7 protein 40Met Thr Ser Asn Gly Arg
Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr
Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly
Phe 35 40 45 Pro
Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110 Leu Ser
Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His 115
120 125 Ser Gln Gly Arg Ile Arg Glu
Leu Phe Ser Gln Ala Glu Ser His Phe 130 135
140 Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr
Glu Val Leu Phe 145 150 155
160 Leu Thr Thr Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg
165 170 175 Asp Val Ser
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile 180
185 190 Asn Ser Arg Tyr Asn Asp Leu Thr
Arg Leu Ile Gly Asn Tyr Thr Asp 195 200
205 His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val
Trp Gly Pro 210 215 220
Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr 225
230 235 240 Leu Thr Val Leu
Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg 245
250 255 Thr Tyr Pro Ile Arg Thr Val Ser Gln
Leu Thr Arg Glu Ile Tyr Thr 260 265
270 Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser
Ala Gln 275 280 285
Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn 290
295 300 Ser Ile Thr Ile Tyr
Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser 305 310
315 320 Gly His Gln Ile Met Ala Ser Pro Val Gly
Phe Ser Gly Pro Glu Phe 325 330
335 Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln
Arg 340 345 350 Ile
Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr 355
360 365 Leu Tyr Arg Arg Pro Phe
Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser 370 375
380 Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr
Ser Ser Asn Leu Pro 385 390 395
400 Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile
405 410 415 Pro Pro
Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg 420
425 430 Leu Ser His Val Ser Met Phe
Arg Ser Gly Phe Ser Asn Ser Ser Val 435 440
445 Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His
Arg Ser Ala Glu 450 455 460
Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr 465
470 475 480 Lys Ser Thr
Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly 485
490 495 Phe Thr Gly Gly Asp Ile Leu Arg
Arg Thr Ser Pro Gly Gln Ile Ser 500 505
510 Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg
Tyr Arg Val 515 520 525
Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile 530
535 540 Asp Gly Arg Pro
Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser 545 550
555 560 Gly Ser Asn Leu Gln Ser Gly Ser Phe
Arg Thr Val Gly Phe Thr Thr 565 570
575 Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser
Ala His 580 585 590
Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val
595 600 605 Pro Ala Glu Val
Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln 610
615 620 Lys Ala Val Asn Glu Leu Phe Thr
Ser Ser Asn Gln Ile Gly Leu Lys 625 630
635 640 Thr Asp Val Thr Asp Tyr 645
411917DNAArtificial Sequence2OL-10 coding sequence 41atgacggccg
acaacaacac cgaggccctg gacagcagca ccaccaagga cgtgatccag 60aagggcatca
gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt cggcggcgcc 120ctggtgagct
tctacaccaa cttcctgaac accatctggc ccagcgagga cccctggaag 180gccttcatgg
agcaggtgga ggccctgatg gaccagaaga tcgccgacta cgccaagaac 240aaggcactgg
ccgagctaca gggcctccag aacaacgtgg aggactatgt gagcgccctg 300agcagctggc
agaagaaccc cgctgcaccg ttccgcaacc cccacagcca gggccgcatc 360cgcgagctgt
tcagccaggc cgagagccac ttccgcaaca gcatgcccag cttcgccatc 420agcggctacg
aggtgctgtt cctgaccacc tacgcccagg ccgccaacac ccacctgttc 480ctgctgaagg
acgcccaaat ctacggagag gagtggggct acgagaagga ggacatcgcc 540gagttctaca
agcgccagct gaagctgacc caggagtaca ccgaccactg cgtgaagtgg 600tacaacgtgg
gtctagacaa gctccgcggc agcagctacg agagctgggt gaacttcaac 660cgctaccgcc
gcgagatgac cctgaccgtg ctggacatcg tgagcctgtt ccccaactac 720gacagccgca
cctaccccat ccgcaccgtg agccagctga cccgcgagat ttacaccaac 780cccgtgctgg
agaacttcga cggcagcttc cgcggcagcg cccagggcat cgagggcagc 840atccgcagcc
cccacctgat ggacatcctg aacagcatca ccatctacac cgacgcccac 900cgcggcgagt
actactggag cggccaccag atcatggcca gccccgtcgg cttcagcggc 960cccgagttca
ccttccccct gtacggcacc atgggcaacg ctgcacctca gcagcgcatc 1020gtggcacagc
tgggccaggg agtgtaccgc accctgagca gcaccctgta ccgtcgacct 1080ttcaacatcg
gcatcaacaa ccagcagctg agcgtgctgg acggcaccga gttcgcctac 1140ggcaccagca
gcaacctgcc cagcgccgtg taccgcaaga gcggcaccgt ggacagcctg 1200gacgagatcc
cccctcagaa caacaacgtg ccacctcgac agggcttcag ccaccgtctg 1260agccacgtga
gcatgttccg cagtggcttc agcaacagca gcgtgagcat catccgtgca 1320cctatgttca
gctggattca ccgcagtgcc gagttcaaca acatcatccc cagcagccag 1380atcacccaga
tccccctgac caagagcacc aacctgggca gcggcaccag cgtggtgaag 1440ggccccggct
tcaccggcgg cgacatcctg cgccgcacca gccccggcca gatcagcacc 1500ctgcgcgtga
acatcaccgc ccccctgagc cagcgctacc gcgtccgcat ccgctacgcc 1560agcaccacca
acctgcagtt ccacaccagc atcgacggcc gccccatcaa ccagggcaac 1620ttcagcgcca
ccatgagcag cggcagcaac ctgcagagcg gcagcttccg caccgtgggc 1680ttcaccaccc
ccttcaactt cagcaacggc agcagcgtgt tcaccctgag cgcccacgtg 1740ttcaacagcg
gcaacgaggt gtacatcgac cgcatcgagt tcgtgcccgc cgaggtgacc 1800ttcgaggccg
agtacgacct ggagagggct cagaaggccg tgaacgagct gttcaccagc 1860agcaaccaga
tcggcctgaa gaccgacgtg accgactacc acatcgatca ggtgtag
191742638PRTArtificial Sequence2OL-10 protein 42Met Thr Ala Asp Asn Asn
Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly
Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn
Phe 35 40 45 Leu
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu Met
Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn
Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110 Asn Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 115
120 125 Ser His Phe Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130 135
140 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe 145 150 155
160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175 Glu Asp Ile
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu 180
185 190 Tyr Thr Asp His Cys Val Lys Trp
Tyr Asn Val Gly Leu Asp Lys Leu 195 200
205 Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg
Tyr Arg Arg 210 215 220
Glu Met Thr Leu Thr Val Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr 225
230 235 240 Asp Ser Arg Thr
Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu 245
250 255 Ile Tyr Thr Asn Pro Val Leu Glu Asn
Phe Asp Gly Ser Phe Arg Gly 260 265
270 Ser Ala Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu
Met Asp 275 280 285
Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr 290
295 300 Tyr Trp Ser Gly His
Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly 305 310
315 320 Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr
Met Gly Asn Ala Ala Pro 325 330
335 Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr
Leu 340 345 350 Ser
Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln 355
360 365 Gln Leu Ser Val Leu Asp
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser 370 375
380 Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly
Thr Val Asp Ser Leu 385 390 395
400 Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe
405 410 415 Ser His
Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn 420
425 430 Ser Ser Val Ser Ile Ile Arg
Ala Pro Met Phe Ser Trp Ile His Arg 435 440
445 Ser Ala Glu Phe Asn Asn Ile Ile Pro Ser Ser Gln
Ile Thr Gln Ile 450 455 460
Pro Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys 465
470 475 480 Gly Pro Gly
Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly 485
490 495 Gln Ile Ser Thr Leu Arg Val Asn
Ile Thr Ala Pro Leu Ser Gln Arg 500 505
510 Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu
Gln Phe His 515 520 525
Thr Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr 530
535 540 Met Ser Ser Gly
Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly 545 550
555 560 Phe Thr Thr Pro Phe Asn Phe Ser Asn
Gly Ser Ser Val Phe Thr Leu 565 570
575 Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp
Arg Ile 580 585 590
Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu
595 600 605 Arg Ala Gln Lys
Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile 610
615 620 Gly Leu Lys Thr Asp Val Thr Asp
Tyr His Ile Asp Gln Val 625 630 635
431956DNAArtificial Sequence5*2OL-10 coding sequence 43atgactagta
acggccgcca gtgtgctggt attcgccctt atgacggccg acaacaacac 60cgaggcctgg
acagcagcac caccaaggac gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg
gcgtggtggg cttccccttc ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca
ccatctggcc cagcgaggac ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg
accagaagat cgccgactac gccaagaaca aggcactggc cgagctacag 300ggcctccaga
acaacgtgga ggactatgtg agcgccctga gcagctggca gaagaacccc 360gctgcaccgt
tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt cagccaggcc 420gagagccact
tccgcaacag catgcccagc ttcgccatca gcggctacga ggtgctgttc 480ctgaccacct
acgcccaggc cgccaacacc cacctgttcc tgctgaagga cgcccaaatc 540tacggagagg
agtggggcta cgagaaggag gacatcgccg agttctacaa gcgccagctg 600aagctgaccc
aggagtacac cgaccactgc gtgaagtggt acaacgtggg tctagacaag 660ctccgcggca
gcagctacga gagctgggtg aacttcaacc gctaccgccg cgagatgacc 720ctgaccgtgc
tggacatcgt gagcctgttc cccaactacg acagccgcac ctaccccatc 780cgcaccgtga
gccagctgac ccgcgagatt tacaccaacc ccgtgctgga gaacttcgac 840ggcagcttcc
gcggcagcgc ccagggcatc gagggcagca tccgcagccc ccacctgatg 900gacatcctga
acagcatcac catctacacc gacgcccacc gcggcgagta ctactggagc 960ggccaccaga
tcatggccag ccccgtcggc ttcagcggcc ccgagttcac cttccccctg 1020tacggcacca
tgggcaacgc tgcacctcag cagcgcatcg tggcacagct gggccaggga 1080gtgtaccgca
ccctgagcag caccctgtac cgtcgacctt tcaacatcgg catcaacaac 1140cagcagctga
gcgtgctgga cggcaccgag ttcgcctacg gcaccagcag caacctgccc 1200agcgccgtgt
accgcaagag cggcaccgtg gacagcctgg acgagatccc ccctcagaac 1260aacaacgtgc
cacctcgaca gggcttcagc caccgtctga gccacgtgag catgttccgc 1320agtggcttca
gcaacagcag cgtgagcatc atccgtgcac ctatgttcag ctggattcac 1380cgcagtgccg
agttcaacaa catcatcccc agcagccaga tcacccagat ccccctgacc 1440aagagcacca
acctgggcag cggcaccagc gtggtgaagg gccccggctt caccggcggc 1500gacatcctgc
gccgcaccag ccccggccag atcagcaccc tgcgcgtgaa catcaccgcc 1560cccctgagcc
agcgctaccg cgtccgcatc cgctacgcca gcaccaccaa cctgcagttc 1620cacaccagca
tcgacggccg ccccatcaac cagggcaact tcagcgccac catgagcagc 1680ggcagcaacc
tgcagagcgg cagcttccgc accgtgggct tcaccacccc cttcaacttc 1740agcaacggca
gcagcgtgtt caccctgagc gcccacgtgt tcaacagcgg caacgaggtg 1800tacatcgacc
gcatcgagtt cgtgcccgcc gaggtgacct tcgaggccga gtacgacctg 1860gagagggctc
agaaggccgt gaacgagctg ttcaccagca gcaaccagat cggcctgaag 1920accgacgtga
ccgactacca catcgatcag gtgtag
195644651PRTArtificial Sequnece5*2OL-10 protein 44Met Thr Ser Asn Gly Arg
Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr
Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly
Phe 35 40 45 Pro
Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110 Leu Ser
Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His 115
120 125 Ser Gln Gly Arg Ile Arg Glu
Leu Phe Ser Gln Ala Glu Ser His Phe 130 135
140 Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr
Glu Val Leu Phe 145 150 155
160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys
165 170 175 Asp Ala Gln
Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile 180
185 190 Ala Glu Phe Tyr Lys Arg Gln Leu
Lys Leu Thr Gln Glu Tyr Thr Asp 195 200
205 His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
Arg Gly Ser 210 215 220
Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225
230 235 240 Leu Thr Val Leu
Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg 245
250 255 Thr Tyr Pro Ile Arg Thr Val Ser Gln
Leu Thr Arg Glu Ile Tyr Thr 260 265
270 Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser
Ala Gln 275 280 285
Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn 290
295 300 Ser Ile Thr Ile Tyr
Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser 305 310
315 320 Gly His Gln Ile Met Ala Ser Pro Val Gly
Phe Ser Gly Pro Glu Phe 325 330
335 Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln
Arg 340 345 350 Ile
Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr 355
360 365 Leu Tyr Arg Arg Pro Phe
Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser 370 375
380 Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr
Ser Ser Asn Leu Pro 385 390 395
400 Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile
405 410 415 Pro Pro
Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg 420
425 430 Leu Ser His Val Ser Met Phe
Arg Ser Gly Phe Ser Asn Ser Ser Val 435 440
445 Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His
Arg Ser Ala Glu 450 455 460
Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr 465
470 475 480 Lys Ser Thr
Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly 485
490 495 Phe Thr Gly Gly Asp Ile Leu Arg
Arg Thr Ser Pro Gly Gln Ile Ser 500 505
510 Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg
Tyr Arg Val 515 520 525
Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile 530
535 540 Asp Gly Arg Pro
Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser 545 550
555 560 Gly Ser Asn Leu Gln Ser Gly Ser Phe
Arg Thr Val Gly Phe Thr Thr 565 570
575 Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser
Ala His 580 585 590
Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val
595 600 605 Pro Ala Glu Val
Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln 610
615 620 Lys Ala Val Asn Glu Leu Phe Thr
Ser Ser Asn Gln Ile Gly Leu Lys 625 630
635 640 Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val
645 650 451821DNAArtificial Sequence2OL-12A
coding sequence 45atggacaaca accccaacat caacgagtgc atcccctaca actgcctgag
caaccccgag 60gtggaggtgc tgggcggcga gcgcatcgag accggctaca cccccatcga
catcagcctg 120agcctgaccc agttcctgct gagcgagttc gtgcccggcg ccggcttcgt
gctgggcctg 180gtggacatca tctggggcat cttcggcccc agccagtggg acgccttcct
ggtgcagatc 240gagcagttga taaaccaacg catagaggaa ttcgcccgca accaggccat
cagccgcctg 300gagggcctga gcaacctgta ccaaatctac gccgagagct tccgcgagtg
ggaggccgac 360cccaccaacc ccgccctgcg cgaggagatg cgcatccagt tcaacgacat
gaacagcgcc 420ctgaccaccg ccatccccct gttcgccgtg cagaactacc aggtgcccct
gctgagcgtg 480tacgtgcagg ccgccaacct gcacctgagc gtgctgcgcg acgtcagcgt
gttcggccag 540cgctggggct tcgacgccgc caccatcaac agccgctaca acgacctgac
ccgcctgatc 600ggcaactaca ccgaccacgc cgtgcgctgg tacaacaccg gcctggagcg
cgtgtggggt 660cccgacagcc gcgactggat caggtacaac cagttccgcc gcgagctgac
cctgaccgtg 720ctggacatcg tgagcctgtt ccccaactac gacagccgca cctaccccat
ccgcaccgtg 780agccagctga cccgcgagat ttacaccaac cccgtgctgg agaacttcga
cggcagcttc 840cgcggcagcg cccagggcat cgagggcagc atccgcagcc cccacctgat
ggacatcctg 900aacagcatca ccatctacac cgacgcccac cgcggcgagt actactggag
cggccaccag 960atcatggcca gccccgtcgg cttcagcggc cccgagttca ccttccccct
gtacggcacc 1020atgggcaacg ctgcacctca gcagcgcatc gtggcacagc tgggccaggg
agtgtaccgc 1080accctgagca gcaccctgta ccgtcgacct ttcaacatcg gcatcaacaa
ccagcagctg 1140agcgtgctgg acggcaccga gttcgcctac ggcaccagca gcaacctgcc
cagcgccgtg 1200taccgcaaga gcggcaccgt ggacagcctg gacgagatcc cccctcagaa
caacaacgtg 1260ccacctcgac agggcttcag ccaccgtctg agccacgtga gcatgttccg
cagtggcttc 1320agcaacagca gcgtgagcat catccgtgca cctatgttca gctggattca
ccgcagtgcc 1380gagttcaaca acatcatccc cagcagccag atcacccaga tccccctggt
gaaggcctac 1440aagctccaga gcggcgccag cgtggtggca ggcccccgct tcaccggcgg
cgacatcatc 1500cagtgcaccg agaacggcag cgccgccacc atctacgtga cccccgacgt
gagctacagc 1560cagaagtacc gcgcccgcat ccactacgcc agcaccagcc agatcacctt
caccctgagc 1620ctggacgggg cccccttcaa ccaatactac ttcgacaaga ccatcaacaa
gggcgacacc 1680ctgacctaca acagcttcaa cctggccagc ttcagcaccc ctttcgagct
gagcggcaac 1740aacctccaga tcggcgtgac cggcctgagc gccggcgaca aggtgtacat
cgacaagatc 1800gagttcatcc ccgtgaacta g
182146606PRTArtificial Sequence2OL-12A protein 46Met Asp Asn
Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5
10 15 Ser Asn Pro Glu Val Glu Val Leu
Gly Gly Glu Arg Ile Glu Thr Gly 20 25
30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe
Leu Leu Ser 35 40 45
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50
55 60 Trp Gly Ile Phe
Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70
75 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu
Glu Phe Ala Arg Asn Gln Ala 85 90
95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr
Ala Glu 100 105 110
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125 Glu Met Arg Ile
Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130
135 140 Ile Pro Leu Phe Ala Val Gln Asn
Tyr Gln Val Pro Leu Leu Ser Val 145 150
155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu
Arg Asp Val Ser 165 170
175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190 Tyr Asn Asp
Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val 195
200 205 Arg Trp Tyr Asn Thr Gly Leu Glu
Arg Val Trp Gly Pro Asp Ser Arg 210 215
220 Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr
Leu Thr Val 225 230 235
240 Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro
245 250 255 Ile Arg Thr Val
Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260
265 270 Leu Glu Asn Phe Asp Gly Ser Phe Arg
Gly Ser Ala Gln Gly Ile Glu 275 280
285 Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser
Ile Thr 290 295 300
Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln 305
310 315 320 Ile Met Ala Ser Pro
Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325
330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro
Gln Gln Arg Ile Val Ala 340 345
350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr
Arg 355 360 365 Arg
Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370
375 380 Gly Thr Glu Phe Ala Tyr
Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390
395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp
Glu Ile Pro Pro Gln 405 410
415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
420 425 430 Val Ser
Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435
440 445 Arg Ala Pro Met Phe Ser Trp
Ile His Arg Ser Ala Glu Phe Asn Asn 450 455
460 Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu
Val Lys Ala Tyr 465 470 475
480 Lys Leu Gln Ser Gly Ala Ser Val Val Ala Gly Pro Arg Phe Thr Gly
485 490 495 Gly Asp Ile
Ile Gln Cys Thr Glu Asn Gly Ser Ala Ala Thr Ile Tyr 500
505 510 Val Thr Pro Asp Val Ser Tyr Ser
Gln Lys Tyr Arg Ala Arg Ile His 515 520
525 Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr Leu Ser Leu
Asp Gly Ala 530 535 540
Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr 545
550 555 560 Leu Thr Tyr Asn
Ser Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu 565
570 575 Leu Ser Gly Asn Asn Leu Gln Ile Gly
Val Thr Gly Leu Ser Ala Gly 580 585
590 Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Asn
595 600 605 47
1791DNAArtificial Sequence2OL-13 coding sequence 47atgacggccg acaacaacac
cgaggccctg gacagcagca ccaccaagga cgtgatccag 60aagggcatca gcgtggtggg
cgacctgctg ggcgtggtgg gcttcccctt cggcggcgcc 120ctggtgagct tctacaccaa
cttcctgaac accatctggc ccagcgagga cccctggaag 180gccttcatgg agcaggtgga
ggccctgatg gaccagaaga tcgccgacta cgccaagaac 240aaggcactgg ccgagctaca
gggcctccag aacaacgtgg aggactatgt gagcgccctg 300agcagctggc agaagaaccc
cgctgcaccg ttccgcaacc cccacagcca gggccgcatc 360cgcgagctgt tcagccaggc
cgagagccac ttccgcaaca gcatgcccag cttcgccatc 420agcggctacg aggtgctgtt
cctgaccacc tacgcccagg ccgccaacac ccacctgagc 480gtgctgcgcg acgtcagcgt
gttcggccag cgctggggct tcgacgccgc caccatcaac 540agccgctaca acgacctgac
ccgcctgatc ggcaactaca ccgaccacgc cgtgcgctgg 600tacaacaccg gcctggagcg
cgtgtggggt cccgacagcc gcgactggat caggtacaac 660cagttccgcc gcgagctgac
cctgaccgtg ctggacatcg tgagcctgtt ccccaactac 720gacagccgca cctaccccat
ccgcaccgtg agccagctga cccgcgagat ttacaccaac 780cccgtgctgg agaacttcga
cggcagcttc cgcggcagcg cccagggcat cgagggcagc 840atccgcagcc cccacctgat
ggacatcctg aacagcatca ccatctacac cgacgcccac 900cgcggcgagt actactggag
cggccaccag atcatggcca gccccgtcgg cttcagcggc 960cccgagttca ccttccccct
gtacggcacc atgggcaacg ctgcacctca gcagcgcatc 1020gtggcacagc tgggccaggg
agtgtaccgc accctgagca gcaccctgta ccgtcgacct 1080ttcaacatcg gcatcaacaa
ccagcagctg agcgtgctgg acggcaccga gttcgcctac 1140ggcaccagca gcaacctgcc
cagcgccgtg taccgcaaga gcggcaccgt ggacagcctg 1200gacgagatcc cccctcagaa
caacaacgtg ccacctcgac agggcttcag ccaccgtctg 1260agccacgtga gcatgttccg
cagtggcttc agcaacagca gcgtgagcat catccgtgca 1320cctatgttca gctggattca
ccgcagtgcc gagttcaaca acatcatccc cagcagccag 1380atcacccaga tccccctgac
caagagcacc aacctgggca gcggcaccag cgtggtgaag 1440ggccccggct tcaccggcgg
cgacatcctg cgccgcacca gccccggcca gatcagcacc 1500ctgcgcgtga acatcaccgc
ccccctgagc cagcgctacc gcgcccgcat ccactacgcc 1560agcaccagcc agatcacctt
caccctgagc ctggacgggg cccccttcaa ccaatactac 1620ttcgacaaga ccatcaacaa
gggcgacacc ctgacctaca acagcttcaa cctggccagc 1680ttcagcaccc ctttcgagct
gagcggcaac aacctccaga tcggcgtgac cggcctgagc 1740gccggcgaca aggtgtacat
cgacaagatc gagttcatcc ccgtgaacta g 179148596PRTArtificial
Sequence2OL-13 protein 48Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser
Ser Thr Thr Lys 1 5 10
15 Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30 Val Gly Phe
Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe 35
40 45 Leu Asn Thr Ile Trp Pro Ser Glu
Asp Pro Trp Lys Ala Phe Met Glu 50 55
60 Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr
Ala Lys Asn 65 70 75
80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95 Val Ser Ala Leu
Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg 100
105 110 Asn Pro His Ser Gln Gly Arg Ile Arg
Glu Leu Phe Ser Gln Ala Glu 115 120
125 Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly
Tyr Glu 130 135 140
Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Ser 145
150 155 160 Val Leu Arg Asp Val
Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala 165
170 175 Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu
Thr Arg Leu Ile Gly Asn 180 185
190 Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg
Val 195 200 205 Trp
Gly Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg 210
215 220 Glu Leu Thr Leu Thr Val
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr 225 230
235 240 Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val Ser
Gln Leu Thr Arg Glu 245 250
255 Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly
260 265 270 Ser Ala
Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp 275
280 285 Ile Leu Asn Ser Ile Thr Ile
Tyr Thr Asp Ala His Arg Gly Glu Tyr 290 295
300 Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val
Gly Phe Ser Gly 305 310 315
320 Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro
325 330 335 Gln Gln Arg
Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu 340
345 350 Ser Ser Thr Leu Tyr Arg Arg Pro
Phe Asn Ile Gly Ile Asn Asn Gln 355 360
365 Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly
Thr Ser Ser 370 375 380
Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu 385
390 395 400 Asp Glu Ile Pro
Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe 405
410 415 Ser His Arg Leu Ser His Val Ser Met
Phe Arg Ser Gly Phe Ser Asn 420 425
430 Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile
His Arg 435 440 445
Ser Ala Glu Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile 450
455 460 Pro Leu Thr Lys Ser
Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys 465 470
475 480 Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu
Arg Arg Thr Ser Pro Gly 485 490
495 Gln Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln
Arg 500 505 510 Tyr
Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr 515
520 525 Leu Ser Leu Asp Gly Ala
Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr 530 535
540 Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser
Phe Asn Leu Ala Ser 545 550 555
560 Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val
565 570 575 Thr Gly
Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe 580
585 590 Ile Pro Val Asn 595
49 1923DNAArtificial SequenceV5&6 coding sequence 49atgacggccg
acaacaacac cgaggccctg gacagcagca ccaccaagga cgtgatccag 60aagggcatca
gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt cggcggcgcc 120ctggtgagct
tctacaccaa cttcctgaac accatctggc ccagcgagga cccctggaag 180gccttcatgg
agcaggtgga ggccctgatg gaccagaaga tcgccgacta cgccaagaac 240aaggcactgg
ccgagctaca gggcctccag aacaacgtgg aggactatgt gagcgccctg 300agcagctggc
agaagaaccc cgctgcaccg ttccgcaacc cccacagcca gggccgcatc 360cgcgagctgt
tcagccaggc cgagagccac ttccgcaaca gcatgcccag cttcgccatc 420agcggctacg
aggtgctgtt cctgaccacc tacgcccagg ccgccaacac ccacctgttc 480ctgctgaagg
acgcccaaat ctacggagag gagtggggct acgagaagga ggacatcgcc 540gagttctaca
agcgccagct gaagctgacc caggagtaca ccgaccactg cgtgaagtgg 600tacaacgtgg
gtctagacaa gctccgcggc agcagctacg agagctgggt gaacttcaac 660cgctaccgcc
gcgagatgac cctgaccgtg ctggacctga tcgccctgtt ccccctgtac 720gacgtgcgcc
tgtaccccaa ggaggtgaag accgagctga cccgcgacgt gctgaccgac 780cccatcgtgg
gcgtgaacaa cctgcgcggc tacggcacca ccttcagcaa catcgagaac 840tacatccgca
agccccacct gttcgactac ctgcaccgca tccagttcca cacgcgtttc 900cagcccggct
actacggcaa cgacagcttc aactactgga gcggcaacta cgtgagcacc 960cgccccagca
tcggcagcaa cgacatcatc accagcccct tctacggcaa caagagcagc 1020gagcccgtgc
agaaccttga gttcaacggc gagaaggtgt accgcgccgt ggctaacacc 1080aacctggccg
tgtggccctc tgcagtgtac agcggcgtga ccaaggtgga gttcagccag 1140tacaacgacc
agaccgacga ggccagcacc cagacctacg acagcaagcg caacgtgggc 1200gccgtgagct
gggacagcat cgaccagctg ccccccgaga ccaccgacga gcccctggag 1260aagggctaca
gccaccagct gaactacgtg atgtgcttcc tgatgcaggg cagccgcggc 1320accatccccg
tgctgacctg gacccacaag agcgtcgact tcttcaacat gatcgacagc 1380aagaagatca
cccagctgcc cctggtgaag gcctacaagc tccagagcgg cgccagcgtg 1440gtggcaggcc
cccgcttcac cggcggcgac atcatccagt gcaccgagaa cggcagcgcc 1500gccaccatct
acgtgacccc cgacgtgagc tacagccaga agtaccgcgc ccgcatccac 1560tacgccagca
ccaccaacct gcagttccac accagcatcg acggccgccc catcaaccag 1620ggcaacttca
gcgccaccat gagcagcggc agcaacctgc agagcggcag cttccgcacc 1680gtgggcttca
ccaccccctt caacttcagc aacggcagca gcgtgttcac cctgagcgcc 1740cacgtgttca
acagcggcaa cgaggtgtac atcgaccgca tcgagttcgt gcccgccgag 1800gtgaccttcg
aggccgagta cgacctggag agggctcaga aggccgtgaa cgagctgttc 1860accagcagca
accagatcgg cctgaagacc gacgtgaccg actaccacat cgatcaggtg 1920tag
192350640PRTArtificial SequenceV5&6 protein 50Met Thr Ala Asp Asn Asn Thr
Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly
Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn
Phe 35 40 45 Leu
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu Met
Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn
Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110 Asn Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 115
120 125 Ser His Phe Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130 135
140 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe 145 150 155
160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175 Glu Asp Ile
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu 180
185 190 Tyr Thr Asp His Cys Val Lys Trp
Tyr Asn Val Gly Leu Asp Lys Leu 195 200
205 Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg
Tyr Arg Arg 210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr 225
230 235 240 Asp Val Arg Leu
Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp 245
250 255 Val Leu Thr Asp Pro Ile Val Gly Val
Asn Asn Leu Arg Gly Tyr Gly 260 265
270 Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His
Leu Phe 275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr 290
295 300 Tyr Gly Asn Asp Ser
Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 305 310
315 320 Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile
Thr Ser Pro Phe Tyr Gly 325 330
335 Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu
Lys 340 345 350 Val
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala 355
360 365 Val Tyr Ser Gly Val Thr
Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 370 375
380 Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Val Gly 385 390 395
400 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415 Glu Pro
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys 420
425 430 Phe Leu Met Gln Gly Ser Arg
Gly Thr Ile Pro Val Leu Thr Trp Thr 435 440
445 His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser
Lys Lys Ile Thr 450 455 460
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val 465
470 475 480 Val Ala Gly
Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu 485
490 495 Asn Gly Ser Ala Ala Thr Ile Tyr
Val Thr Pro Asp Val Ser Tyr Ser 500 505
510 Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Thr
Asn Leu Gln 515 520 525
Phe His Thr Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser 530
535 540 Ala Thr Met Ser
Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr 545 550
555 560 Val Gly Phe Thr Thr Pro Phe Asn Phe
Ser Asn Gly Ser Ser Val Phe 565 570
575 Thr Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr
Ile Asp 580 585 590
Arg Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp
595 600 605 Leu Glu Arg Ala
Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn 610
615 620 Gln Ile Gly Leu Lys Thr Asp Val
Thr Asp Tyr His Ile Asp Gln Val 625 630
635 640 511962DNAArtificial Sequence5*V5&6 coding
sequence 51atgactagta acggccgcca gtgtgctggt attcgccctt atgacggccg
acaacaacac 60cgaggcctgg acagcagcac caccaaggac gtgatccaga agggcatcag
cgtggtgggc 120gacctgctgg gcgtggtggg cttccccttc ggcggcgccc tggtgagctt
ctacaccaac 180ttcctgaaca ccatctggcc cagcgaggac ccctggaagg ccttcatgga
gcaggtggag 240gccctgatgg accagaagat cgccgactac gccaagaaca aggcactggc
cgagctacag 300ggcctccaga acaacgtgga ggactatgtg agcgccctga gcagctggca
gaagaacccc 360gctgcaccgt tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt
cagccaggcc 420gagagccact tccgcaacag catgcccagc ttcgccatca gcggctacga
ggtgctgttc 480ctgaccacct acgcccaggc cgccaacacc cacctgttcc tgctgaagga
cgcccaaatc 540tacggagagg agtggggcta cgagaaggag gacatcgccg agttctacaa
gcgccagctg 600aagctgaccc aggagtacac cgaccactgc gtgaagtggt acaacgtggg
tctagacaag 660ctccgcggca gcagctacga gagctgggtg aacttcaacc gctaccgccg
cgagatgacc 720ctgaccgtgc tggacctgat cgccctgttc cccctgtacg acgtgcgcct
gtaccccaag 780gaggtgaaga ccgagctgac ccgcgacgtg ctgaccgacc ccatcgtggg
cgtgaacaac 840ctgcgcggct acggcaccac cttcagcaac atcgagaact acatccgcaa
gccccacctg 900ttcgactacc tgcaccgcat ccagttccac acgcgtttcc agcccggcta
ctacggcaac 960gacagcttca actactggag cggcaactac gtgagcaccc gccccagcat
cggcagcaac 1020gacatcatca ccagcccctt ctacggcaac aagagcagcg agcccgtgca
gaaccttgag 1080ttcaacggcg agaaggtgta ccgcgccgtg gctaacacca acctggccgt
gtggccctct 1140gcagtgtaca gcggcgtgac caaggtggag ttcagccagt acaacgacca
gaccgacgag 1200gccagcaccc agacctacga cagcaagcgc aacgtgggcg ccgtgagctg
ggacagcatc 1260gaccagctgc cccccgagac caccgacgag cccctggaga agggctacag
ccaccagctg 1320aactacgtga tgtgcttcct gatgcagggc agccgcggca ccatccccgt
gctgacctgg 1380acccacaaga gcgtcgactt cttcaacatg atcgacagca agaagatcac
ccagctgccc 1440ctggtgaagg cctacaagct ccagagcggc gccagcgtgg tggcaggccc
ccgcttcacc 1500ggcggcgaca tcatccagtg caccgagaac ggcagcgccg ccaccatcta
cgtgaccccc 1560gacgtgagct acagccagaa gtaccgcgcc cgcatccact acgccagcac
caccaacctg 1620cagttccaca ccagcatcga cggccgcccc atcaaccagg gcaacttcag
cgccaccatg 1680agcagcggca gcaacctgca gagcggcagc ttccgcaccg tgggcttcac
cacccccttc 1740aacttcagca acggcagcag cgtgttcacc ctgagcgccc acgtgttcaa
cagcggcaac 1800gaggtgtaca tcgaccgcat cgagttcgtg cccgccgagg tgaccttcga
ggccgagtac 1860gacctggaga gggctcagaa ggccgtgaac gagctgttca ccagcagcaa
ccagatcggc 1920ctgaagaccg acgtgaccga ctaccacatc gatcaggtgt ag
196252653PRTArtificial Sequence5*V5&6 protein 52Met Thr Ser
Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp
Ser Ser Thr Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
Val Gly Phe 35 40 45
Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser
Glu Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp
Tyr Ala Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val
Ser Ala 100 105 110
Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His
115 120 125 Ser Gln Gly Arg
Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe 130
135 140 Arg Asn Ser Met Pro Ser Phe Ala
Ile Ser Gly Tyr Glu Val Leu Phe 145 150
155 160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Phe Leu Leu Lys 165 170
175 Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile
180 185 190 Ala Glu Phe
Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp 195
200 205 His Cys Val Lys Trp Tyr Asn Val
Gly Leu Asp Lys Leu Arg Gly Ser 210 215
220 Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg
Glu Met Thr 225 230 235
240 Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg
245 250 255 Leu Tyr Pro Lys
Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr 260
265 270 Asp Pro Ile Val Gly Val Asn Asn Leu
Arg Gly Tyr Gly Thr Thr Phe 275 280
285 Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp
Tyr Leu 290 295 300
His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn 305
310 315 320 Asp Ser Phe Asn Tyr
Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser 325
330 335 Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro
Phe Tyr Gly Asn Lys Ser 340 345
350 Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
Arg 355 360 365 Ala
Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser 370
375 380 Gly Val Thr Lys Val Glu
Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu 385 390
395 400 Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Val Gly Ala Val Ser 405 410
415 Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu
420 425 430 Glu Lys
Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu Met 435
440 445 Gln Gly Ser Arg Gly Thr Ile
Pro Val Leu Thr Trp Thr His Lys Ser 450 455
460 Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile
Thr Gln Leu Pro 465 470 475
480 Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala Gly
485 490 495 Pro Arg Phe
Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly Ser 500
505 510 Ala Ala Thr Ile Tyr Val Thr Pro
Asp Val Ser Tyr Ser Gln Lys Tyr 515 520
525 Arg Ala Arg Ile His Tyr Ala Ser Thr Thr Asn Leu Gln
Phe His Thr 530 535 540
Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met 545
550 555 560 Ser Ser Gly Ser
Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe 565
570 575 Thr Thr Pro Phe Asn Phe Ser Asn Gly
Ser Ser Val Phe Thr Leu Ser 580 585
590 Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg
Ile Glu 595 600 605
Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg 610
615 620 Ala Gln Lys Ala Val
Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly 625 630
635 640 Leu Lys Thr Asp Val Thr Asp Tyr His Ile
Asp Gln Val 645 650
531845DNAArtificial Sequence88A-dm3 coding sequence 53atgactagta
acggccgcca gtgtgctggt attcgccctt atgacggccg acaacaacac 60cgaggcctgg
acagcagcac caccaaggac gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg
gcgtggtggg cttccccttc ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca
ccatctggcc cagcgaggac ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg
accagaagat cgccgactac gccaagaaca aggcactggc cgagctacag 300ggcctccaga
acaacgtgga ggactatgtg agcgccctga gcagctggca gaagaacccc 360gctgcaccgt
tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt cagccaggcc 420gagagccact
tccgcaacag catgcccagc ttcgccatca gcggctacga ggtgctgttc 480ctgaccacct
acgcccaggc cgccaacacc cacctgttcc tgctgaagga cgcccaaatc 540tacggagagg
agtggggcta cgagaaggag gacatcgccg agttctacaa gcgccagctg 600aagctgaccc
aggagtacac cgaccactgc gtgaagtggt acaacgtggg tctagacaag 660ctccgcggca
gcagctacga gagctgggtg aacttcaacc gctaccgccg cgagatgacc 720ctgaccgtgc
tggacctgat cgccctgttc cccctgtacg acgtgcgcct gtaccccaag 780gaggtgaaga
ccgagctgac ccgcgacgtg ctgaccgacc ccatcgtggg cgtgaacaac 840ctgcgcggct
acggcaccac cttcagcaac atcgagaact acatccgcaa gccccacctg 900ttcgactacc
tgcaccgcat ccagttccac acgcgtttcc agcccggcta ctacggcaac 960gacagcttca
actactggag cggcaactac gtgagcaccc gccccagcat cggcagcaac 1020gacatcatca
ccagcccctt ctacggcaac aagagcagcg agcccgtgca gaaccttgag 1080ttcaacggcg
agaaggtgta ccgcgccgtg gctaacacca acctggccgt gtggccctct 1140gcagtgtaca
gcggcgtgac caaggtggag ttcagccagt acaacgacca gaccgacgag 1200gccagcaccc
agacctacga cagcaagcgc aacgtgggcg ccgtgagctg ggacagcatc 1260gaccagctgc
cccccgagac caccgacgag cccctggaga agggctacag ccaccagctg 1320aactacgtga
tgtgcttcct gatgcagggc agccgcggca ccatccccgt gctgacctgg 1380acccacaaga
gcgtcgactt cttcaacatg atcgacagca agaagatcac ccagctgccc 1440ctggtaaagg
gagacatgtt atatctaggg ggttccgtag tacagggtcc tggatttaca 1500ggaggagata
tattaaaaag aaccaatcct agcatattag ggacctttgc ggttacagta 1560aatgggtcgt
tatcacaaag atatcgtgta agaattcgct atgcctctac aacagatttt 1620gaatttactc
tataccttgg cgacacaata gaaaaaaata gatttaacaa aactatggat 1680aatggggcat
ctttaacgta tgaaacattt aaattcgcaa gtttcattac tgatttccaa 1740ttcagagaaa
cacaagataa aatactccta tccatgggtg attttagctc cggtcaagaa 1800gtttatatag
accgaatcga attcatccca gtagatgaga catag
184554614PRTArtificial Sequence88A-dm3 protein 54Met Thr Ser Asn Gly Arg
Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr
Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly
Phe 35 40 45 Pro
Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110 Leu Ser
Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His 115
120 125 Ser Gln Gly Arg Ile Arg Glu
Leu Phe Ser Gln Ala Glu Ser His Phe 130 135
140 Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr
Glu Val Leu Phe 145 150 155
160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys
165 170 175 Asp Ala Gln
Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile 180
185 190 Ala Glu Phe Tyr Lys Arg Gln Leu
Lys Leu Thr Gln Glu Tyr Thr Asp 195 200
205 His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
Arg Gly Ser 210 215 220
Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225
230 235 240 Leu Thr Val Leu
Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg 245
250 255 Leu Tyr Pro Lys Glu Val Lys Thr Glu
Leu Thr Arg Asp Val Leu Thr 260 265
270 Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
Thr Phe 275 280 285
Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu 290
295 300 His Arg Ile Gln Phe
His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn 305 310
315 320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr
Val Ser Thr Arg Pro Ser 325 330
335 Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
Ser 340 345 350 Ser
Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg 355
360 365 Ala Val Ala Asn Thr Asn
Leu Ala Val Trp Pro Ser Ala Val Tyr Ser 370 375
380 Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn
Asp Gln Thr Asp Glu 385 390 395
400 Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser
405 410 415 Trp Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu 420
425 430 Glu Lys Gly Tyr Ser His Gln
Leu Asn Tyr Val Met Cys Phe Leu Met 435 440
445 Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp
Thr His Lys Ser 450 455 460
Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro 465
470 475 480 Leu Val Lys
Gly Asp Met Leu Tyr Leu Gly Gly Ser Val Val Gln Gly 485
490 495 Pro Gly Phe Thr Gly Gly Asp Ile
Leu Lys Arg Thr Asn Pro Ser Ile 500 505
510 Leu Gly Thr Phe Ala Val Thr Val Asn Gly Ser Leu Ser
Gln Arg Tyr 515 520 525
Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Phe Glu Phe Thr Leu 530
535 540 Tyr Leu Gly Asp
Thr Ile Glu Lys Asn Arg Phe Asn Lys Thr Met Asp 545 550
555 560 Asn Gly Ala Ser Leu Thr Tyr Glu Thr
Phe Lys Phe Ala Ser Phe Ile 565 570
575 Thr Asp Phe Gln Phe Arg Glu Thr Gln Asp Lys Ile Leu Leu
Ser Met 580 585 590
Gly Asp Phe Ser Ser Gly Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe
595 600 605 Ile Pro Val Asp
Glu Thr 610 551986DNAArtificial SequenceFR(1Fa)
coding sequence 55atgactagta acggccgcca gtgtgctggt attcgccctt atgacggccg
acaacaacac 60cgaggcctgg acagcagcac caccaaggac gtgatccaga agggcatcag
cgtggtgggc 120gacctgctgg gcgtggtggg cttccccttc ggcggcgccc tggtgagctt
ctacaccaac 180ttcctgaaca ccatctggcc cagcgaggac ccctggaagg ccttcatgga
gcaggtggag 240gccctgatgg accagaagat cgccgactac gccaagaaca aggcactggc
cgagctacag 300ggcctccaga acaacgtgga ggactatgtg agcgccctga gcagctggca
gaagaacccc 360gctgcaccgt tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt
cagccaggcc 420gagagccact tccgcaacag catgcccagc ttcgccatca gcggctacga
ggtgctgttc 480ctgaccacct acgcccaggc cgccaacacc cacctgttcc tgctgaagga
cgcccaaatc 540tacggagagg agtggggcta cgagaaggag gacatcgccg agttctacaa
gcgccagctg 600aagctgaccc aggagtacac cgaccactgc gtgaagtggt acaacgtggg
tctagacaag 660ctccgcggca gcagctacga gagctgggtg aacttcaacc gctaccgccg
cgagatgacc 720ctgaccgtgc tggacctgat cgccctgttc cccctgtacg acgtgcgcct
gtaccccaag 780gaggtgaaga ccgagctgac ccgcgacgtg ctgaccgacc ccatcgtggg
cgtgaacaac 840ctgcgcggct acggcaccac cttcagcaac atcgagaact acatccgcaa
gccccacctg 900ttcgactacc tgcaccgcat ccagttccac acgcgtttcc agcccggcta
ctacggcaac 960gacagcttca actactggag cggcaactac gtgagcaccc gccccagcat
cggcagcaac 1020gacatcatca ccagcccctt ctacggcaac aagagcagcg agcccgtgca
gaaccttgag 1080ttcaacggcg agaaggtgta ccgcgccgtg gctaacacca acctggccgt
gtggccctct 1140gcagtgtaca gcggcgtgac caaggtggag ttcagccagt acaacgacca
gaccgacgag 1200gccagcaccc agacctacga cagcaagcgc aacgtgggcg ccgtgagctg
ggacagcatc 1260gaccagctgc cccccgagac caccgacgag cccctggaga agggctacag
ccaccagctg 1320aactacgtga tgtgcttcct gatgcagggc agccgcggca ccatccccgt
gctgacctgg 1380acccacaaga gcgtcgactt cttcaacatg atcgacagca agaagatcac
ccagctgccc 1440ctggtgaagg cccacaccct ccagtccggc accaccgtgg tgcgcggccc
gggcttcacc 1500ggcggcgaca tcctccgccg cacctccggc ggcccgttcg cctacaccat
cgtgaacatc 1560aacggccagc tcccgcagcg ctaccgcgcc cgcatccgct acgcctccac
caccaacctc 1620cgcatctacg tgaccgtggc cggcgagcgc atcttcgccg gccagttcaa
caagaccatg 1680gacaccggcg acccgctcac cttccagtcc ttctcctacg ccaccatcaa
caccgccttc 1740accttcccga tgtcccagtc ctccttcacc gtgggcgccg acaccttctc
ctccggcaac 1800gaggtgtaca tcgaccgctt cgagctgatc ccggtgaccg ccaccttcga
ggccgagtac 1860gacctggagc gcgcccagaa ggccgtgaac gccctcttca cctccatcaa
ccagatcggc 1920atcaagaccg acgtgaccga ctaccacatc gaccaggtgt ccaacctcgt
ggactgctta 1980agctag
198656661PRTArtificial SequenceFR(1Fa) protein 56Met Thr Ser
Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp
Ser Ser Thr Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
Val Gly Phe 35 40 45
Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser
Glu Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp
Tyr Ala Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val
Ser Ala 100 105 110
Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His
115 120 125 Ser Gln Gly Arg
Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe 130
135 140 Arg Asn Ser Met Pro Ser Phe Ala
Ile Ser Gly Tyr Glu Val Leu Phe 145 150
155 160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
Phe Leu Leu Lys 165 170
175 Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile
180 185 190 Ala Glu Phe
Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp 195
200 205 His Cys Val Lys Trp Tyr Asn Val
Gly Leu Asp Lys Leu Arg Gly Ser 210 215
220 Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg
Glu Met Thr 225 230 235
240 Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg
245 250 255 Leu Tyr Pro Lys
Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr 260
265 270 Asp Pro Ile Val Gly Val Asn Asn Leu
Arg Gly Tyr Gly Thr Thr Phe 275 280
285 Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp
Tyr Leu 290 295 300
His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn 305
310 315 320 Asp Ser Phe Asn Tyr
Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser 325
330 335 Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro
Phe Tyr Gly Asn Lys Ser 340 345
350 Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
Arg 355 360 365 Ala
Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser 370
375 380 Gly Val Thr Lys Val Glu
Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu 385 390
395 400 Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn
Val Gly Ala Val Ser 405 410
415 Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu
420 425 430 Glu Lys
Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu Met 435
440 445 Gln Gly Ser Arg Gly Thr Ile
Pro Val Leu Thr Trp Thr His Lys Ser 450 455
460 Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile
Thr Gln Leu Pro 465 470 475
480 Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly
485 490 495 Pro Gly Phe
Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro 500
505 510 Phe Ala Tyr Thr Ile Val Asn Ile
Asn Gly Gln Leu Pro Gln Arg Tyr 515 520
525 Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg
Ile Tyr Val 530 535 540
Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met 545
550 555 560 Asp Thr Gly Asp
Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile 565
570 575 Asn Thr Ala Phe Thr Phe Pro Met Ser
Gln Ser Ser Phe Thr Val Gly 580 585
590 Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg
Phe Glu 595 600 605
Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg 610
615 620 Ala Gln Lys Ala Val
Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly 625 630
635 640 Ile Lys Thr Asp Val Thr Asp Tyr His Ile
Asp Gln Val Ser Asn Leu 645 650
655 Val Asp Cys Leu Ser 660 571842DNAArtificial
SequenceFR(1Ac) coding sequence 57atgactagta acggccgcca gtgtgctggt
attcgccctt atgacggccg acaacaacac 60cgaggcctgg acagcagcac caccaaggac
gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg gcgtggtggg cttccccttc
ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca ccatctggcc cagcgaggac
ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg accagaagat cgccgactac
gccaagaaca aggcactggc cgagctacag 300ggcctccaga acaacgtgga ggactatgtg
agcgccctga gcagctggca gaagaacccc 360gctgcaccgt tccgcaaccc ccacagccag
ggccgcatcc gcgagctgtt cagccaggcc 420gagagccact tccgcaacag catgcccagc
ttcgccatca gcggctacga ggtgctgttc 480ctgaccacct acgcccaggc cgccaacacc
cacctgttcc tgctgaagga cgcccaaatc 540tacggagagg agtggggcta cgagaaggag
gacatcgccg agttctacaa gcgccagctg 600aagctgaccc aggagtacac cgaccactgc
gtgaagtggt acaacgtggg tctagacaag 660ctccgcggca gcagctacga gagctgggtg
aacttcaacc gctaccgccg cgagatgacc 720ctgaccgtgc tggacctgat cgccctgttc
cccctgtacg acgtgcgcct gtaccccaag 780gaggtgaaga ccgagctgac ccgcgacgtg
ctgaccgacc ccatcgtggg cgtgaacaac 840ctgcgcggct acggcaccac cttcagcaac
atcgagaact acatccgcaa gccccacctg 900ttcgactacc tgcaccgcat ccagttccac
acgcgtttcc agcccggcta ctacggcaac 960gacagcttca actactggag cggcaactac
gtgagcaccc gccccagcat cggcagcaac 1020gacatcatca ccagcccctt ctacggcaac
aagagcagcg agcccgtgca gaaccttgag 1080ttcaacggcg agaaggtgta ccgcgccgtg
gctaacacca acctggccgt gtggccctct 1140gcagtgtaca gcggcgtgac caaggtggag
ttcagccagt acaacgacca gaccgacgag 1200gccagcaccc agacctacga cagcaagcgc
aacgtgggcg ccgtgagctg ggacagcatc 1260gaccagctgc cccccgagac caccgacgag
cccctggaga agggctacag ccaccagctg 1320aactacgtga tgtgcttcct gatgcagggc
agccgcggca ccatccccgt gctgacctgg 1380acccacaaga gcgtcgactt cttcaacatg
atcgacagca agaagatcac ccagctgccc 1440ctggtgaagg gaaactttct ttttaatggt
tctgtaattt caggaccagg atttactggt 1500ggggacttag ttagattaaa tagtagtgga
aataacattc agaatagagg gtatattgaa 1560gttccaattc acttcccatc gacatctacc
agatatcgag ttcgtgtacg gtatgcttct 1620gtaaccccga ttcacctcaa cgttaattgg
ggtaattcat ccattttttc caatacagta 1680ccagctacag ctacgtcatt agataatcta
caatcaagtg attttggtta ttttgaaagt 1740gccaatgctt ttacatcttc attaggtaat
atagtaggtg ttagaaattt tagtgggact 1800gcaggagtga taatagacag atttgaattt
attccagttt ag 184258613PRTArtificial SequenceFR(1Ac)
protein 58Met Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly
1 5 10 15 Arg Gln
Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp Val Ile 20
25 30 Gln Lys Gly Ile Ser Val Val
Gly Asp Leu Leu Gly Val Val Gly Phe 35 40
45 Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn
Phe Leu Asn Thr 50 55 60
Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65
70 75 80 Ala Leu Met
Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu 85
90 95 Ala Glu Leu Gln Gly Leu Gln Asn
Asn Val Glu Asp Tyr Val Ser Ala 100 105
110 Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
Asn Pro His 115 120 125
Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe 130
135 140 Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe 145 150
155 160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe Leu Leu Lys 165 170
175 Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
Asp Ile 180 185 190
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp
195 200 205 His Cys Val Lys
Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser 210
215 220 Ser Tyr Glu Ser Trp Val Asn Phe
Asn Arg Tyr Arg Arg Glu Met Thr 225 230
235 240 Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu
Tyr Asp Val Arg 245 250
255 Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr
260 265 270 Asp Pro Ile
Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe 275
280 285 Ser Asn Ile Glu Asn Tyr Ile Arg
Lys Pro His Leu Phe Asp Tyr Leu 290 295
300 His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
Tyr Gly Asn 305 310 315
320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser
325 330 335 Ile Gly Ser Asn
Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser 340
345 350 Ser Glu Pro Val Gln Asn Leu Glu Phe
Asn Gly Glu Lys Val Tyr Arg 355 360
365 Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
Tyr Ser 370 375 380
Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu 385
390 395 400 Ala Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser 405
410 415 Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu
Thr Thr Asp Glu Pro Leu 420 425
430 Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
Met 435 440 445 Gln
Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser 450
455 460 Val Asp Phe Phe Asn Met
Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro 465 470
475 480 Leu Val Lys Gly Asn Phe Leu Phe Asn Gly Ser
Val Ile Ser Gly Pro 485 490
495 Gly Phe Thr Gly Gly Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn
500 505 510 Ile Gln
Asn Arg Gly Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr 515
520 525 Ser Thr Arg Tyr Arg Val Arg
Val Arg Tyr Ala Ser Val Thr Pro Ile 530 535
540 His Leu Asn Val Asn Trp Gly Asn Ser Ser Ile Phe
Ser Asn Thr Val 545 550 555
560 Pro Ala Thr Ala Thr Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly
565 570 575 Tyr Phe Glu
Ser Ala Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val 580
585 590 Gly Val Arg Asn Phe Ser Gly Thr
Ala Gly Val Ile Ile Asp Arg Phe 595 600
605 Glu Phe Ile Pro Val 610
592067DNAArtificial SequenceFR(1Ia) coding sequence 59atgactagta
acggccgcca gtgtgctggt attcgccctt atgacggccg acaacaacac 60cgaggcctgg
acagcagcac caccaaggac gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg
gcgtggtggg cttccccttc ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca
ccatctggcc cagcgaggac ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg
accagaagat cgccgactac gccaagaaca aggcactggc cgagctacag 300ggcctccaga
acaacgtgga ggactatgtg agcgccctga gcagctggca gaagaacccc 360gctgcaccgt
tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt cagccaggcc 420gagagccact
tccgcaacag catgcccagc ttcgccatca gcggctacga ggtgctgttc 480ctgaccacct
acgcccaggc cgccaacacc cacctgttcc tgctgaagga cgcccaaatc 540tacggagagg
agtggggcta cgagaaggag gacatcgccg agttctacaa gcgccagctg 600aagctgaccc
aggagtacac cgaccactgc gtgaagtggt acaacgtggg tctagacaag 660ctccgcggca
gcagctacga gagctgggtg aacttcaacc gctaccgccg cgagatgacc 720ctgaccgtgc
tggacctgat cgccctgttc cccctgtacg acgtgcgcct gtaccccaag 780gaggtgaaga
ccgagctgac ccgcgacgtg ctgaccgacc ccatcgtggg cgtgaacaac 840ctgcgcggct
acggcaccac cttcagcaac atcgagaact acatccgcaa gccccacctg 900ttcgactacc
tgcaccgcat ccagttccac acgcgtttcc agcccggcta ctacggcaac 960gacagcttca
actactggag cggcaactac gtgagcaccc gccccagcat cggcagcaac 1020gacatcatca
ccagcccctt ctacggcaac aagagcagcg agcccgtgca gaaccttgag 1080ttcaacggcg
agaaggtgta ccgcgccgtg gctaacacca acctggccgt gtggccctct 1140gcagtgtaca
gcggcgtgac caaggtggag ttcagccagt acaacgacca gaccgacgag 1200gccagcaccc
agacctacga cagcaagcgc aacgtgggcg ccgtgagctg ggacagcatc 1260gaccagctgc
cccccgagac caccgacgag cccctggaga agggctacag ccaccagctg 1320aactacgtga
tgtgcttcct gatgcagggc agccgcggca ccatccccgt gctgacctgg 1380acccacaaga
gcgtcgactt cttcaacatg atcgacagca agaagatcac ccagctgccc 1440ctggtaaaag
ctttcaatct gtcttcaggt gccgctgtag tgagaggacc aggatttaca 1500ggtggggata
tccttcgaag aacgaatact ggtacatttg gggatatacg agtaaatatt 1560aatccaccat
ttgcacaaag atatcgcgtg aggattcgct atgcttctac cacagattta 1620caattccata
cgtcaattaa cggtaaagct attaatcaag gtaatttttc agcaactatg 1680aatagaggag
aggacttaga ctataaaacc tttagaactg taggctttac cactccattt 1740agctttttag
atgtacaaag tacattcaca ataggtgctt ggaacttctc ttcaggtaac 1800gaagtttata
tagatagaat tgaatttgtt ccggtagaag taacatatga ggcagaatat 1860gattttgaaa
aagcgcaaga gaaggttact gcactgttta catctacgaa tccaagagga 1920ttaaaaacag
atgtaaagga ttatcatatt gaccaggtat caaatttagt agagtctcta 1980tcagatgaat
tctatcttga tgaaaagaga gaattattcg agatagttaa atacgcgaag 2040caactccata
ttgagcgtaa catgtag
206760688PRTArtificial SequenceFR(1Ia) protein 60Met Thr Ser Asn Gly Arg
Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr
Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly
Phe 35 40 45 Pro
Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110 Leu Ser
Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His 115
120 125 Ser Gln Gly Arg Ile Arg Glu
Leu Phe Ser Gln Ala Glu Ser His Phe 130 135
140 Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr
Glu Val Leu Phe 145 150 155
160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys
165 170 175 Asp Ala Gln
Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile 180
185 190 Ala Glu Phe Tyr Lys Arg Gln Leu
Lys Leu Thr Gln Glu Tyr Thr Asp 195 200
205 His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
Arg Gly Ser 210 215 220
Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225
230 235 240 Leu Thr Val Leu
Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg 245
250 255 Leu Tyr Pro Lys Glu Val Lys Thr Glu
Leu Thr Arg Asp Val Leu Thr 260 265
270 Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
Thr Phe 275 280 285
Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu 290
295 300 His Arg Ile Gln Phe
His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn 305 310
315 320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr
Val Ser Thr Arg Pro Ser 325 330
335 Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
Ser 340 345 350 Ser
Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg 355
360 365 Ala Val Ala Asn Thr Asn
Leu Ala Val Trp Pro Ser Ala Val Tyr Ser 370 375
380 Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn
Asp Gln Thr Asp Glu 385 390 395
400 Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser
405 410 415 Trp Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu 420
425 430 Glu Lys Gly Tyr Ser His Gln
Leu Asn Tyr Val Met Cys Phe Leu Met 435 440
445 Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp
Thr His Lys Ser 450 455 460
Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro 465
470 475 480 Leu Val Lys
Ala Phe Asn Leu Ser Ser Gly Ala Ala Val Val Arg Gly 485
490 495 Pro Gly Phe Thr Gly Gly Asp Ile
Leu Arg Arg Thr Asn Thr Gly Thr 500 505
510 Phe Gly Asp Ile Arg Val Asn Ile Asn Pro Pro Phe Ala
Gln Arg Tyr 515 520 525
Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe His Thr 530
535 540 Ser Ile Asn Gly
Lys Ala Ile Asn Gln Gly Asn Phe Ser Ala Thr Met 545 550
555 560 Asn Arg Gly Glu Asp Leu Asp Tyr Lys
Thr Phe Arg Thr Val Gly Phe 565 570
575 Thr Thr Pro Phe Ser Phe Leu Asp Val Gln Ser Thr Phe Thr
Ile Gly 580 585 590
Ala Trp Asn Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu
595 600 605 Phe Val Pro Val
Glu Val Thr Tyr Glu Ala Glu Tyr Asp Phe Glu Lys 610
615 620 Ala Gln Glu Lys Val Thr Ala Leu
Phe Thr Ser Thr Asn Pro Arg Gly 625 630
635 640 Leu Lys Thr Asp Val Lys Asp Tyr His Ile Asp Gln
Val Ser Asn Leu 645 650
655 Val Glu Ser Leu Ser Asp Glu Phe Tyr Leu Asp Glu Lys Arg Glu Leu
660 665 670 Phe Glu Ile
Val Lys Tyr Ala Lys Gln Leu His Ile Glu Arg Asn Met 675
680 685 61 1962DNAArtificial
SequenceDM23A coding sequence 61atgactagta acggccgcca gtgtgctggt
attcgccctt atgacggccg acaacaacac 60cgaggcctgg acagcagcac caccaaggac
gtgatccaga agggcatcag cgtggtgggc 120gacctgctgg gcgtggtggg cttccccttc
ggcggcgccc tggtgagctt ctacaccaac 180ttcctgaaca ccatctggcc cagcgaggac
ccctggaagg ccttcatgga gcaggtggag 240gccctgatgg accagaagat cgccgactac
gccaagaaca aggcactggc cgagctacag 300ggcctccaga acaacgtgga ggactatgtg
agcgccctga gcagctggca gaagaacccc 360gctgcaccgt tccgcaaccc ccacagccag
ggccgcatcc gcgagctgtt cagccaggcc 420gagagccact tccgcaacag catgcccagc
ttcgccatca gcggctacga ggtgctgttc 480ctgaccacct acgcccaggc cgccaacacc
cacctgttcc tgctgaagga cgcccaaatc 540tacggagagg agtggggcta cgagaaggag
gacatcgccg agttctacaa gcgccagctg 600aagctgaccc aggagtacac cgaccactgc
gtgaagtggt acaacgtggg tctagacaag 660ctccgcggca gcagctacga gagctgggtg
aacttcaacc gctaccgccg cgagatgacc 720ctgaccgtgc tggacctgat cgccctgttc
cccctgtacg acgtgcgcct gtaccccaag 780gaggtgaaga ccgagctgac ccgcgacgtg
ctgaccgacc ccatcgtggg cgtgaacaac 840ctgcgcggct acggcaccac cttcagcaac
atcgagaact acatccgcaa gccccacctg 900ttcgactacc tgcaccgcat ccagttccac
acgcgtttcc agcccggcta ctacggcaac 960gacagcttca actactggag cggcaactac
gtgagcaccc gccccagcat cggcagcaac 1020gacatcatca ccagcccctt ctacggcaac
aagagcagcg agcccgtgca gaaccttgag 1080ttcaacggcg agaaggtgta ccgcgccgtg
gctaacacca acctggccgt gtggccctct 1140gcagtgtaca gcggcgtgac caaggtggag
ttcagccagt acaacgacca gaccgacgag 1200gccagcaccc agacctacga cagcaagcgc
aacgtgggcg ccgtgagctg ggacagcatc 1260gaccagctgc cccccgagac caccgacgag
cccctggaga agggctacag ccaccagctg 1320aactacgtga tgtgcttcct gatgcagggc
agccgcggca ccatccccgt gctgacctgg 1380acccacaaga gcgccgagtt caacaacatc
atccccagca gccagatcac ccagatcccc 1440ctgaccaaga gcaccaacct gggcagcggc
accagcgtgg tgaagggccc cggcttcacc 1500ggcggcgaca tcctgcgccg caccagcccc
ggccagatca gcaccctgcg cgtgaacatc 1560accgcccccc tgagccagcg ctaccgcgtc
cgcatccgct acgccagcac caccaacctg 1620cagttccaca ccagcatcga cggccgcccc
atcaaccagg gcaacttcag cgccaccatg 1680agcagcggca gcaacctgca gagcggcagc
ttccgcaccg tgggcttcac cacccccttc 1740aacttcagca acggcagcag cgtgttcacc
ctgagcgccc acgtgttcaa cagcggcaac 1800gaggtgtaca tcgaccgcat cgagttcgtg
cccgccgagg tgaccttcga ggccgagtac 1860gacctggaga gggctcagaa ggccgtgaac
gagctgttca ccagcagcaa ccagatcggc 1920ctgaagaccg acgtgaccga ctaccacatc
gatcaggtgt ag 196262653PRTArtificial SequenceDM23A
protein 62Met Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly
1 5 10 15 Arg Gln
Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp Val Ile 20
25 30 Gln Lys Gly Ile Ser Val Val
Gly Asp Leu Leu Gly Val Val Gly Phe 35 40
45 Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn
Phe Leu Asn Thr 50 55 60
Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65
70 75 80 Ala Leu Met
Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu 85
90 95 Ala Glu Leu Gln Gly Leu Gln Asn
Asn Val Glu Asp Tyr Val Ser Ala 100 105
110 Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
Asn Pro His 115 120 125
Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe 130
135 140 Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe 145 150
155 160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe Leu Leu Lys 165 170
175 Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
Asp Ile 180 185 190
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp
195 200 205 His Cys Val Lys
Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser 210
215 220 Ser Tyr Glu Ser Trp Val Asn Phe
Asn Arg Tyr Arg Arg Glu Met Thr 225 230
235 240 Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu
Tyr Asp Val Arg 245 250
255 Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr
260 265 270 Asp Pro Ile
Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe 275
280 285 Ser Asn Ile Glu Asn Tyr Ile Arg
Lys Pro His Leu Phe Asp Tyr Leu 290 295
300 His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
Tyr Gly Asn 305 310 315
320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser
325 330 335 Ile Gly Ser Asn
Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser 340
345 350 Ser Glu Pro Val Gln Asn Leu Glu Phe
Asn Gly Glu Lys Val Tyr Arg 355 360
365 Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
Tyr Ser 370 375 380
Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu 385
390 395 400 Ala Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser 405
410 415 Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu
Thr Thr Asp Glu Pro Leu 420 425
430 Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
Met 435 440 445 Gln
Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser 450
455 460 Ala Glu Phe Asn Asn Ile
Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro 465 470
475 480 Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr
Ser Val Val Lys Gly 485 490
495 Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln
500 505 510 Ile Ser
Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr 515
520 525 Arg Val Arg Ile Arg Tyr Ala
Ser Thr Thr Asn Leu Gln Phe His Thr 530 535
540 Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe
Ser Ala Thr Met 545 550 555
560 Ser Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe
565 570 575 Thr Thr Pro
Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser 580
585 590 Ala His Val Phe Asn Ser Gly Asn
Glu Val Tyr Ile Asp Arg Ile Glu 595 600
605 Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp
Leu Glu Arg 610 615 620
Ala Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly 625
630 635 640 Leu Lys Thr Asp
Val Thr Asp Tyr His Ile Asp Gln Val 645
650 631923DNAArtificial Sequence8AF coding sequence
63atgacggccg acaacaacac cgaggccctg gacagcagca ccaccaagga cgtgatccag
60aagggcatca gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt cggcggcgcc
120ctggtgagct tctacaccaa cttcctgaac accatctggc ccagcgagga cccctggaag
180gccttcatgg agcaggtgga ggccctgatg gaccagaaga tcgccgacta cgccaagaac
240aaggcactgg ccgagctaca gggcctccag aacaacgtgg aggactatgt gagcgccctg
300agcagctggc agaagaaccc cgctgcaccg ttccgcaacc cccacagcca gggccgcatc
360cgcgagctgt tcagccaggc cgagagccac ttccgcaaca gcatgcccag cttcgccatc
420agcggctacg aggtgctgtt cctgaccacc tacgcccagg ccgccaacac ccacctgttc
480ctgctgaagg acgcccaaat ctacggagag gagtggggct acgagaagga ggacatcgcc
540gagttctaca agcgccagct gaagctgacc caggagtaca ccgaccactg cgtgaagtgg
600tacaacgtgg gtctagacaa gctccgcggc agcagctacg agagctgggt gaacttcaac
660cgctaccgcc gcgagatgac cctgaccgtg ctggacctga tcgccctgtt ccccctgtac
720gacgtgcgcc tgtaccccaa ggaggtgaag accgagctga cccgcgacgt gctgaccgac
780cccatcgtgg gcgtgaacaa cctgcgcggc tacggcacca ccttcagcaa catcgagaac
840tacatccgca agccccacct gttcgactac ctgcaccgca tccagttcca cacgcgtttc
900cagcccggct actacggcaa cgacagcttc aactactgga gcggcaacta cgtgagcacc
960cgccccagca tcggcagcaa cgacatcatc accagcccct tctacggcaa caagagcagc
1020gagcccgtgc agaaccttga gttcaacggc gagaaggtgt accgcgccgt ggctaacacc
1080aacctggccg tgtggccctc tgcagtgtac agcggcgtga ccaaggtgga gttcagccag
1140tacaacgacc agaccgacga ggccagcacc cagacctacg acagcaagcg caacgtgggc
1200gccgtgagct gggacagcat cgaccagctg ccccccgaga ccaccgacga gcccctggag
1260aagggctaca gccaccagct gaactacgtg atgtgcttcc tgatgcaggg cagccgcggc
1320accatccccg tgctgacctg gacccacaag agcgtcgact tcttcaacat gatcgacagc
1380aagaagatca cccagctgcc cctgaccaag agcaccaacc tgggcagcgg caccagcgtg
1440gtgaagggcc ccggcttcac cggcggcgac atcctgcgcc gcaccagccc cggccagatc
1500agcaccctgc gcgtgaacat caccgccccc ctgagccagc gctaccgcgt ccgcatccgc
1560tacgccagca ccaccaacct gcagttccac accagcatcg acggccgccc catcaaccag
1620ggcaacttca gcgccaccat gagcagcggc agcaacctgc agagcggcag cttccgcacc
1680gtgggcttca ccaccccctt caacttcagc aacggcagca gcgtgttcac cctgagcgcc
1740cacgtgttca acagcggcaa cgaggtgtac atcgaccgca tcgagttcgt gcccgccgag
1800gtgaccttcg aggccgagta cgacctggag agggctcaga aggccgtgaa cgagctgttc
1860accagcagca accagatcgg cctgaagacc gacgtgaccg actaccacat cgatcaggtg
1920tag
192364640PRTArtificial Sequence8AF protein 64Met Thr Ala Asp Asn Asn Thr
Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly
Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn
Phe 35 40 45 Leu
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu Met
Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn
Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110 Asn Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 115
120 125 Ser His Phe Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130 135
140 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe 145 150 155
160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175 Glu Asp Ile
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu 180
185 190 Tyr Thr Asp His Cys Val Lys Trp
Tyr Asn Val Gly Leu Asp Lys Leu 195 200
205 Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg
Tyr Arg Arg 210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr 225
230 235 240 Asp Val Arg Leu
Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp 245
250 255 Val Leu Thr Asp Pro Ile Val Gly Val
Asn Asn Leu Arg Gly Tyr Gly 260 265
270 Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His
Leu Phe 275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr 290
295 300 Tyr Gly Asn Asp Ser
Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 305 310
315 320 Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile
Thr Ser Pro Phe Tyr Gly 325 330
335 Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu
Lys 340 345 350 Val
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala 355
360 365 Val Tyr Ser Gly Val Thr
Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 370 375
380 Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Val Gly 385 390 395
400 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415 Glu Pro
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys 420
425 430 Phe Leu Met Gln Gly Ser Arg
Gly Thr Ile Pro Val Leu Thr Trp Thr 435 440
445 His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser
Lys Lys Ile Thr 450 455 460
Gln Leu Pro Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val 465
470 475 480 Val Lys Gly
Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser 485
490 495 Pro Gly Gln Ile Ser Thr Leu Arg
Val Asn Ile Thr Ala Pro Leu Ser 500 505
510 Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr
Asn Leu Gln 515 520 525
Phe His Thr Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser 530
535 540 Ala Thr Met Ser
Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr 545 550
555 560 Val Gly Phe Thr Thr Pro Phe Asn Phe
Ser Asn Gly Ser Ser Val Phe 565 570
575 Thr Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr
Ile Asp 580 585 590
Arg Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp
595 600 605 Leu Glu Arg Ala
Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn 610
615 620 Gln Ile Gly Leu Lys Thr Asp Val
Thr Asp Tyr His Ile Asp Gln Val 625 630
635 640 651836DNAArtificial Sequence5*cry3A055 coding
sequence 65atgactagta acggccgcca gtgtgctgga attcgccctt atgacggccg
acaacaacac 60cgaggcctgg acagcagcac caccaaggac gtgatccaga agggcatcag
cgtggtgggc 120gacctgctgg gcgtggtggg cttccccttc ggcggcgccc tggtgagctt
ctacaccaac 180ttcctgaaca ccatctggcc cagcgaggac ccctggaagg ccttcatgga
gcaggtggag 240gccctgatgg accagaagat cgccgactac gccaagaaca aggcactggc
cgagctacag 300ggcctccaga acaacgtgga ggactatgtg agcgccctga gcagctggca
gaagaacccc 360gctgcaccgt tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt
cagccaggcc 420gagagccact tccgcaacag catgcccagc ttcgccatca gcggctacga
ggtgctgttc 480ctgaccacct acgcccaggc cgccaacacc cacctgttcc tgctgaagga
cgcccaaatc 540tacggagagg agtggggcta cgagaaggag gacatcgccg agttctacaa
gcgccagctg 600aagctgaccc aggagtacac cgaccactgc gtgaagtggt acaacgtggg
tctagacaag 660ctccgcggca gcagctacga gagctgggtg aacttcaacc gctaccgccg
cgagatgacc 720ctgaccgtgc tggacctgat cgccctgttc cccctgtacg acgtgcgcct
gtaccccaag 780gaggtgaaga ccgagctgac ccgcgacgtg ctgaccgacc ccatcgtggg
cgtgaacaac 840ctgcgcggct acggcaccac cttcagcaac atcgagaact acatccgcaa
gccccacctg 900ttcgactacc tgcaccgcat ccagttccac acgcgtttcc agcccggcta
ctacggcaac 960gacagcttca actactggag cggcaactac gtgagcaccc gccccagcat
cggcagcaac 1020gacatcatca ccagcccctt ctacggcaac aagagcagcg agcccgtgca
gaaccttgag 1080ttcaacggcg agaaggtgta ccgcgccgtg gctaacacca acctggccgt
gtggccctct 1140gcagtgtaca gcggcgtgac caaggtggag ttcagccagt acaacgacca
gaccgacgag 1200gccagcaccc agacctacga cagcaagcgc aacgtgggcg ccgtgagctg
ggacagcatc 1260gaccagctgc cccccgagac caccgacgag cccctggaga agggctacag
ccaccagctg 1320aactacgtga tgtgcttcct gatgcagggc agccgcggca ccatccccgt
gctgacctgg 1380acccacaaga gcgtcgactt cttcaacatg atcgacagca agaagatcac
ccagctgccc 1440ctggtgaagg cctacaagct ccagagcggc gccagcgtgg tggcaggccc
ccgcttcacc 1500ggcggcgaca tcatccagtg caccgagaac ggcagcgccg ccaccatcta
cgtgaccccc 1560gacgtgagct acagccagaa gtaccgcgcc cgcatccact acgccagcac
cagccagatc 1620accttcaccc tgagcctgga cggggccccc ttcaaccaat actacttcga
caagaccatc 1680aacaagggcg acaccctgac ctacaacagc ttcaacctgg ccagcttcag
cacccctttc 1740gagctgagcg gcaacaacct ccagatcggc gtgaccggcc tgagcgccgg
cgacaaggtg 1800tacatcgaca agatcgagtt catccccgtg aactag
183666611PRTArtificial Sequence5*Cry3A055 protein 66Met Thr
Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu
Asp Ser Ser Thr Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly
Val Val Gly Phe 35 40 45
Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr
50 55 60 Ile Trp Pro
Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65
70 75 80 Ala Leu Met Asp Gln Lys Ile
Ala Asp Tyr Ala Lys Asn Lys Ala Leu 85
90 95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu
Asp Tyr Val Ser Ala 100 105
110 Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro
His 115 120 125 Ser
Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe 130
135 140 Arg Asn Ser Met Pro Ser
Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe 145 150
155 160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His
Leu Phe Leu Leu Lys 165 170
175 Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile
180 185 190 Ala Glu
Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp 195
200 205 His Cys Val Lys Trp Tyr Asn
Val Gly Leu Asp Lys Leu Arg Gly Ser 210 215
220 Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg
Arg Glu Met Thr 225 230 235
240 Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg
245 250 255 Leu Tyr Pro
Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr 260
265 270 Asp Pro Ile Val Gly Val Asn Asn
Leu Arg Gly Tyr Gly Thr Thr Phe 275 280
285 Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe
Asp Tyr Leu 290 295 300
His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn 305
310 315 320 Asp Ser Phe Asn
Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser 325
330 335 Ile Gly Ser Asn Asp Ile Ile Thr Ser
Pro Phe Tyr Gly Asn Lys Ser 340 345
350 Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val
Tyr Arg 355 360 365
Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser 370
375 380 Gly Val Thr Lys Val
Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu 385 390
395 400 Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg
Asn Val Gly Ala Val Ser 405 410
415 Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
Leu 420 425 430 Glu
Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu Met 435
440 445 Gln Gly Ser Arg Gly Thr
Ile Pro Val Leu Thr Trp Thr His Lys Ser 450 455
460 Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys
Ile Thr Gln Leu Pro 465 470 475
480 Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala Gly
485 490 495 Pro Arg
Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly Ser 500
505 510 Ala Ala Thr Ile Tyr Val Thr
Pro Asp Val Ser Tyr Ser Gln Lys Tyr 515 520
525 Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile
Thr Phe Thr Leu 530 535 540
Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr Ile 545
550 555 560 Asn Lys Gly
Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser Phe 565
570 575 Ser Thr Pro Phe Glu Leu Ser Gly
Asn Asn Leu Gln Ile Gly Val Thr 580 585
590 Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile
Glu Phe Ile 595 600 605
Pro Val Asn 610 671803DNAArtificial Sequencemocry3A coding
sequence 67atgacggccg acaacaacac cgaggccctg gacagcagca ccaccaagga
cgtgatccag 60aagggcatca gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt
cggcggcgcc 120ctggtgagct tctacaccaa cttcctgaac accatctggc ccagcgagga
cccctggaag 180gccttcatgg agcaggtgga ggccctgatg gaccagaaga tcgccgacta
cgccaagaac 240aaggcactgg ccgagctaca gggcctccag aacaacgtgg aggactatgt
gagcgccctg 300agcagctggc agaagaaccc cgtctcgagc cgcaaccccc acagccaggg
ccgcatccgc 360gagctgttca gccaggccga gagccacttc cgcaacagca tgcccagctt
cgccatcagc 420ggctacgagg tgctgttcct gaccacctac gcccaggccg ccaacaccca
cctgttcctg 480ctgaaggacg cccaaatcta cggagaggag tggggctacg agaaggagga
catcgccgag 540ttctacaagc gccagctgaa gctgacccag gagtacaccg accactgcgt
gaagtggtac 600aacgtgggtc tagacaagct ccgcggcagc agctacgaga gctgggtgaa
cttcaaccgc 660taccgccgcg agatgaccct gaccgtgctg gacctgatcg ccctgttccc
cctgtacgac 720gtgcgcctgt accccaagga ggtgaagacc gagctgaccc gcgacgtgct
gaccgacccc 780atcgtgggcg tgaacaacct gcgcggctac ggcaccacct tcagcaacat
cgagaactac 840atccgcaagc cccacctgtt cgactacctg caccgcatcc agttccacac
gcgtttccag 900cccggctact acggcaacga cagcttcaac tactggagcg gcaactacgt
gagcacccgc 960cccagcatcg gcagcaacga catcatcacc agccccttct acggcaacaa
gagcagcgag 1020cccgtgcaga accttgagtt caacggcgag aaggtgtacc gcgccgtggc
taacaccaac 1080ctggccgtgt ggccctctgc agtgtacagc ggcgtgacca aggtggagtt
cagccagtac 1140aacgaccaga ccgacgaggc cagcacccag acctacgaca gcaagcgcaa
cgtgggcgcc 1200gtgagctggg acagcatcga ccagctgccc cccgagacca ccgacgagcc
cctggagaag 1260ggctacagcc accagctgaa ctacgtgatg tgcttcctga tgcagggcag
ccgcggcacc 1320atccccgtgc tgacctggac ccacaagagc gtcgacttct tcaacatgat
cgacagcaag 1380aagatcaccc agctgcccct ggtgaaggcc tacaagctcc agagcggcgc
cagcgtggtg 1440gcaggccccc gcttcaccgg cggcgacatc atccagtgca ccgagaacgg
cagcgccgcc 1500accatctacg tgacccccga cgtgagctac agccagaagt accgcgcccg
catccactac 1560gccagcacca gccagatcac cttcaccctg agcctggacg gggccccctt
caaccaatac 1620tacttcgaca agaccatcaa caagggcgac accctgacct acaacagctt
caacctggcc 1680agcttcagca cccctttcga gctgagcggc aacaacctcc agatcggcgt
gaccggcctg 1740agcgccggcg acaaggtgta catcgacaag atcgagttca tccccgtgaa
ctagatctga 1800gct
180368597PRTBacillus thuringiensismoCry3A 68Met Thr Ala Asp
Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val
Val Gly Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr
Asn Phe 35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu
Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln
Asn Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg
Asn 100 105 110 Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 115
120 125 His Phe Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val 130 135
140 Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe Leu 145 150 155
160 Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
165 170 175 Asp Ile
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr 180
185 190 Thr Asp His Cys Val Lys Trp
Tyr Asn Val Gly Leu Asp Lys Leu Arg 195 200
205 Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg
Tyr Arg Arg Glu 210 215 220
Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp 225
230 235 240 Val Arg Leu
Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val 245
250 255 Leu Thr Asp Pro Ile Val Gly Val
Asn Asn Leu Arg Gly Tyr Gly Thr 260 265
270 Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His
Leu Phe Asp 275 280 285
Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr 290
295 300 Gly Asn Asp Ser
Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg 305 310
315 320 Pro Ser Ile Gly Ser Asn Asp Ile Ile
Thr Ser Pro Phe Tyr Gly Asn 325 330
335 Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu
Lys Val 340 345 350
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
355 360 365 Tyr Ser Gly Val
Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr 370
375 380 Asp Glu Ala Ser Thr Gln Thr Tyr
Asp Ser Lys Arg Asn Val Gly Ala 385 390
395 400 Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu
Thr Thr Asp Glu 405 410
415 Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
420 425 430 Leu Met Gln
Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His 435
440 445 Lys Ser Val Asp Phe Phe Asn Met
Ile Asp Ser Lys Lys Ile Thr Gln 450 455
460 Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala
Ser Val Val 465 470 475
480 Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn
485 490 495 Gly Ser Ala Ala
Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln 500
505 510 Lys Tyr Arg Ala Arg Ile His Tyr Ala
Ser Thr Ser Gln Ile Thr Phe 515 520
525 Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe
Asp Lys 530 535 540
Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala 545
550 555 560 Ser Phe Ser Thr Pro
Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly 565
570 575 Val Thr Gly Leu Ser Ala Gly Asp Lys Val
Tyr Ile Asp Lys Ile Glu 580 585
590 Phe Ile Pro Val Asn 595 69
1807DNAArtificial Sequencecry3A055 coding sequence 69atgacggccg
acaacaacac cgaggccctg gacagcagca ccaccaagga cgtgatccag 60aagggcatca
gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt cggcggcgcc 120ctggtgagct
tctacaccaa cttcctgaac accatctggc ccagcgagga cccctggaag 180gccttcatgg
agcaggtgga ggccctgatg gaccagaaga tcgccgacta cgccaagaac 240aaggcactgg
ccgagctaca gggcctccag aacaacgtgg aggactatgt gagcgccctg 300agcagctggc
agaagaaccc cgctgcaccg ttccgcaacc cccacagcca gggccgcatc 360cgcgagctgt
tcagccaggc cgagagccac ttccgcaaca gcatgcccag cttcgccatc 420agcggctacg
aggtgctgtt cctgaccacc tacgcccagg ccgccaacac ccacctgttc 480ctgctgaagg
acgcccaaat ctacggagag gagtggggct acgagaagga ggacatcgcc 540gagttctaca
agcgccagct gaagctgacc caggagtaca ccgaccactg cgtgaagtgg 600tacaacgtgg
gtctagacaa gctccgcggc agcagctacg agagctgggt gaacttcaac 660cgctaccgcc
gcgagatgac cctgaccgtg ctggacctga tcgccctgtt ccccctgtac 720gacgtgcgcc
tgtaccccaa ggaggtgaag accgagctga cccgcgacgt gctgaccgac 780cccatcgtgg
gcgtgaacaa cctgcgcggc tacggcacca ccttcagcaa catcgagaac 840tacatccgca
agccccacct gttcgactac ctgcaccgca tccagttcca cacgcgtttc 900cagcccggct
actacggcaa cgacagcttc aactactgga gcggcaacta cgtgagcacc 960cgccccagca
tcggcagcaa cgacatcatc accagcccct tctacggcaa caagagcagc 1020gagcccgtgc
agaaccttga gttcaacggc gagaaggtgt accgcgccgt ggctaacacc 1080aacctggccg
tgtggccctc tgcagtgtac agcggcgtga ccaaggtgga gttcagccag 1140tacaacgacc
agaccgacga ggccagcacc cagacctacg acagcaagcg caacgtgggc 1200gccgtgagct
gggacagcat cgaccagctg ccccccgaga ccaccgacga gcccctggag 1260aagggctaca
gccaccagct gaactacgtg atgtgcttcc tgatgcaggg cagccgcggc 1320accatccccg
tgctgacctg gacccacaag agcgtcgact tcttcaacat gatcgacagc 1380aagaagatca
cccagctgcc cctggtgaag gcctacaagc tccagagcgg cgccagcgtg 1440gtggcaggcc
cccgcttcac cggcggcgac atcatccagt gcaccgagaa cggcagcgcc 1500gccaccatct
acgtgacccc cgacgtgagc tacagccaga agtaccgcgc ccgcatccac 1560tacgccagca
ccagccagat caccttcacc ctgagcctgg acggggcccc cttcaaccaa 1620tactacttcg
acaagaccat caacaagggc gacaccctga cctacaacag cttcaacctg 1680gccagcttca
gcaccccttt cgagctgagc ggcaacaacc tccagatcgg cgtgaccggc 1740ctgagcgccg
gcgacaaggt gtacatcgac aagatcgagt tcatccccgt gaactagatc 1800tgagctc
180770598PRTArtificial SequenceCry3A055 protein 70Met Thr Ala Asp Asn Asn
Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly
Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn
Phe 35 40 45 Leu
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu Met
Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn
Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110 Asn Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 115
120 125 Ser His Phe Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130 135
140 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe 145 150 155
160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175 Glu Asp Ile
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu 180
185 190 Tyr Thr Asp His Cys Val Lys Trp
Tyr Asn Val Gly Leu Asp Lys Leu 195 200
205 Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg
Tyr Arg Arg 210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr 225
230 235 240 Asp Val Arg Leu
Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp 245
250 255 Val Leu Thr Asp Pro Ile Val Gly Val
Asn Asn Leu Arg Gly Tyr Gly 260 265
270 Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His
Leu Phe 275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr 290
295 300 Tyr Gly Asn Asp Ser
Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 305 310
315 320 Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile
Thr Ser Pro Phe Tyr Gly 325 330
335 Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu
Lys 340 345 350 Val
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala 355
360 365 Val Tyr Ser Gly Val Thr
Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 370 375
380 Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Val Gly 385 390 395
400 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415 Glu Pro
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys 420
425 430 Phe Leu Met Gln Gly Ser Arg
Gly Thr Ile Pro Val Leu Thr Trp Thr 435 440
445 His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser
Lys Lys Ile Thr 450 455 460
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val 465
470 475 480 Val Ala Gly
Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu 485
490 495 Asn Gly Ser Ala Ala Thr Ile Tyr
Val Thr Pro Asp Val Ser Tyr Ser 500 505
510 Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser
Gln Ile Thr 515 520 525
Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp 530
535 540 Lys Thr Ile Asn
Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu 545 550
555 560 Ala Ser Phe Ser Thr Pro Phe Glu Leu
Ser Gly Asn Asn Leu Gln Ile 565 570
575 Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp
Lys Ile 580 585 590
Glu Phe Ile Pro Val Asn 595 71 1947DNAArtificial
Sequencemocry1Ab coding sequence 71atggacaaca accccaacat caacgagtgc
atcccctaca actgcctgag caaccccgag 60gtggaggtgc tgggcggcga gcgcatcgag
accggctaca cccccatcga catcagcctg 120agcctgaccc agttcctgct gagcgagttc
gtgcccggcg ccggcttcgt gctgggcctg 180gtggacatca tctggggcat cttcggcccc
agccagtggg acgccttcct ggtgcagatc 240gagcagttga taaaccaacg catagaggaa
ttcgcccgca accaggccat cagccgcctg 300gagggcctga gcaacctgta ccaaatctac
gccgagagct tccgcgagtg ggaggccgac 360cccaccaacc ccgccctgcg cgaggagatg
cgcatccagt tcaacgacat gaacagcgcc 420ctgaccaccg ccatccccct gttcgccgtg
cagaactacc aggtgcccct gctgagcgtg 480tacgtgcagg ccgccaacct gcacctgagc
gtgctgcgcg acgtcagcgt gttcggccag 540cgctggggct tcgacgccgc caccatcaac
agccgctaca acgacctgac ccgcctgatc 600ggcaactaca ccgaccacgc cgtgcgctgg
tacaacaccg gcctggagcg cgtgtggggt 660cccgacagcc gcgactggat caggtacaac
cagttccgcc gcgagctgac cctgaccgtg 720ctggacatcg tgagcctgtt ccccaactac
gacagccgca cctaccccat ccgcaccgtg 780agccagctga cccgcgagat ttacaccaac
cccgtgctgg agaacttcga cggcagcttc 840cgcggcagcg cccagggcat cgagggcagc
atccgcagcc cccacctgat ggacatcctg 900aacagcatca ccatctacac cgacgcccac
cgcggcgagt actactggag cggccaccag 960atcatggcca gccccgtcgg cttcagcggc
cccgagttca ccttccccct gtacggcacc 1020atgggcaacg ctgcacctca gcagcgcatc
gtggcacagc tgggccaggg agtgtaccgc 1080accctgagca gcaccctgta ccgtcgacct
ttcaacatcg gcatcaacaa ccagcagctg 1140agcgtgctgg acggcaccga gttcgcctac
ggcaccagca gcaacctgcc cagcgccgtg 1200taccgcaaga gcggcaccgt ggacagcctg
gacgagatcc cccctcagaa caacaacgtg 1260ccacctcgac agggcttcag ccaccgtctg
agccacgtga gcatgttccg cagtggcttc 1320agcaacagca gcgtgagcat catccgtgca
cctatgttca gctggattca ccgcagtgcc 1380gagttcaaca acatcatccc cagcagccag
atcacccaga tccccctgac caagagcacc 1440aacctgggca gcggcaccag cgtggtgaag
ggccccggct tcaccggcgg cgacatcctg 1500cgccgcacca gccccggcca gatcagcacc
ctgcgcgtga acatcaccgc ccccctgagc 1560cagcgctacc gcgtccgcat ccgctacgcc
agcaccacca acctgcagtt ccacaccagc 1620atcgacggcc gccccatcaa ccagggcaac
ttcagcgcca ccatgagcag cggcagcaac 1680ctgcagagcg gcagcttccg caccgtgggc
ttcaccaccc ccttcaactt cagcaacggc 1740agcagcgtgt tcaccctgag cgcccacgtg
ttcaacagcg gcaacgaggt gtacatcgac 1800cgcatcgagt tcgtgcccgc cgaggtgacc
ttcgaggccg agtacgacct ggagagggct 1860cagaaggccg tgaacgagct gttcaccagc
agcaaccaga tcggcctgaa gaccgacgtg 1920accgactacc acatcgatca ggtgtag
194772648PRTBacillus thuringiensisCry1Ab
protein 72Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15 Ser Asn
Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20
25 30 Tyr Thr Pro Ile Asp Ile Ser
Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40
45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu
Val Asp Ile Ile 50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65
70 75 80 Glu Gln Leu
Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85
90 95 Ile Ser Arg Leu Glu Gly Leu Ser
Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105
110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala
Leu Arg Glu 115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130
135 140 Ile Pro Leu Phe
Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150
155 160 Tyr Val Gln Ala Ala Asn Leu His Leu
Ser Val Leu Arg Asp Val Ser 165 170
175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn
Ser Arg 180 185 190
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205 Arg Trp Tyr Asn
Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210
215 220 Asp Trp Ile Arg Tyr Asn Gln Phe
Arg Arg Glu Leu Thr Leu Thr Val 225 230
235 240 Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser
Arg Thr Tyr Pro 245 250
255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270 Leu Glu Asn
Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275
280 285 Gly Ser Ile Arg Ser Pro His Leu
Met Asp Ile Leu Asn Ser Ile Thr 290 295
300 Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser
Gly His Gln 305 310 315
320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335 Leu Tyr Gly Thr
Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340
345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr
Leu Ser Ser Thr Leu Tyr Arg 355 360
365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val
Leu Asp 370 375 380
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385
390 395 400 Tyr Arg Lys Ser Gly
Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405
410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe
Ser His Arg Leu Ser His 420 425
430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile
Ile 435 440 445 Arg
Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450
455 460 Ile Ile Pro Ser Ser Gln
Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465 470
475 480 Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly
Pro Gly Phe Thr Gly 485 490
495 Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg
500 505 510 Val Asn
Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515
520 525 Tyr Ala Ser Thr Thr Asn Leu
Gln Phe His Thr Ser Ile Asp Gly Arg 530 535
540 Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser
Ser Gly Ser Asn 545 550 555
560 Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn
565 570 575 Phe Ser Asn
Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn 580
585 590 Ser Gly Asn Glu Val Tyr Ile Asp
Arg Ile Glu Phe Val Pro Ala Glu 595 600
605 Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln
Lys Ala Val 610 615 620
Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val 625
630 635 640 Thr Asp Tyr His
Ile Asp Gln Val 645 731971DNAArtificial
Sequencemocry1Ba coding sequence 73atgaccagca accgcaagaa cgagaacgag
atcatcaacg ccgtgagcaa ccacagcgcc 60cagatggacc tgctgcccga cgcccgcatc
gaggacagcc tgtgcatcgc cgagggcaac 120aacatcgacc ccttcgtgag cgctagcacc
gtgcagaccg gtatcaacat cgctggccgc 180atcctgggcg tgctgggcgt gcccttcgcc
ggccagctgg ctagcttcta cagcttcctg 240gtcggtgagc tgtggccacg cggccgcgac
cagtgggaaa tcttcctgga gcacgtggag 300cagctgatca accagcagat caccgagaac
gcccgcaaca ccgctcttgc ccgcctgcag 360ggtctgggcg acagcttccg cgcctaccag
cagagcctgg aggactggct ggagaaccgc 420gacgacgccc gcacccgcag cgtgctgtac
acccagtaca tcgccctgga gctggacttc 480ctgaacgcca tgcccctgtt cgccattcga
aaccaggagg tgcccctgct gatggtgtac 540gcccaggccg ccaacctgca cctgctgctg
ctgcgcgacg ccagcctgtt cggcagcgag 600ttcggcctga ccagccagga gatccagcgg
tactacgagc gccaggtgga gcgcacccgc 660gactacagcg actactgcgt ggagtggtac
aacaccggcc tgaacagctt aaggggcacc 720aacgccgcca gctgggtgcg ctacaaccag
ttccgccgcg acctgaccct gggcgtgctg 780gacctggtgg ccctgttccc cagctacgac
acccgcacct accccatcaa caccagcgcc 840cagctgaccc gcgaggtgta caccgacgcc
atcggcgcca ccggcgtgaa catggccagc 900atgaactggt acaacaacaa cgcccccagc
ttcagcgcca tcgaggccgc cgccatccgc 960agcccccacc tgctggactt cctggagcag
ctgaccatct tcagtgccag cagccgctgg 1020agcaacaccc gccacatgac ctactggcgc
ggccacacca tccagtctag acccatcggc 1080ggcggcctga acaccagcac ccacggcgcc
accaacacca gcatcaaccc cgtgaccctg 1140cgcttcgcct cccgagacgt ctaccgcacc
gagagctacg ccggcgtgct gctgtggggc 1200atctacctgg agcccatcca tggcgtgccc
accgtgcgct tcaacttcac caacccccag 1260aacatcagcg accgcggcac cgccaactac
agccagccct acgagagccc cgggttgcag 1320ctgaaggaca gcgagaccga gctgcccccc
gagaccaccg agcgccccaa ctacgagagc 1380tacagccacc gcctgagcca catcggcatc
atcttgcaga gccgcgtgaa cgtgcccgtg 1440tacagctgga cccaccgcag cgccgaccgc
accaacacca tcggccccaa ccgcatcacc 1500cagatcccca tggtgaaggc cagcgagctg
ccccagggca ccaccgtggt tcgcggcccc 1560ggcttcaccg gaggcgacat cctgcgacgc
accaacaccg gcggcttcgg ccccatccgc 1620gtgaccgtga acggccccct gacccagcgc
taccgcatcg gcttccgcta cgccagcacc 1680gtggacttcg acttcttcgt gagccgcggc
ggcaccaccg tgaacaactt ccgcttcctg 1740cgcaccatga acagcggcga cgagctgaag
tacggcaact tcgtgcgccg cgccttcacc 1800acccccttca ccttcaccca gatccaggac
atcatccgca ccagcatcca gggcctgagc 1860ggcaacggcg aggtgtacat cgacaagatc
gagatcatcc ccgtgaccgc caccttcgag 1920gccgagtacg acctagagcg cgcccaggag
gccgtgaacg ccctgttcta g 197174656PRTBacillus
thuringiensisCry1B protein 74Met Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile
Ile Asn Ala Val Ser 1 5 10
15 Asn His Ser Ala Gln Met Asp Leu Leu Pro Asp Ala Arg Ile Glu Asp
20 25 30 Ser Leu
Cys Ile Ala Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Ala 35
40 45 Ser Thr Val Gln Thr Gly Ile
Asn Ile Ala Gly Arg Ile Leu Gly Val 50 55
60 Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser Phe
Tyr Ser Phe Leu 65 70 75
80 Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gln Trp Glu Ile Phe Leu
85 90 95 Glu His Val
Glu Gln Leu Ile Asn Gln Gln Ile Thr Glu Asn Ala Arg 100
105 110 Asn Thr Ala Leu Ala Arg Leu Gln
Gly Leu Gly Asp Ser Phe Arg Ala 115 120
125 Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp
Asp Ala Arg 130 135 140
Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala Leu Glu Leu Asp Phe 145
150 155 160 Leu Asn Ala Met
Pro Leu Phe Ala Ile Arg Asn Gln Glu Val Pro Leu 165
170 175 Leu Met Val Tyr Ala Gln Ala Ala Asn
Leu His Leu Leu Leu Leu Arg 180 185
190 Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gln
Glu Ile 195 200 205
Gln Arg Tyr Tyr Glu Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser Asp 210
215 220 Tyr Cys Val Glu Trp
Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr 225 230
235 240 Asn Ala Ala Ser Trp Val Arg Tyr Asn Gln
Phe Arg Arg Asp Leu Thr 245 250
255 Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr
Arg 260 265 270 Thr
Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr Thr 275
280 285 Asp Ala Ile Gly Ala Thr
Gly Val Asn Met Ala Ser Met Asn Trp Tyr 290 295
300 Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu
Ala Ala Ala Ile Arg 305 310 315
320 Ser Pro His Leu Leu Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser Ala
325 330 335 Ser Ser
Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly His 340
345 350 Thr Ile Gln Ser Arg Pro Ile
Gly Gly Gly Leu Asn Thr Ser Thr His 355 360
365 Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu
Arg Phe Ala Ser 370 375 380
Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly 385
390 395 400 Ile Tyr Leu
Glu Pro Ile His Gly Val Pro Thr Val Arg Phe Asn Phe 405
410 415 Thr Asn Pro Gln Asn Ile Ser Asp
Arg Gly Thr Ala Asn Tyr Ser Gln 420 425
430 Pro Tyr Glu Ser Pro Gly Leu Gln Leu Lys Asp Ser Glu
Thr Glu Leu 435 440 445
Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg 450
455 460 Leu Ser His Ile
Gly Ile Ile Leu Gln Ser Arg Val Asn Val Pro Val 465 470
475 480 Tyr Ser Trp Thr His Arg Ser Ala Asp
Arg Thr Asn Thr Ile Gly Pro 485 490
495 Asn Arg Ile Thr Gln Ile Pro Met Val Lys Ala Ser Glu Leu
Pro Gln 500 505 510
Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu
515 520 525 Arg Arg Thr Asn
Thr Gly Gly Phe Gly Pro Ile Arg Val Thr Val Asn 530
535 540 Gly Pro Leu Thr Gln Arg Tyr Arg
Ile Gly Phe Arg Tyr Ala Ser Thr 545 550
555 560 Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr
Thr Val Asn Asn 565 570
575 Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly
580 585 590 Asn Phe Val
Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln Ile 595
600 605 Gln Asp Ile Ile Arg Thr Ser Ile
Gln Gly Leu Ser Gly Asn Gly Glu 610 615
620 Val Tyr Ile Asp Lys Ile Glu Ile Ile Pro Val Thr Ala
Thr Phe Glu 625 630 635
640 Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu Ala Val Asn Ala Leu Phe
645 650 655
751950DNAArtificial Sequencemocry1Fa coding sequence 75atggagaaca
acatccagaa ccagtgcgtg ccgtacaact gcctcaacaa cccggaggtg 60gagatcctca
acgaggagcg ctccaccggc cgcctcccgc tcgacatctc cctctccctc 120acccgcttcc
tcctctccga gttcgtgccg ggcgtgggcg tggccttcgg cctcttcgac 180ctcatctggg
gcttcatcac cccgtccgac tggtccctct tcctcctcca gatcgagcag 240ctcatcgagc
agcgcatcga gaccctggag cgcaaccgcg ccatcaccac cctccgcggc 300ctcgccgact
cctacgaaat ctacatcgag gccctccgcg agtgggaggc caacccgaac 360aacgcccagc
tccgcgagga cgtgcgcatc cgcttcgcca acaccgacga cgccctcatc 420accgccatca
acaacttcac cctcacctcc ttcgagatcc cgctcctctc cgtgtacgtg 480caggccgcca
acctccacct ctccctcctc cgcgacgccg tgtccttcgg ccagggctgg 540ggcctcgaca
tcgccaccgt gaacaaccac tacaaccgcc tcatcaacct catccaccgc 600tacaccaagc
actgcctcga cacctacaac cagggcctgg agaacctccg cggcaccaac 660acccgccagt
gggcccgctt caaccagttc cgccgcgacc tcaccctcac cgtgctcgac 720atcgtggccc
tcttcccgaa ctacgacgtg cgcacctacc cgatccagac ctcctcccag 780ctcacccgcg
aaatctacac ctcctccgtg atcgaggact ccccggtgtc cgccaacatc 840ccgaacggct
tcaaccgcgc cgagttcggc gtgcgcccgc cgcacctcat ggacttcatg 900aactccctct
tcgtgaccgc cgagaccgtg cgctcccaga ccgtgtgggg cggccacctc 960gtgtcctccc
gcaacaccgc cggcaaccgc atcaacttcc cgtcctacgg cgtgttcaac 1020ccgggcggcg
ccatctggat cgccgacgag gacccgcgcc cgttctaccg caccctctcc 1080gacccggtgt
tcgtgcgcgg cggcttcggc aacccgcact acgtgctcgg cctccgcggc 1140gtggccttcc
agcagaccgg caccaaccac acccgcacct tccgcaactc cggcaccatc 1200gactccctcg
acgagatccc gccgcaggac aactccggcg ccccgtggaa cgactactcc 1260cacgtgctca
accacgtgac cttcgtgcgc tggccgggcg agatatccgg ctccgactcc 1320tggcgtgcac
cgatgttctc ctggacccac cgctccgcca ccccgaccaa caccatcgac 1380ccggagcgca
tcacccagat cccgctcgtg aaggcccaca ccctccagtc cggcaccacc 1440gtggtgcgcg
gcccgggctt caccggcggc gacatcctcc gccgcacctc cggcggcccg 1500ttcgcctaca
ccatcgtgaa catcaacggc cagctcccgc agcgctaccg cgcccgcatc 1560cgctacgcct
ccaccaccaa cctccgcatc tacgtgaccg tggccggcga gcgcatcttc 1620gccggccagt
tcaacaagac catggacacc ggcgacccgc tcaccttcca gtccttctcc 1680tacgccacca
tcaacaccgc cttcaccttc ccgatgtccc agtcctcctt caccgtgggc 1740gccgacacct
tctcctccgg caacgaggtg tacatcgacc gcttcgagct gatcccggtg 1800accgccacct
tcgaggccga gtacgacctg gagcgcgccc agaaggccgt gaacgccctc 1860ttcacctcca
tcaaccagat cggcatcaag accgacgtga ccgactacca catcgaccag 1920gtgtccaacc
tcgtggactg cttaagctag
195076649PRTBacillus thguringiensisCry1F protein 76Met Glu Asn Asn Ile
Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5
10 15 Asn Pro Glu Val Glu Ile Leu Asn Glu Glu
Arg Ser Thr Gly Arg Leu 20 25
30 Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu
Phe 35 40 45 Val
Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50
55 60 Phe Ile Thr Pro Ser Asp
Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70
75 80 Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg
Asn Arg Ala Ile Thr 85 90
95 Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu
100 105 110 Arg Glu
Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115
120 125 Arg Ile Arg Phe Ala Asn Thr
Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135
140 Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu
Ser Val Tyr Val 145 150 155
160 Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe
165 170 175 Gly Gln Gly
Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180
185 190 Arg Leu Ile Asn Leu Ile His Arg
Tyr Thr Lys His Cys Leu Asp Thr 195 200
205 Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr
Arg Gln Trp 210 215 220
Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225
230 235 240 Ile Val Ala Leu
Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245
250 255 Thr Ser Ser Gln Leu Thr Arg Glu Ile
Tyr Thr Ser Ser Val Ile Glu 260 265
270 Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg
Ala Glu 275 280 285
Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290
295 300 Val Thr Ala Glu Thr
Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310
315 320 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg
Ile Asn Phe Pro Ser Tyr 325 330
335 Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp
Pro 340 345 350 Arg
Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355
360 365 Phe Gly Asn Pro His Tyr
Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375
380 Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg
Asn Ser Gly Thr Ile 385 390 395
400 Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp
405 410 415 Asn Asp
Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420
425 430 Gly Glu Ile Ser Gly Ser Asp
Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440
445 Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp
Pro Glu Arg Ile 450 455 460
Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465
470 475 480 Val Val Arg
Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485
490 495 Ser Gly Gly Pro Phe Ala Tyr Thr
Ile Val Asn Ile Asn Gly Gln Leu 500 505
510 Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr
Thr Asn Leu 515 520 525
Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530
535 540 Asn Lys Thr Met
Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550
555 560 Tyr Ala Thr Ile Asn Thr Ala Phe Thr
Phe Pro Met Ser Gln Ser Ser 565 570
575 Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val
Tyr Ile 580 585 590
Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr
595 600 605 Asp Leu Glu Arg
Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile 610
615 620 Asn Gln Ile Gly Ile Lys Thr Asp
Val Thr Asp Tyr His Ile Asp Gln 625 630
635 640 Val Ser Asn Leu Val Asp Cys Leu Ser
645 773469DNABacillus
thuringiensismisc_feature(1)..(3469)cry8Aa coding sequence 77atgagtccaa
ataatcaaaa tgaatatgaa attatagatg cgacaccttc tacatctgta 60tccagtgatt
ctaacagata cccttttgcg aatgagccaa cagatgcgtt acaaaatatg 120aattataaag
attatctgaa aatgtctggg ggagagaatc ctgaattatt tggaaatccg 180gagacgttta
ttagttcatc cacgattcaa actggaattg gcattgttgg tcgaatacta 240ggagctttag
gggttccatt tgctagtcag atagctagtt tctatagttt cattgttggt 300caattatggc
cgtcaaagag cgtagatata tggggagaaa ttatggaacg agtggaagaa 360ctcgttgatc
aaaaaataga aaaatatgta aaagataagg ctcttgctga attaaaaggg 420ctaggaaatg
ctttggatgt atatcagcag tcacttgaag attggctgga aaatcgcaat 480gatgcaagaa
ctagaagtgt tgtttctaat caatttatag ctttagatct taactttgtt 540agttcaattc
catcttttgc agtatccgga cacgaagtac tattattagc agtatatgca 600caggctgtga
acctacattt attgttatta agagatgctt ctatttttgg agaagagtgg 660ggatttacac
caggtgaaat ttctagattt tataatcgtc aagtgcaact taccgctgaa 720tattcagact
attgtgtaaa gtggtataaa atcggcttag ataaattgaa aggtaccact 780tctaaaagtt
ggctgaatta tcatcagttc cgtagagaga tgacattact ggtattagat 840ttggtggcgt
tatttccaaa ctatgacaca catatgtatc caatcgaaac aacagctcaa 900cttacacggg
atgtgtatac agatccgata gcatttaaca tagtgacaag tactggattc 960tgcaaccctt
ggtcaaccca cagtggtatt cttttttatg aagttgaaaa caacgtaatt 1020cgtccgccac
acttgtttga tatactcagc tcagtagaaa ttaatacaag tagagggggt 1080attacgttaa
ataatgatgc atatataaac tactggtcag gacataccct aaaatatcgt 1140agaacagctg
attcgaccgt aacatacaca gctaattacg gtcgaatcac ttcagaaaag 1200aattcatttg
cacttgagga tagggatatt tttgaaatta attcaactgt ggcaaaccta 1260gctaattact
accaaaaggc atatggtgtg ccgggatctt ggttccatat ggtaaaaagg 1320ggaacctcat
caacaacagc gtatttatat tcaaaaacac atacagctct ccaagggtgt 1380acacaggttt
atgaatcaag tgatgaaata cctctagata gaactgtacc ggtagctgaa 1440agctatagtc
atagattatc tcatattacc tcccattctt tctctaaaaa tgggagtgca 1500tactatggga
gtttccctgt atttgtttgg acacatacta gtgcggattt aaataataca 1560atatattcag
ataaaatcac tcaaattcca gcggtaaagg gagacatgtt atatctaggg 1620ggttccgtag
tacagggtcc tggatttaca ggaggagata tattaaaaag aaccaatcct 1680agcatattag
ggacctttgc ggttacagta aatgggtcgt tatcacaaag atatcgtgta 1740agaattcgct
atgcctctac aacagatttt gaatttactc tataccttgg cgacacaata 1800gaaaaaaata
gatttaacaa aactatggat aatggggcat ctttaacgta tgaaacattt 1860aaattcgcaa
gtttcattac tgatttccaa ttcagagaaa cacaagataa aatactccta 1920tccatgggtg
attttagctc cggtcaagaa gtttatatag accgaatcga attcatccca 1980gtagatgaga
catatgaggc ggaacaagat ttagaagcgg cgaagaaagc agtgaatgcc 2040ttgtttacga
atacaaaaga tggcttacga ccaggtgtaa cggattatga agtaaatcaa 2100gcggcaaact
tagtggaatg cctatcggat gatttatatc caaatgaaaa acgattgtta 2160tttgatgcgg
tgagagaggc aaaacgcctc agtggggcac gtaacttact acaagatcca 2220gatttccaag
agataaacgg agaaaatgga tgggcggcaa gtacgggaat tgagattgta 2280gaaggggatg
ctgtatttaa aggacgttat ctacgcctac caggtgcacg agaaattgat 2340acggaaacgt
atccaacgta tctgtatcaa aaagtagagg aaggtgtatt aaaaccatac 2400acaagatata
gactgagagg gtttgtggga agtagtcaag gattagaaat ttatacgata 2460cgtcaccaaa
cgaatcgaat tgtaaagaat gtaccagatg atttattgcc agatgtatct 2520cctgtaaact
ctgatggcag tatcaatcga tgcagcgaac aaaagtatgt gaatagccgt 2580ttagaaggag
aaaaccgttc tggtgatgca catgagttct cgctccctat cgatatagga 2640gagctggatt
acaatgaaaa tgcaggaata tgggttggat ttaagattac ggacccagag 2700ggatacgcaa
cacttggaaa tcttgaatta gtcgaagagg gacctttgtc aggagacgca 2760ttagagcgct
tgcaaagaga agaacaacag tggaagattc aaatgacaag aagacgtgaa 2820gagacagata
gaagatacat ggcatcgaaa caagcggtag atcgtttata tgccgattat 2880caggatcaac
aactgaatcc tgatgtagag attacagatc ttactgcggc tcaagatctg 2940atacagtcca
ttccttacgt atataacgaa atgttcccag aaataccagg gatgaactat 3000acgaagttta
cagaattaac agatcgactc caacaagcgt ggaatttgta tgatcagcga 3060aatgccatac
caaatggtga ttttcgaaat gggttaagta attggaatgc aacgcctggc 3120gtagaagtac
aacaaatcaa tcatacatct gtccttgtga ttccaaactg ggatgaacaa 3180gtttcacaac
agtttacagt tcaaccgaat caaagatatg tattacgagt tactgcaaga 3240aaagaagggg
taggaaatgg atatgtaagt attcgtgatg gtggaaatca atcagaaacg 3300cttactttta
gtgcaagcga ttatgataca aatggtgtgt ataatgacca aaccggctat 3360atcacaaaaa
cagtgacatt catcccgtat acagatcaaa tgtggattga aataagtgaa 3420acagaaggta
cgttctatat agaaagtgta gaattgattg tagacgtag
3469781156PRTBacillus thuringiensisMISC_FEATURE(1)..(1156)Cry8Aa protein
78Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1
5 10 15 Ser Thr Ser Val
Ser Ser Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20
25 30 Pro Thr Asp Ala Leu Gln Asn Met Asn
Tyr Lys Asp Tyr Leu Lys Met 35 40
45 Ser Gly Gly Glu Asn Pro Glu Leu Phe Gly Asn Pro Glu Thr
Phe Ile 50 55 60
Ser Ser Ser Thr Ile Gln Thr Gly Ile Gly Ile Val Gly Arg Ile Leu 65
70 75 80 Gly Ala Leu Gly Val
Pro Phe Ala Ser Gln Ile Ala Ser Phe Tyr Ser 85
90 95 Phe Ile Val Gly Gln Leu Trp Pro Ser Lys
Ser Val Asp Ile Trp Gly 100 105
110 Glu Ile Met Glu Arg Val Glu Glu Leu Val Asp Gln Lys Ile Glu
Lys 115 120 125 Tyr
Val Lys Asp Lys Ala Leu Ala Glu Leu Lys Gly Leu Gly Asn Ala 130
135 140 Leu Asp Val Tyr Gln Gln
Ser Leu Glu Asp Trp Leu Glu Asn Arg Asn 145 150
155 160 Asp Ala Arg Thr Arg Ser Val Val Ser Asn Gln
Phe Ile Ala Leu Asp 165 170
175 Leu Asn Phe Val Ser Ser Ile Pro Ser Phe Ala Val Ser Gly His Glu
180 185 190 Val Leu
Leu Leu Ala Val Tyr Ala Gln Ala Val Asn Leu His Leu Leu 195
200 205 Leu Leu Arg Asp Ala Ser Ile
Phe Gly Glu Glu Trp Gly Phe Thr Pro 210 215
220 Gly Glu Ile Ser Arg Phe Tyr Asn Arg Gln Val Gln
Leu Thr Ala Glu 225 230 235
240 Tyr Ser Asp Tyr Cys Val Lys Trp Tyr Lys Ile Gly Leu Asp Lys Leu
245 250 255 Lys Gly Thr
Thr Ser Lys Ser Trp Leu Asn Tyr His Gln Phe Arg Arg 260
265 270 Glu Met Thr Leu Leu Val Leu Asp
Leu Val Ala Leu Phe Pro Asn Tyr 275 280
285 Asp Thr His Met Tyr Pro Ile Glu Thr Thr Ala Gln Leu
Thr Arg Asp 290 295 300
Val Tyr Thr Asp Pro Ile Ala Phe Asn Ile Val Thr Ser Thr Gly Phe 305
310 315 320 Cys Asn Pro Trp
Ser Thr His Ser Gly Ile Leu Phe Tyr Glu Val Glu 325
330 335 Asn Asn Val Ile Arg Pro Pro His Leu
Phe Asp Ile Leu Ser Ser Val 340 345
350 Glu Ile Asn Thr Ser Arg Gly Gly Ile Thr Leu Asn Asn Asp
Ala Tyr 355 360 365
Ile Asn Tyr Trp Ser Gly His Thr Leu Lys Tyr Arg Arg Thr Ala Asp 370
375 380 Ser Thr Val Thr Tyr
Thr Ala Asn Tyr Gly Arg Ile Thr Ser Glu Lys 385 390
395 400 Asn Ser Phe Ala Leu Glu Asp Arg Asp Ile
Phe Glu Ile Asn Ser Thr 405 410
415 Val Ala Asn Leu Ala Asn Tyr Tyr Gln Lys Ala Tyr Gly Val Pro
Gly 420 425 430 Ser
Trp Phe His Met Val Lys Arg Gly Thr Ser Ser Thr Thr Ala Tyr 435
440 445 Leu Tyr Ser Lys Thr His
Thr Ala Leu Gln Gly Cys Thr Gln Val Tyr 450 455
460 Glu Ser Ser Asp Glu Ile Pro Leu Asp Arg Thr
Val Pro Val Ala Glu 465 470 475
480 Ser Tyr Ser His Arg Leu Ser His Ile Thr Ser His Ser Phe Ser Lys
485 490 495 Asn Gly
Ser Ala Tyr Tyr Gly Ser Phe Pro Val Phe Val Trp Thr His 500
505 510 Thr Ser Ala Asp Leu Asn Asn
Thr Ile Tyr Ser Asp Lys Ile Thr Gln 515 520
525 Ile Pro Ala Val Lys Gly Asp Met Leu Tyr Leu Gly
Gly Ser Val Val 530 535 540
Gln Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Lys Arg Thr Asn Pro 545
550 555 560 Ser Ile Leu
Gly Thr Phe Ala Val Thr Val Asn Gly Ser Leu Ser Gln 565
570 575 Arg Tyr Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr Asp Phe Glu Phe 580 585
590 Thr Leu Tyr Leu Gly Asp Thr Ile Glu Lys Asn Arg Phe
Asn Lys Thr 595 600 605
Met Asp Asn Gly Ala Ser Leu Thr Tyr Glu Thr Phe Lys Phe Ala Ser 610
615 620 Phe Ile Thr Asp
Phe Gln Phe Arg Glu Thr Gln Asp Lys Ile Leu Leu 625 630
635 640 Ser Met Gly Asp Phe Ser Ser Gly Gln
Glu Val Tyr Ile Asp Arg Ile 645 650
655 Glu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu Gln Asp
Leu Glu 660 665 670
Ala Ala Lys Lys Ala Val Asn Ala Leu Phe Thr Asn Thr Lys Asp Gly
675 680 685 Leu Arg Pro Gly
Val Thr Asp Tyr Glu Val Asn Gln Ala Ala Asn Leu 690
695 700 Val Glu Cys Leu Ser Asp Asp Leu
Tyr Pro Asn Glu Lys Arg Leu Leu 705 710
715 720 Phe Asp Ala Val Arg Glu Ala Lys Arg Leu Ser Gly
Ala Arg Asn Leu 725 730
735 Leu Gln Asp Pro Asp Phe Gln Glu Ile Asn Gly Glu Asn Gly Trp Ala
740 745 750 Ala Ser Thr
Gly Ile Glu Ile Val Glu Gly Asp Ala Val Phe Lys Gly 755
760 765 Arg Tyr Leu Arg Leu Pro Gly Ala
Arg Glu Ile Asp Thr Glu Thr Tyr 770 775
780 Pro Thr Tyr Leu Tyr Gln Lys Val Glu Glu Gly Val Leu
Lys Pro Tyr 785 790 795
800 Thr Arg Tyr Arg Leu Arg Gly Phe Val Gly Ser Ser Gln Gly Leu Glu
805 810 815 Ile Tyr Thr Ile
Arg His Gln Thr Asn Arg Ile Val Lys Asn Val Pro 820
825 830 Asp Asp Leu Leu Pro Asp Val Ser Pro
Val Asn Ser Asp Gly Ser Ile 835 840
845 Asn Arg Cys Ser Glu Gln Lys Tyr Val Asn Ser Arg Leu Glu
Gly Glu 850 855 860
Asn Arg Ser Gly Asp Ala His Glu Phe Ser Leu Pro Ile Asp Ile Gly 865
870 875 880 Glu Leu Asp Tyr Asn
Glu Asn Ala Gly Ile Trp Val Gly Phe Lys Ile 885
890 895 Thr Asp Pro Glu Gly Tyr Ala Thr Leu Gly
Asn Leu Glu Leu Val Glu 900 905
910 Glu Gly Pro Leu Ser Gly Asp Ala Leu Glu Arg Leu Gln Arg Glu
Glu 915 920 925 Gln
Gln Trp Lys Ile Gln Met Thr Arg Arg Arg Glu Glu Thr Asp Arg 930
935 940 Arg Tyr Met Ala Ser Lys
Gln Ala Val Asp Arg Leu Tyr Ala Asp Tyr 945 950
955 960 Gln Asp Gln Gln Leu Asn Pro Asp Val Glu Ile
Thr Asp Leu Thr Ala 965 970
975 Ala Gln Asp Leu Ile Gln Ser Ile Pro Tyr Val Tyr Asn Glu Met Phe
980 985 990 Pro Glu
Ile Pro Gly Met Asn Tyr Thr Lys Phe Thr Glu Leu Thr Asp 995
1000 1005 Arg Leu Gln Gln Ala
Trp Asn Leu Tyr Asp Gln Arg Asn Ala Ile 1010 1015
1020 Pro Asn Gly Asp Phe Arg Asn Gly Leu Ser
Asn Trp Asn Ala Thr 1025 1030 1035
Pro Gly Val Glu Val Gln Gln Ile Asn His Thr Ser Val Leu Val
1040 1045 1050 Ile Pro
Asn Trp Asp Glu Gln Val Ser Gln Gln Phe Thr Val Gln 1055
1060 1065 Pro Asn Gln Arg Tyr Val Leu
Arg Val Thr Ala Arg Lys Glu Gly 1070 1075
1080 Val Gly Asn Gly Tyr Val Ser Ile Arg Asp Gly Gly
Asn Gln Ser 1085 1090 1095
Glu Thr Leu Thr Phe Ser Ala Ser Asp Tyr Asp Thr Asn Gly Val 1100
1105 1110 Tyr Asn Asp Gln Thr
Gly Tyr Ile Thr Lys Thr Val Thr Phe Ile 1115 1120
1125 Pro Tyr Thr Asp Gln Met Trp Ile Glu Ile
Ser Glu Thr Glu Gly 1130 1135 1140
Thr Phe Tyr Ile Glu Ser Val Glu Leu Ile Val Asp Val 1145
1150 1155 793537DNABacillus
thuringiensismisc_feature(1)..(3537)cry1Ac coding sequence 79atggataaca
atccgaacat caatgaatgc attccttata attgtttaag taaccctgaa 60gtagaagtat
taggtggaga aagaatagaa actggttaca ccccaatcga tatttccttg 120tcgctaacgc
aatttctttt gagtgaattt gttcccggtg ctggatttgt gttaggacta 180gttgatataa
tatggggaat ttttggtccc tctcaatggg acgcatttct tgtacaaatt 240gaacagttaa
ttaaccaaag aatagaagaa ttcgctagga accaagccat ttctagatta 300gaaggactaa
gcaatcttta tcaaatttac gcagaatctt ttagagagtg ggaagcagat 360cctactaatc
cagcattaag agaagagatg cgtattcaat tcaatgacat gaacagtgcc 420cttacaaccg
ctattcctct ttttgcagtt caaaattatc aagttcctct tttatcagta 480tatgttcaag
ctgcaaattt acatttatca gttttgagag atgtttcagt gtttggacaa 540aggtggggat
ttgatgccgc gactatcaat agtcgttata atgatttaac taggcttatt 600ggcaactata
cagattatgc tgtacgctgg tacaatacgg gattagaacg tgtatgggga 660ccggattcta
gagattgggt aaggtataat caatttagaa gagaattaac actaactgta 720ttagatatcg
ttgctctgtt cccgaattat gatagtagaa gatatccaat tcgaacagtt 780tcccaattaa
caagagaaat ttatacaaac ccagtattag aaaattttga tggtagtttt 840cgaggctcgg
ctcagggcat agaaagaagt attaggagtc cacatttgat ggatatactt 900aacagtataa
ccatctatac ggatgctcat aggggttatt attattggtc agggcatcaa 960ataatggctt
ctcctgtagg gttttcgggg ccagaattca cttttccgct atatggaact 1020atgggaaatg
cagctccaca acaacgtatt gttgctcaac taggtcaggg cgtgtataga 1080acattatcgt
ccactttata tagaagacct tttaatatag ggataaataa tcaacaacta 1140tctgttcttg
acgggacaga atttgcttat ggaacctcct caaatttgcc atccgctgta 1200tacagaaaaa
gcggaacggt agattcgctg gatgaaatac cgccacagaa taacaacgtg 1260ccacctaggc
aaggatttag tcatcgatta agccatgttt caatgtttcg ttcaggcttt 1320agtaatagta
gtgtaagtat aataagagct cctatgttct cttggataca tcgtagtgct 1380gaatttaata
atataattgc atcggatagt attactcaaa tccctgcagt gaagggaaac 1440tttcttttta
atggttctgt aatttcagga ccaggattta ctggtgggga cttagttaga 1500ttaaatagta
gtggaaataa cattcagaat agagggtata ttgaagttcc aattcacttc 1560ccatcgacat
ctaccagata tcgagttcgt gtacggtatg cttctgtaac cccgattcac 1620ctcaacgtta
attggggtaa ttcatccatt ttttccaata cagtaccagc tacagctacg 1680tcattagata
atctacaatc aagtgatttt ggttattttg aaagtgccaa tgcttttaca 1740tcttcattag
gtaatatagt aggtgttaga aattttagtg ggactgcagg agtgataata 1800gacagatttg
aatttattcc agttactgca acactcgagg ctgaatataa tctggaaaga 1860gcgcagaagg
cggtgaatgc gctgtttacg tctacaaacc aactagggct aaaaacaaat 1920gtaacggatt
atcatattga tcaagtgtcc aatttagtta cgtatttatc ggatgaattt 1980tgtctggatg
aaaagcgaga attgtccgag aaagtcaaac atgcgaagcg actcagtgat 2040gaacgcaatt
tactccaaga ttcaaatttc aaagacatta ataggcaacc agaacgtggg 2100tggggcggaa
gtacagggat taccatccaa ggaggggatg acgtatttaa agaaaattac 2160gtcacactat
caggtacctt tgatgagtgc tatccaacat atttgtatca aaaaatcgat 2220gaatcaaaat
taaaagcctt tacccgttat caattaagag ggtatatcga agatagtcaa 2280gacttagaaa
tctatttaat tcgctacaat gcaaaacatg aaacagtaaa tgtgccaggt 2340acgggttcct
tatggccgct ttcagcccaa agtccaatcg gaaagtgtgg agagccgaat 2400cgatgcgcgc
cacaccttga atggaatcct gacttagatt gttcgtgtag ggatggagaa 2460aagtgtgccc
atcattcgca tcatttctcc ttagacattg atgtaggatg tacagactta 2520aatgaggacc
taggtgtatg ggtgatcttt aagattaaga cgcaagatgg gcacgcaaga 2580ctagggaatc
tagagtttct cgaagagaaa ccattagtag gagaagcgct agctcgtgtg 2640aaaagagcgg
agaaaaaatg gagagacaaa cgtgaaaaat tggaatggga aacaaatatc 2700gtttataaag
aggcaaaaga atctgtagat gctttatttg taaactctca atatgatcaa 2760ttacaagcgg
atacgaatat tgccatgatt catgcggcag ataaacgtgt tcatagcatt 2820cgagaagctt
atctgcctga gctgtctgtg attccgggtg tcaatgcggc tatttttgaa 2880gaattagaag
ggcgtatttt cactgcattc tccctatatg atgcgagaaa tgtcattaaa 2940aatggtgatt
ttaataatgg cttatcctgc tggaacgtga aagggcatgt agatgtagaa 3000gaacaaaaca
accaacgttc ggtccttgtt gttccggaat gggaagcaga agtgtcacaa 3060gaagttcgtg
tctgtccggg tcgtggctat atccttcgtg tcacagcgta caaggaggga 3120tatggagaag
gttgcgtaac cattcatgag atcgagaaca atacagacga actgaagttt 3180agcaactgcg
tagaagagga aatctatcca aataacacgg taacgtgtaa tgattatact 3240gtaaatcaag
aagaatacgg aggtgcgtac acttctcgta atcgaggata taacgaagct 3300ccttccgtac
cagctgatta tgcgtcagtc tatgaagaaa aatcgtatac agatggacga 3360agagagaatc
cttgtgaatt taacagaggg tatagggatt acacgccact accagttggt 3420tatgtgacaa
aagaattaga atacttccca gaaaccgata aggtatggat tgagattgga 3480gaaacggaag
gaacatttat cgtggacagc gtggaattac tccttatgga ggaatag
3537801178PRTBacillus thuringiensisMISC_FEATURE(1)..(1178)Cry1Ac protein
80Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1
5 10 15 Ser Asn Pro Glu
Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20
25 30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser
Leu Thr Gln Phe Leu Leu Ser 35 40
45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp
Ile Ile 50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65
70 75 80 Glu Gln Leu Ile Asn
Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85
90 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu
Tyr Gln Ile Tyr Ala Glu 100 105
110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg
Glu 115 120 125 Glu
Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130
135 140 Ile Pro Leu Phe Ala Val
Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150
155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val
Leu Arg Asp Val Ser 165 170
175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190 Tyr Asn
Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195
200 205 Arg Trp Tyr Asn Thr Gly Leu
Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215
220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu
Thr Leu Thr Val 225 230 235
240 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro
245 250 255 Ile Arg Thr
Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260
265 270 Leu Glu Asn Phe Asp Gly Ser Phe
Arg Gly Ser Ala Gln Gly Ile Glu 275 280
285 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn
Ser Ile Thr 290 295 300
Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305
310 315 320 Ile Met Ala Ser
Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325
330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala
Pro Gln Gln Arg Ile Val Ala 340 345
350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu
Tyr Arg 355 360 365
Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370
375 380 Gly Thr Glu Phe Ala
Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390
395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu
Asp Glu Ile Pro Pro Gln 405 410
415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser
His 420 425 430 Val
Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435
440 445 Arg Ala Pro Met Phe Ser
Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450 455
460 Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro
Ala Val Lys Gly Asn 465 470 475
480 Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly
485 490 495 Asp Leu
Val Arg Leu Asn Ser Ser Gly Asn Asn Ile Gln Asn Arg Gly 500
505 510 Tyr Ile Glu Val Pro Ile His
Phe Pro Ser Thr Ser Thr Arg Tyr Arg 515 520
525 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His
Leu Asn Val Asn 530 535 540
Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr 545
550 555 560 Ser Leu Asp
Asn Leu Gln Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala 565
570 575 Asn Ala Phe Thr Ser Ser Leu Gly
Asn Ile Val Gly Val Arg Asn Phe 580 585
590 Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu Phe
Ile Pro Val 595 600 605
Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala 610
615 620 Val Asn Ala Leu
Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn 625 630
635 640 Val Thr Asp Tyr His Ile Asp Gln Val
Ser Asn Leu Val Thr Tyr Leu 645 650
655 Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu
Lys Val 660 665 670
Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser
675 680 685 Asn Phe Lys Asp
Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser 690
695 700 Thr Gly Ile Thr Ile Gln Gly Gly
Asp Asp Val Phe Lys Glu Asn Tyr 705 710
715 720 Val Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro
Thr Tyr Leu Tyr 725 730
735 Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu
740 745 750 Arg Gly Tyr
Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg 755
760 765 Tyr Asn Ala Lys His Glu Thr Val
Asn Val Pro Gly Thr Gly Ser Leu 770 775
780 Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly
Glu Pro Asn 785 790 795
800 Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys
805 810 815 Arg Asp Gly Glu
Lys Cys Ala His His Ser His His Phe Ser Leu Asp 820
825 830 Ile Asp Val Gly Cys Thr Asp Leu Asn
Glu Asp Leu Gly Val Trp Val 835 840
845 Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly
Asn Leu 850 855 860
Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val 865
870 875 880 Lys Arg Ala Glu Lys
Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp 885
890 895 Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys
Glu Ser Val Asp Ala Leu 900 905
910 Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile
Ala 915 920 925 Met
Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr 930
935 940 Leu Pro Glu Leu Ser Val
Ile Pro Gly Val Asn Ala Ala Ile Phe Glu 945 950
955 960 Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser
Leu Tyr Asp Ala Arg 965 970
975 Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn
980 985 990 Val Lys
Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val 995
1000 1005 Leu Val Val Pro Glu
Trp Glu Ala Glu Val Ser Gln Glu Val Arg 1010 1015
1020 Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg
Val Thr Ala Tyr Lys 1025 1030 1035
Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn
1040 1045 1050 Asn Thr
Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile 1055
1060 1065 Tyr Pro Asn Asn Thr Val Thr
Cys Asn Asp Tyr Thr Val Asn Gln 1070 1075
1080 Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg
Gly Tyr Asn 1085 1090 1095
Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu 1100
1105 1110 Lys Ser Tyr Thr Asp
Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn 1115 1120
1125 Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro
Val Gly Tyr Val Thr 1130 1135 1140
Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu
1145 1150 1155 Ile Gly
Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu 1160
1165 1170 Leu Leu Met Glu Glu 1175
812160DNABacillus thuringiensismisc_feature(1)..(2160)cry1Ia
coding sequence 81atgaaactaa agaatcaaga taagcatcaa agtttttcta gcaatgcgaa
agtagataaa 60atctctacgg attcactaaa aaatgaaaca gatatagaat tacaaaacat
taatcatgaa 120gattgtttga aaatgtctga gtatgaaaat gtagagccgt ttgttagtgc
atcaacaatt 180caaacaggta ttggtattgc gggtaaaata cttggtaccc taggcgttcc
ttttgcagga 240caagtagcta gtctttatag ttttatctta ggtgagctat ggcctaaggg
gaaaaatcaa 300tgggaaatct ttatggaaca tgtagaagag attattaatc aaaaaatatc
aacttatgca 360agaaataaag cacttacaga cttgaaagga ttaggagatg ccttagctgt
ctaccatgat 420tcgcttgaaa gttgggttgg aaatcgtaat aacacaaggg ctaggagtgt
tgtcaagagc 480caatatatcg cattagaatt gatgttcgtt cagaaactac cttcttttgc
agtgtctgga 540gaggaggtac cattattacc gatatatgcc caagctgcaa atttacattt
gttgctatta 600agagatgcat ctatttttgg aaaagagtgg ggattatcat cttcagaaat
ttcaacattt 660tataaccgtc aagtcgaacg agcaggagat tattcctacc attgtgtgaa
atggtatagc 720acaggtctaa ataacttgag gggtacaaat gccgaaagtt gggtacgata
taatcaattc 780cgtagagaca tgactttaat ggtactagat ttagtggcac tatttccaag
ctatgataca 840caaatgtatc caattaaaac tacagcccaa cttacaagag aagtatatac
agacgcaatt 900gggacagtac atccgcatcc aagttttaca agtacgactt ggtataataa
taatgcacct 960tcgttctctg ccatagaggc tgctgttgtt cgaaacccgc atctactcga
ttttctagaa 1020caagttacaa tttacagctt attaagtcga tggagtaaca ctcagtatat
gaatatgtgg 1080ggaggacata aactagaatt ccgaacaata ggaggaacgt taaatatctc
aacacaagga 1140tctactaata cttctattaa tcctgtaaca ttaccgttca cttctcgaga
cgtctatagg 1200actgaatcat tggcagggct gaatctattt ttaactcaac ctgttaatgg
agtacctagg 1260gttgattttc attggaaatt cgtcacacat ccgatcgcat ctgataattt
ctattatcca 1320gggtatgctg gaattgggac gcaattacag gattcagaaa atgaattacc
acctgaagca 1380acaggacagc caaattatga atcttatagt catagattat ctcatatagg
actcatttca 1440gcatcacatg tgaaagcatt ggtatattct tggacgcatc gtagtgcaga
tcgtacaaat 1500acaattgagc caaatagcat tacacaaata ccattagtaa aagctttcaa
tctgtcttca 1560ggtgccgctg tagtgagagg accaggattt acaggtgggg atatccttcg
aagaacgaat 1620actggtacat ttggggatat acgagtaaat attaatccac catttgcaca
aagatatcgc 1680gtgaggattc gctatgcttc taccacagat ttacaattcc atacgtcaat
taacggtaaa 1740gctattaatc aaggtaattt ttcagcaact atgaatagag gagaggactt
agactataaa 1800acctttagaa ctgtaggctt taccactcca tttagctttt tagatgtaca
aagtacattc 1860acaataggtg cttggaactt ctcttcaggt aacgaagttt atatagatag
aattgaattt 1920gttccggtag aagtaacata tgaggcagaa tatgattttg aaaaagcgca
agagaaggtt 1980actgcactgt ttacatctac gaatccaaga ggattaaaaa cagatgtaaa
ggattatcat 2040attgaccagg tatcaaattt agtagagtct ctatcagatg aattctatct
tgatgaaaag 2100agagaattat tcgagatagt taaatacgcg aagcaactcc atattgagcg
taacatgtag 216082719PRTBacillus
thuringiensisMISC_FEATURE(1)..(719)Cry1Ia protein 82Met Lys Leu Lys Asn
Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala 1 5
10 15 Lys Val Asp Lys Ile Ser Thr Asp Ser Leu
Lys Asn Glu Thr Asp Ile 20 25
30 Glu Leu Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met Ser Glu
Tyr 35 40 45 Glu
Asn Val Glu Pro Phe Val Ser Ala Ser Thr Ile Gln Thr Gly Ile 50
55 60 Gly Ile Ala Gly Lys Ile
Leu Gly Thr Leu Gly Val Pro Phe Ala Gly 65 70
75 80 Gln Val Ala Ser Leu Tyr Ser Phe Ile Leu Gly
Glu Leu Trp Pro Lys 85 90
95 Gly Lys Asn Gln Trp Glu Ile Phe Met Glu His Val Glu Glu Ile Ile
100 105 110 Asn Gln
Lys Ile Ser Thr Tyr Ala Arg Asn Lys Ala Leu Thr Asp Leu 115
120 125 Lys Gly Leu Gly Asp Ala Leu
Ala Val Tyr His Asp Ser Leu Glu Ser 130 135
140 Trp Val Gly Asn Arg Asn Asn Thr Arg Ala Arg Ser
Val Val Lys Ser 145 150 155
160 Gln Tyr Ile Ala Leu Glu Leu Met Phe Val Gln Lys Leu Pro Ser Phe
165 170 175 Ala Val Ser
Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala Gln Ala 180
185 190 Ala Asn Leu His Leu Leu Leu Leu
Arg Asp Ala Ser Ile Phe Gly Lys 195 200
205 Glu Trp Gly Leu Ser Ser Ser Glu Ile Ser Thr Phe Tyr
Asn Arg Gln 210 215 220
Val Glu Arg Ala Gly Asp Tyr Ser Tyr His Cys Val Lys Trp Tyr Ser 225
230 235 240 Thr Gly Leu Asn
Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Val Arg 245
250 255 Tyr Asn Gln Phe Arg Arg Asp Met Thr
Leu Met Val Leu Asp Leu Val 260 265
270 Ala Leu Phe Pro Ser Tyr Asp Thr Gln Met Tyr Pro Ile Lys
Thr Thr 275 280 285
Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Thr Val His 290
295 300 Pro His Pro Ser Phe
Thr Ser Thr Thr Trp Tyr Asn Asn Asn Ala Pro 305 310
315 320 Ser Phe Ser Ala Ile Glu Ala Ala Val Val
Arg Asn Pro His Leu Leu 325 330
335 Asp Phe Leu Glu Gln Val Thr Ile Tyr Ser Leu Leu Ser Arg Trp
Ser 340 345 350 Asn
Thr Gln Tyr Met Asn Met Trp Gly Gly His Lys Leu Glu Phe Arg 355
360 365 Thr Ile Gly Gly Thr Leu
Asn Ile Ser Thr Gln Gly Ser Thr Asn Thr 370 375
380 Ser Ile Asn Pro Val Thr Leu Pro Phe Thr Ser
Arg Asp Val Tyr Arg 385 390 395
400 Thr Glu Ser Leu Ala Gly Leu Asn Leu Phe Leu Thr Gln Pro Val Asn
405 410 415 Gly Val
Pro Arg Val Asp Phe His Trp Lys Phe Val Thr His Pro Ile 420
425 430 Ala Ser Asp Asn Phe Tyr Tyr
Pro Gly Tyr Ala Gly Ile Gly Thr Gln 435 440
445 Leu Gln Asp Ser Glu Asn Glu Leu Pro Pro Glu Ala
Thr Gly Gln Pro 450 455 460
Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly Leu Ile Ser 465
470 475 480 Ala Ser His
Val Lys Ala Leu Val Tyr Ser Trp Thr His Arg Ser Ala 485
490 495 Asp Arg Thr Asn Thr Ile Glu Pro
Asn Ser Ile Thr Gln Ile Pro Leu 500 505
510 Val Lys Ala Phe Asn Leu Ser Ser Gly Ala Ala Val Val
Arg Gly Pro 515 520 525
Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 530
535 540 Gly Asp Ile Arg
Val Asn Ile Asn Pro Pro Phe Ala Gln Arg Tyr Arg 545 550
555 560 Val Arg Ile Arg Tyr Ala Ser Thr Thr
Asp Leu Gln Phe His Thr Ser 565 570
575 Ile Asn Gly Lys Ala Ile Asn Gln Gly Asn Phe Ser Ala Thr
Met Asn 580 585 590
Arg Gly Glu Asp Leu Asp Tyr Lys Thr Phe Arg Thr Val Gly Phe Thr
595 600 605 Thr Pro Phe Ser
Phe Leu Asp Val Gln Ser Thr Phe Thr Ile Gly Ala 610
615 620 Trp Asn Phe Ser Ser Gly Asn Glu
Val Tyr Ile Asp Arg Ile Glu Phe 625 630
635 640 Val Pro Val Glu Val Thr Tyr Glu Ala Glu Tyr Asp
Phe Glu Lys Ala 645 650
655 Gln Glu Lys Val Thr Ala Leu Phe Thr Ser Thr Asn Pro Arg Gly Leu
660 665 670 Lys Thr Asp
Val Lys Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val 675
680 685 Glu Ser Leu Ser Asp Glu Phe Tyr
Leu Asp Glu Lys Arg Glu Leu Phe 690 695
700 Glu Ile Val Lys Tyr Ala Lys Gln Leu His Ile Glu Arg
Asn Met 705 710 715
8334DNAArtificial Sequence53A-1-bam 83ccggatccat gacggccgac aacaacaccg
aggc 348420DNAArtificial SequenceC3-3a-6
primer 84caggggcagc tgggtgatct
208520DNAArtificial SequenceC3-1Ab-3 primer 85agatcaccca gatccccctg
208639DNAArtificial
Sequence1Ab-6-sac primer 86ccgagctcag ctcctacacc tgatcgatgt ggtagtcgg
398756DNAArtificial Sequence8a-atg-delri primer
87ccggatccac catgactagt aacggccgcc agtgtgctgg tattcgccct tatgac
568820DNAArtificial SequenceC2-3A-4 primer 88gtccagcacg gtcagggtca
208920DNAArtificial
Sequencereverse primer 89gcgtgcagtc aagtcagatc
209038DNAArtificial SequenceFR8a-OL-1 primer
90ggtgttgttg tcggccgtca tagggcgaat accagcac
389139DNAArtificial SequenceFR8a-OL-2 primer 91gccgacaaca acaccgaggc
cctggacagc agcaccacc 399227DNAArtificial
SequenceC1-3a-2 primer 92caggtgggtg ttggcggcct gggcgta
279321DNAArtificial Sequence5'FR8a primer
93ggatccacca tgactagtaa c
219436DNAArtificial Sequence5'fr8a-12aa primer 94ccggatccac catgtatgac
ggccgacaac aacacc 369520DNAArtificial
SequenceC2-3A-3 primer 95tgaccctgac cgtgctggac
209627DNAArtificial Sequence3'1Ab-dm3 primer
96gagctcctag gtcacctcgg cgggcac
279732DNAArtificial Sequence5'FR-del6 primer 97ggatccacca tgtgtgctgg
tattcgccct at 329832DNAArtificial
Sequence5'1Ab-bam primer 98ccggatccat ggacaacaac cccaacatca ac
329920DNAArtificial SequenceC3-3a-7 primer
99gcttcaccgg cggcgacatc
2010020DNAArtificial SequenceC3-3a-8 primer 100gatgtcgccg ccggtgaagc
2010123DNAArtificial
SequenceC4-3a-9 primer 101ccgcatccac tacgccagca cca
2310223DNAArtificial SequenceC4-3a-10 primer
102tggtgctggc gtagtggatg cgg
2310344DNAArtificial Sequence3a-12-sac primer 103ccgagctcag ctcagatcta
gttcacgggg atgaactcga tctt 4410427DNAArtificial
Sequence3a-22 primer 104ggccttcacc aggggcagct gggtgat
2710531DNAArtificial Sequence1B-5 primer
105ccgccgcgac ctgaccctgg gcgtgctgga c
3110627DNAArtificial Sequence1B-7 primer 106atcacccaga tccccatggt gaaggcc
2710726DNAArtificial
Sequence1B-10 primer 107ccgagctcct agaacagggc gttcac
2610820DNAArtificial SequenceC3-1Ab-2 primer
108cagggggatc tgggtgatct
2010920DNAArtificial SequenceC3-3A-5 primer 109agatcaccca gctgcccctg
2011027DNAArtificial
SequenceC1-1Ab-1 primer 110tacgtgcagg ccgccaacct gcacctg
2711142DNAArtificial Sequence5'8Aa-dm3 primer
111agatcaccca gctgcccctg gtaaagggag acatgttata tc
4211230DNAArtificial Sequence3'8Aa-dm3 primer 112gagctcctat gtctcatcta
ctgggatgaa 3011333DNAArtificial
SequenceTant-OL-1 primer 113acccagctgc ccctggtgaa ggcccacacc ctc
3311433DNAArtificial SequenceTant-OL-2 primer
114gagggtgtgg gccttcacca ggggcagctg ggt
3311529DNAArtificial SequenceTant-3'sac primer 115gagctctagc ttaagcagtc
cacgaggtt 2911637DNAArtificial
Sequence1Ac-OL-1 primer 116acccagctgc ccctggtgaa gggaaacttt cttttta
3711737DNAArtificial Sequence1Ac-OL-2 primer
117taaaaagaaa gtttcccttc accaggggca gctgggt
3711830DNAArtificial Sequnce1Ac-3'sac primer 118gagctcctat gttgcagtaa
ctggaataaa 3011938DNAArtificial
Sequence1Ia-OL-1 primer 119acccagctgc ccctgagtaa aagctttcaa tctgtctt
3812038DNAArtificial Sequence1Ia-OL-2 primer
120aagacagatt gaaagctttt actcaggggc agctgggt
3812131DNAArtificial Sequence1Ia-3'sac primer 121gagctcctac atgttacgct
caatatggag t 3112233DNAArtificial
SequenceFR-1Ab-1 primer 122tggacccaca agagcgccga gttcaacaac atc
3312333DNAArtificial SequenceFR-1Ab-2 primer
123gatgttgttg aactcggcgc tcttgtgggt cca
3312440DNAArtificial SequenceFR-1Ab-3 primer 124ccacaagagc gtcgacttca
acacatcatc cccagcagcc 4012541DNAArtificial
SequenceFR-1Ab-4 primer 125ggctcgtggg gatgatgttg ttgaagtcga cgctcttgtg g
4112635PRTArtificial SequencePeptidyl fragment 1
126Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Thr Ser 1
5 10 15 Asn Gly Arg Gln
Cys Ala Gly Ile Arg Pro Tyr Asp Gly Arg Gln Gln 20
25 30 His Arg Gly 35
12722PRTArtificial SequencePeptidyl Fragment 2 127Met Thr Ser Asn Gly Arg
Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly 20
12810PRTArtificial SequencePeptidyl Fragment 3 128Met Tyr Asp Gly Arg
Gln Gln His Arg Gly 1 5 10
12913PRTArtificial SequencePeptidyl Fragment 4 129Met Thr Ser Asn Gly Arg
Gln Cys Ala Gly Ile Arg Pro 1 5 10
1307PRTArtificial SequencePeptidyl fragment 5 130Met Cys Ala Gly
Ile Arg Pro 1 5 13155PRTArtificial
SequencePeptidyl fragment 6 131Met Lys Glu Thr Ala Ala Ala Lys Phe Glu
Arg Gln His Met Asp Ser 1 5 10
15 Pro Asp Leu Gly Thr Leu Val Pro Arg Gly Ser Met Ala Asp Ile
Gly 20 25 30 Ser
Thr Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp 35
40 45 Gly Arg Gln Gln His Arg
Gly 50 55 13214PRTArtificial SequenceChemically
synthesized peptidyl fragment 7 132Met Ala Ser Met Thr Gly Gly Gln Gln
Met Gly Arg Gly Ser 1 5 10
1339PRTArtificial SequencePeptidyl fragment 8 133Tyr Asp Gly Arg Gln
Gln His Arg Gly 1 5 13412PRTArtificial
SequencePeptidyl fragment 9 134Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile
Arg Pro 1 5 10 135644PRTBacillus
thuringiensisFull-length Cry3A protein 135Met Asn Pro Asn Asn Arg Ser Glu
His Asp Thr Ile Lys Thr Thr Glu 1 5 10
15 Asn Asn Glu Val Pro Thr Asn His Val Gln Tyr Pro Leu
Ala Glu Thr 20 25 30
Pro Asn Pro Thr Leu Glu Asp Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45 Thr Ala Asp Asn
Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys Asp 50
55 60 Val Ile Gln Lys Gly Ile Ser Val
Val Gly Asp Leu Leu Gly Val Val 65 70
75 80 Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr
Thr Asn Phe Leu 85 90
95 Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln
100 105 110 Val Glu Ala
Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys 115
120 125 Ala Leu Ala Glu Leu Gln Gly Leu
Gln Asn Asn Val Glu Asp Tyr Val 130 135
140 Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser
Arg Asn Pro 145 150 155
160 His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
165 170 175 Phe Arg Asn Ser
Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu 180
185 190 Phe Leu Thr Thr Tyr Ala Gln Ala Ala
Asn Thr His Leu Phe Leu Leu 195 200
205 Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
Glu Asp 210 215 220
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr 225
230 235 240 Asp His Cys Val Lys
Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly 245
250 255 Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn
Arg Tyr Arg Arg Glu Met 260 265
270 Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp
Val 275 280 285 Arg
Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu 290
295 300 Thr Asp Pro Ile Val Gly
Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr 305 310
315 320 Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro
His Leu Phe Asp Tyr 325 330
335 Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly
340 345 350 Asn Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro 355
360 365 Ser Ile Gly Ser Asn Asp Ile
Ile Thr Ser Pro Phe Tyr Gly Asn Lys 370 375
380 Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly
Glu Lys Val Tyr 385 390 395
400 Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr
405 410 415 Ser Gly Val
Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp 420
425 430 Glu Ala Ser Thr Gln Thr Tyr Asp
Ser Lys Arg Asn Val Gly Ala Val 435 440
445 Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr
Asp Glu Pro 450 455 460
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu 465
470 475 480 Met Gln Gly Ser
Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys 485
490 495 Ser Val Asp Phe Phe Asn Met Ile Asp
Ser Lys Lys Ile Thr Gln Leu 500 505
510 Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val
Val Ala 515 520 525
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly 530
535 540 Ser Ala Ala Thr Ile
Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys 545 550
555 560 Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr
Ser Gln Ile Thr Phe Thr 565 570
575 Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys
Thr 580 585 590 Ile
Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser 595
600 605 Phe Ser Thr Pro Phe Glu
Leu Ser Gly Asn Asn Leu Gln Ile Gly Val 610 615
620 Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile
Asp Lys Ile Glu Phe 625 630 635
640 Ile Pro Val Asn 13631DNAArtificial SequenceChemically
synthesized CMS94 primer 136ggcgcgccac catggctagc atgactggtg g
3113720DNAArtificial SequenceChemically
synthesized CMS95 primer 137gcaggaacag gtgggtgttg
2013820DNAArtificial sequenceChemically
synthesized CMS96 primer 138cctgaacacc atctggccca
2013939DNAArtificial SequenceChemically
synthesized CMS97 primer 139ctggctgctg gggatgatgt tgttgaagtc gacgctctt
3914021DNAArtificial SequenceChemically
synthesized CMS98 primer 140gagctcttag gtcacctcgg c
2114139DNAArtificial SequenceChemically
synthesized CMS99 primer 141aagagcgtcg acttcaacaa catcatcccc agcagccag
3914240DNAArtificial SequenceChemically
synthesized CMS100 primer 142gaagtaccgc gcccgcatcc gctacgccag caccaccaac
4014340DNAArtificial SequenceChemically
synthesized CMS101 primer 143gttggtggtg ctggcgtagc ggatgcgggc gcggtacttc
401441966DNAArtificial SequenceT7-8AF coding
sequence 144atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgacggc
cgacaacaac 60accgaggccc tggacagcag caccaccaag gacgtgatcc agaagggcat
cagcgtggtg 120ggcgacctgc tgggcgtggt gggcttcccc ttcggcggcg ccctggtgag
cttctacacc 180aacttcctga acaccatctg gcccagcgag gacccctgga aggccttcat
ggagcaggtg 240gaggccctga tggaccagaa gatcgccgac tacgccaaga acaaggcact
ggccgagcta 300cagggcctcc agaacaacgt ggaggactat gtgagcgccc tgagcagctg
gcagaagaac 360cccgctgcac cgttccgcaa cccccacagc cagggccgca tccgcgagct
gttcagccag 420gccgagagcc acttccgcaa cagcatgccc agcttcgcca tcagcggcta
cgaggtgctg 480ttcctgacca cctacgccca ggccgccaac acccacctgt tcctgctgaa
ggacgcccaa 540atctacggag aggagtgggg ctacgagaag gaggacatcg ccgagttcta
caagcgccag 600ctgaagctga cccaggagta caccgaccac tgcgtgaagt ggtacaacgt
gggtctagac 660aagctccgcg gcagcagcta cgagagctgg gtgaacttca accgctaccg
ccgcgagatg 720accctgaccg tgctggacct gatcgccctg ttccccctgt acgacgtgcg
cctgtacccc 780aaggaggtga agaccgagct gacccgcgac gtgctgaccg accccatcgt
gggcgtgaac 840aacctgcgcg gctacggcac caccttcagc aacatcgaga actacatccg
caagccccac 900ctgttcgact acctgcaccg catccagttc cacacgcgtt tccagcccgg
ctactacggc 960aacgacagct tcaactactg gagcggcaac tacgtgagca cccgccccag
catcggcagc 1020aacgacatca tcaccagccc cttctacggc aacaagagca gcgagcccgt
gcagaacctt 1080gagttcaacg gcgagaaggt gtaccgcgcc gtggctaaca ccaacctggc
cgtgtggccc 1140tctgcagtgt acagcggcgt gaccaaggtg gagttcagcc agtacaacga
ccagaccgac 1200gaggccagca cccagaccta cgacagcaag cgcaacgtgg gcgccgtgag
ctgggacagc 1260atcgaccagc tgccccccga gaccaccgac gagcccctgg agaagggcta
cagccaccag 1320ctgaactacg tgatgtgctt cctgatgcag ggcagccgcg gcaccatccc
cgtgctgacc 1380tggacccaca agagcgtcga cttcttcaac atgatcgaca gcaagaagat
cacccagctg 1440cccctgacca agagcaccaa cctgggcagc ggcaccagcg tggtgaaggg
ccccggcttc 1500accggcggcg acatcctgcg ccgcaccagc cccggccaga tcagcaccct
gcgcgtgaac 1560atcaccgccc ccctgagcca gcgctaccgc gtccgcatcc gctacgccag
caccaccaac 1620ctgcagttcc acaccagcat cgacggccgc cccatcaacc agggcaactt
cagcgccacc 1680atgagcagcg gcagcaacct gcagagcggc agcttccgca ccgtgggctt
caccaccccc 1740ttcaacttca gcaacggcag cagcgtgttc accctgagcg cccacgtgtt
caacagcggc 1800aacgaggtgt acatcgaccg catcgagttc gtgcccgccg aggtgacctt
cgaggccgag 1860tacgacctgg agagggctca gaaggccgtg aacgagctgt tcaccagcag
caaccagatc 1920ggcctgaaga ccgacgtgac cgactaccac atcgatcagg tgtagg
1966145654PRTArtificial SequenceT7-8AF protein 145Met Ala Ser
Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Thr 1 5
10 15 Ala Asp Asn Asn Thr Glu Ala Leu
Asp Ser Ser Thr Thr Lys Asp Val 20 25
30 Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly
Val Val Gly 35 40 45
Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn 50
55 60 Thr Ile Trp Pro
Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln Val 65 70
75 80 Glu Ala Leu Met Asp Gln Lys Ile Ala
Asp Tyr Ala Lys Asn Lys Ala 85 90
95 Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
Val Ser 100 105 110
Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro
115 120 125 His Ser Gln Gly
Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His 130
135 140 Phe Arg Asn Ser Met Pro Ser Phe
Ala Ile Ser Gly Tyr Glu Val Leu 145 150
155 160 Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His
Leu Phe Leu Leu 165 170
175 Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp
180 185 190 Ile Ala Glu
Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr 195
200 205 Asp His Cys Val Lys Trp Tyr Asn
Val Gly Leu Asp Lys Leu Arg Gly 210 215
220 Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg
Arg Glu Met 225 230 235
240 Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val
245 250 255 Arg Leu Tyr Pro
Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu 260
265 270 Thr Asp Pro Ile Val Gly Val Asn Asn
Leu Arg Gly Tyr Gly Thr Thr 275 280
285 Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe
Asp Tyr 290 295 300
Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly 305
310 315 320 Asn Asp Ser Phe Asn
Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro 325
330 335 Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser
Pro Phe Tyr Gly Asn Lys 340 345
350 Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val
Tyr 355 360 365 Arg
Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr 370
375 380 Ser Gly Val Thr Lys Val
Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp 385 390
395 400 Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg
Asn Val Gly Ala Val 405 410
415 Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
420 425 430 Leu Glu
Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu 435
440 445 Met Gln Gly Ser Arg Gly Thr
Ile Pro Val Leu Thr Trp Thr His Lys 450 455
460 Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys
Ile Thr Gln Leu 465 470 475
480 Pro Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys
485 490 495 Gly Pro Gly
Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly 500
505 510 Gln Ile Ser Thr Leu Arg Val Asn
Ile Thr Ala Pro Leu Ser Gln Arg 515 520
525 Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu
Gln Phe His 530 535 540
Thr Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr 545
550 555 560 Met Ser Ser Gly
Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly 565
570 575 Phe Thr Thr Pro Phe Asn Phe Ser Asn
Gly Ser Ser Val Phe Thr Leu 580 585
590 Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp
Arg Ile 595 600 605
Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu 610
615 620 Arg Ala Gln Lys Ala
Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile 625 630
635 640 Gly Leu Lys Thr Asp Val Thr Asp Tyr His
Ile Asp Gln Val 645 650
1461920DNAArtificial Sequence-catG8AF coding sequence 146atgacggccg
acaacaacac cgaggccctg gacagcagca ccaccaagga cgtgatccag 60aagggcatca
gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt cggcggcgcc 120ctggtgagct
tctacaccaa cttcctgaac accatctggc ccagcgagga cccctggaag 180gccttcatgg
agcaggtgga ggccctgatg gaccagaaga tcgccgacta cgccaagaac 240aaggcactgg
ccgagctaca gggcctccag aacaacgtgg aggactatgt gagcgccctg 300agcagctggc
agaagaaccc cgtctcgagc cgcaaccccc acagccaggg ccgcatccgc 360gagctgttca
gccaggccga gagccacttc cgcaacagca tgcccagctt cgccatcagc 420ggctacgagg
tgctgttcct gaccacctac gcccaggccg ccaacaccca cctgttcctg 480ctgaaggacg
cccaaatcta cggagaggag tggggctacg agaaggagga catcgccgag 540ttctacaagc
gccagctgaa gctgacccag gagtacaccg accactgcgt gaagtggtac 600aacgtgggtc
tagacaagct ccgcggcagc agctacgaga gctgggtgaa cttcaaccgc 660taccgccgcg
agatgaccct gaccgtgctg gacctgatcg ccctgttccc cctgtacgac 720gtgcgcctgt
accccaagga ggtgaagacc gagctgaccc gcgacgtgct gaccgacccc 780atcgtgggcg
tgaacaacct gcgcggctac ggcaccacct tcagcaacat cgagaactac 840atccgcaagc
cccacctgtt cgactacctg caccgcatcc agttccacac gcgtttccag 900cccggctact
acggcaacga cagcttcaac tactggagcg gcaactacgt gagcacccgc 960cccagcatcg
gcagcaacga catcatcacc agccccttct acggcaacaa gagcagcgag 1020cccgtgcaga
accttgagtt caacggcgag aaggtgtacc gcgccgtggc taacaccaac 1080ctggccgtgt
ggccctctgc agtgtacagc ggcgtgacca aggtggagtt cagccagtac 1140aacgaccaga
ccgacgaggc cagcacccag acctacgaca gcaagcgcaa cgtgggcgcc 1200gtgagctggg
acagcatcga ccagctgccc cccgagacca ccgacgagcc cctggagaag 1260ggctacagcc
accagctgaa ctacgtgatg tgcttcctga tgcagggcag ccgcggcacc 1320atccccgtgc
tgacctggac ccacaagagc gtcgacttct tcaacatgat cgacagcaag 1380aagatcaccc
agctgcccct gaccaagagc accaacctgg gcagcggcac cagcgtggtg 1440aagggccccg
gcttcaccgg cggcgacatc ctgcgccgca ccagccccgg ccagatcagc 1500accctgcgcg
tgaacatcac cgcccccctg agccagcgct accgcgtccg catccgctac 1560gccagcacca
ccaacctgca gttccacacc agcatcgacg gccgccccat caaccagggc 1620aacttcagcg
ccaccatgag cagcggcagc aacctgcaga gcggcagctt ccgcaccgtg 1680ggcttcacca
cccccttcaa cttcagcaac ggcagcagcg tgttcaccct gagcgcccac 1740gtgttcaaca
gcggcaacga ggtgtacatc gaccgcatcg agttcgtgcc cgccgaggtg 1800accttcgagg
ccgagtacga cctggagagg gctcagaagg ccgtgaacga gctgttcacc 1860agcagcaacc
agatcggcct gaagaccgac gtgaccgact accacatcga tcaggtgtag
1920147639PRTArtificial Sequence-catG8AF protein 147Met Thr Ala Asp Asn
Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val Val
Gly Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn
Phe 35 40 45 Leu
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu Met
Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn
Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn
100 105 110 Pro His
Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 115
120 125 His Phe Arg Asn Ser Met Pro
Ser Phe Ala Ile Ser Gly Tyr Glu Val 130 135
140 Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr
His Leu Phe Leu 145 150 155
160 Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
165 170 175 Asp Ile Ala
Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr 180
185 190 Thr Asp His Cys Val Lys Trp Tyr
Asn Val Gly Leu Asp Lys Leu Arg 195 200
205 Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr
Arg Arg Glu 210 215 220
Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp 225
230 235 240 Val Arg Leu Tyr
Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val 245
250 255 Leu Thr Asp Pro Ile Val Gly Val Asn
Asn Leu Arg Gly Tyr Gly Thr 260 265
270 Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu
Phe Asp 275 280 285
Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr 290
295 300 Gly Asn Asp Ser Phe
Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg 305 310
315 320 Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr
Ser Pro Phe Tyr Gly Asn 325 330
335 Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys
Val 340 345 350 Tyr
Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val 355
360 365 Tyr Ser Gly Val Thr Lys
Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr 370 375
380 Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys
Arg Asn Val Gly Ala 385 390 395
400 Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu
405 410 415 Pro Leu
Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe 420
425 430 Leu Met Gln Gly Ser Arg Gly
Thr Ile Pro Val Leu Thr Trp Thr His 435 440
445 Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys
Lys Ile Thr Gln 450 455 460
Leu Pro Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val 465
470 475 480 Lys Gly Pro
Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro 485
490 495 Gly Gln Ile Ser Thr Leu Arg Val
Asn Ile Thr Ala Pro Leu Ser Gln 500 505
510 Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn
Leu Gln Phe 515 520 525
His Thr Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala 530
535 540 Thr Met Ser Ser
Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val 545 550
555 560 Gly Phe Thr Thr Pro Phe Asn Phe Ser
Asn Gly Ser Ser Val Phe Thr 565 570
575 Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile
Asp Arg 580 585 590
Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu
595 600 605 Glu Arg Ala Gln
Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln 610
615 620 Ile Gly Leu Lys Thr Asp Val Thr
Asp Tyr His Ile Asp Gln Val 625 630 635
1481809DNAArtificial Sequence8AFdm3 coding sequence
148atgacggccg acaacaacac cgaggccctg gacagcagca ccaccaagga cgtgatccag
60aagggcatca gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt cggcggcgcc
120ctggtgagct tctacaccaa cttcctgaac accatctggc ccagcgagga cccctggaag
180gccttcatgg agcaggtgga ggccctgatg gaccagaaga tcgccgacta cgccaagaac
240aaggcactgg ccgagctaca gggcctccag aacaacgtgg aggactatgt gagcgccctg
300agcagctggc agaagaaccc cgctgcaccg ttccgcaacc cccacagcca gggccgcatc
360cgcgagctgt tcagccaggc cgagagccac ttccgcaaca gcatgcccag cttcgccatc
420agcggctacg aggtgctgtt cctgaccacc tacgcccagg ccgccaacac ccacctgttc
480ctgctgaagg acgcccaaat ctacggagag gagtggggct acgagaagga ggacatcgcc
540gagttctaca agcgccagct gaagctgacc caggagtaca ccgaccactg cgtgaagtgg
600tacaacgtgg gtctagacaa gctccgcggc agcagctacg agagctgggt gaacttcaac
660cgctaccgcc gcgagatgac cctgaccgtg ctggacctga tcgccctgtt ccccctgtac
720gacgtgcgcc tgtaccccaa ggaggtgaag accgagctga cccgcgacgt gctgaccgac
780cccatcgtgg gcgtgaacaa cctgcgcggc tacggcacca ccttcagcaa catcgagaac
840tacatccgca agccccacct gttcgactac ctgcaccgca tccagttcca cacgcgtttc
900cagcccggct actacggcaa cgacagcttc aactactgga gcggcaacta cgtgagcacc
960cgccccagca tcggcagcaa cgacatcatc accagcccct tctacggcaa caagagcagc
1020gagcccgtgc agaaccttga gttcaacggc gagaaggtgt accgcgccgt ggctaacacc
1080aacctggccg tgtggccctc tgcagtgtac agcggcgtga ccaaggtgga gttcagccag
1140tacaacgacc agaccgacga ggccagcacc cagacctacg acagcaagcg caacgtgggc
1200gccgtgagct gggacagcat cgaccagctg ccccccgaga ccaccgacga gcccctggag
1260aagggctaca gccaccagct gaactacgtg atgtgcttcc tgatgcaggg cagccgcggc
1320accatccccg tgctgacctg gacccacaag agcgtcgact tcaacaacat catccccagc
1380agccagatca cccagatccc cctgaccaag agcaccaacc tgggcagcgg caccagcgtg
1440gtgaagggcc ccggcttcac cggcggcgac atcctgcgcc gcaccagccc cggccagatc
1500agcaccctgc gcgtgaacat caccgccccc ctgagccagc gctaccgcgt ccgcatccgc
1560tacgccagca ccaccaacct gcagttccac accagcatcg acggccgccc catcaaccag
1620ggcaacttca gcgccaccat gagcagcggc agcaacctgc agagcggcag cttccgcacc
1680gtgggcttca ccaccccctt caacttcagc aacggcagca gcgtgttcac cctgagcgcc
1740cacgtgttca acagcggcaa cgaggtgtac atcgaccgca tcgagttcgt gcccgccgag
1800gtgacctaa
1809149602PRTArtificial Sequence8AFdm3 protein 149Met Thr Ala Asp Asn Asn
Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly
Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn
Phe 35 40 45 Leu
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu Met
Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn
Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110 Asn Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 115
120 125 Ser His Phe Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130 135
140 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe 145 150 155
160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175 Glu Asp Ile
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu 180
185 190 Tyr Thr Asp His Cys Val Lys Trp
Tyr Asn Val Gly Leu Asp Lys Leu 195 200
205 Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg
Tyr Arg Arg 210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr 225
230 235 240 Asp Val Arg Leu
Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp 245
250 255 Val Leu Thr Asp Pro Ile Val Gly Val
Asn Asn Leu Arg Gly Tyr Gly 260 265
270 Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His
Leu Phe 275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr 290
295 300 Tyr Gly Asn Asp Ser
Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 305 310
315 320 Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile
Thr Ser Pro Phe Tyr Gly 325 330
335 Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu
Lys 340 345 350 Val
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala 355
360 365 Val Tyr Ser Gly Val Thr
Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 370 375
380 Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Val Gly 385 390 395
400 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415 Glu Pro
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys 420
425 430 Phe Leu Met Gln Gly Ser Arg
Gly Thr Ile Pro Val Leu Thr Trp Thr 435 440
445 His Lys Ser Val Asp Phe Asn Asn Ile Ile Pro Ser
Ser Gln Ile Thr 450 455 460
Gln Ile Pro Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val 465
470 475 480 Val Lys Gly
Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser 485
490 495 Pro Gly Gln Ile Ser Thr Leu Arg
Val Asn Ile Thr Ala Pro Leu Ser 500 505
510 Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr
Asn Leu Gln 515 520 525
Phe His Thr Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser 530
535 540 Ala Thr Met Ser
Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr 545 550
555 560 Val Gly Phe Thr Thr Pro Phe Asn Phe
Ser Asn Gly Ser Ser Val Phe 565 570
575 Thr Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr
Ile Asp 580 585 590
Arg Ile Glu Phe Val Pro Ala Glu Val Thr 595 600
1501809DNAArtificial Sequence8AFlomgdm3 coding sequence
150atgacggccg acaacaacac cgaggccctg gacagcagca ccaccaagga cgtgatccag
60aagggcatca gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt cggcggcgcc
120ctggtgagct tctacaccaa cttcctgaac accatctggc ccagcgagga cccctggaag
180gccttcatgg agcaggtgga ggccctgatg gaccagaaga tcgccgacta cgccaagaac
240aaggcactgg ccgagctaca gggcctccag aacaacgtgg aggactatgt gagcgccctg
300agcagctggc agaagaaccc cgctgcaccg ttccgcaacc cccacagcca gggccgcatc
360cgcgagctgt tcagccaggc cgagagccac ttccgcaaca gcatgcccag cttcgccatc
420agcggctacg aggtgctgtt cctgaccacc tacgcccagg ccgccaacac ccacctgttc
480ctgctgaagg acgcccaaat ctacggagag gagtggggct acgagaagga ggacatcgcc
540gagttctaca agcgccagct gaagctgacc caggagtaca ccgaccactg cgtgaagtgg
600tacaacgtgg gtctagacaa gctccgcggc agcagctacg agagctgggt gaacttcaac
660cgctaccgcc gcgagatgac cctgaccgtg ctggacctga tcgccctgtt ccccctgtac
720gacgtgcgcc tgtaccccaa ggaggtgaag accgagctga cccgcgacgt gctgaccgac
780cccatcgtgg gcgtgaacaa cctgcgcggc tacggcacca ccttcagcaa catcgagaac
840tacatccgca agccccacct gttcgactac ctgcaccgca tccagttcca cacgcgtttc
900cagcccggct actacggcaa cgacagcttc aactactgga gcggcaacta cgtgagcacc
960cgccccagca tcggcagcaa cgacatcatc accagcccct tctacggcaa caagagcagc
1020gagcccgtgc agaaccttga gttcaacggc gagaaggtgt accgcgccgt ggctaacacc
1080aacctggccg tgtggccctc tgcagtgtac agcggcgtga ccaaggtgga gttcagccag
1140tacaacgacc agaccgacga ggccagcacc cagacctacg acagcaagcg caacgtgggc
1200gccgtgagct gggacagcat cgaccagctg ccccccgaga ccaccgacga gcccctggag
1260aagggctaca gccaccagct gaactacgtg atgtgcttcc tgatgcaggg cagccgcggc
1320accatccccg tgctgacctg gacccacaag agcgtcgact tcttcaacat gatcgacagc
1380aagaagatca cccagctgcc cctggtgaag gcctacaagc tccagagcgg cgccagcgtg
1440gtggcaggcc cccgcttcac cggcggcgac atcatccagt gcaccgagaa cggcagcgcc
1500gccaccatct acgtgacccc cgacgtgagc tacagccaga agtaccgcgc ccgcatccgc
1560tacgccagca ccaccaacct gcagttccac accagcatcg acggccgccc catcaaccag
1620ggcaacttca gcgccaccat gagcagcggc agcaacctgc agagcggcag cttccgcacc
1680gtgggcttca ccaccccctt caacttcagc aacggcagca gcgtgttcac cctgagcgcc
1740cacgtgttca acagcggcaa cgaggtgtac atcgaccgca tcgagttcgt gcccgccgag
1800gtgacctaa
1809151602PRTArtificial Sequence8AFlongdm3 protein 151Met Thr Ala Asp Asn
Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val Val
Gly Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn
Phe 35 40 45 Leu
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu Met
Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn
Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110 Asn Pro
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 115
120 125 Ser His Phe Arg Asn Ser Met
Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130 135
140 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn
Thr His Leu Phe 145 150 155
160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175 Glu Asp Ile
Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu 180
185 190 Tyr Thr Asp His Cys Val Lys Trp
Tyr Asn Val Gly Leu Asp Lys Leu 195 200
205 Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg
Tyr Arg Arg 210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr 225
230 235 240 Asp Val Arg Leu
Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp 245
250 255 Val Leu Thr Asp Pro Ile Val Gly Val
Asn Asn Leu Arg Gly Tyr Gly 260 265
270 Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His
Leu Phe 275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr 290
295 300 Tyr Gly Asn Asp Ser
Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 305 310
315 320 Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile
Thr Ser Pro Phe Tyr Gly 325 330
335 Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu
Lys 340 345 350 Val
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala 355
360 365 Val Tyr Ser Gly Val Thr
Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 370 375
380 Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser
Lys Arg Asn Val Gly 385 390 395
400 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415 Glu Pro
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys 420
425 430 Phe Leu Met Gln Gly Ser Arg
Gly Thr Ile Pro Val Leu Thr Trp Thr 435 440
445 His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser
Lys Lys Ile Thr 450 455 460
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val 465
470 475 480 Val Ala Gly
Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu 485
490 495 Asn Gly Ser Ala Ala Thr Ile Tyr
Val Thr Pro Asp Val Ser Tyr Ser 500 505
510 Gln Lys Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr
Asn Leu Gln 515 520 525
Phe His Thr Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser 530
535 540 Ala Thr Met Ser
Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr 545 550
555 560 Val Gly Phe Thr Thr Pro Phe Asn Phe
Ser Asn Gly Ser Ser Val Phe 565 570
575 Thr Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr
Ile Asp 580 585 590
Arg Ile Glu Phe Val Pro Ala Glu Val Thr 595 600
1521848DNAArtificial Sequencecap8AFdm3 coding sequence
152atgactagta acggccgcca gtgtgctggt attcgccctt atgacggccg acaacaacac
60cgaggcctgg acagcagcac caccaaggac gtgatccaga agggcatcag cgtggtgggc
120gacctgctgg gcgtggtggg cttccccttc ggcggcgccc tggtgagctt ctacaccaac
180ttcctgaaca ccatctggcc cagcgaggac ccctggaagg ccttcatgga gcaggtggag
240gccctgatgg accagaagat cgccgactac gccaagaaca aggcactggc cgagctacag
300ggcctccaga acaacgtgga ggactatgtg agcgccctga gcagctggca gaagaacccc
360gctgcaccgt tccgcaaccc ccacagccag ggccgcatcc gcgagctgtt cagccaggcc
420gagagccact tccgcaacag catgcccagc ttcgccatca gcggctacga ggtgctgttc
480ctgaccacct acgcccaggc cgccaacacc cacctgttcc tgctgaagga cgcccaaatc
540tacggagagg agtggggcta cgagaaggag gacatcgccg agttctacaa gcgccagctg
600aagctgaccc aggagtacac cgaccactgc gtgaagtggt acaacgtggg tctagacaag
660ctccgcggca gcagctacga gagctgggtg aacttcaacc gctaccgccg cgagatgacc
720ctgaccgtgc tggacctgat cgccctgttc cccctgtacg acgtgcgcct gtaccccaag
780gaggtgaaga ccgagctgac ccgcgacgtg ctgaccgacc ccatcgtggg cgtgaacaac
840ctgcgcggct acggcaccac cttcagcaac atcgagaact acatccgcaa gccccacctg
900ttcgactacc tgcaccgcat ccagttccac acgcgtttcc agcccggcta ctacggcaac
960gacagcttca actactggag cggcaactac gtgagcaccc gccccagcat cggcagcaac
1020gacatcatca ccagcccctt ctacggcaac aagagcagcg agcccgtgca gaaccttgag
1080ttcaacggcg agaaggtgta ccgcgccgtg gctaacacca acctggccgt gtggccctct
1140gcagtgtaca gcggcgtgac caaggtggag ttcagccagt acaacgacca gaccgacgag
1200gccagcaccc agacctacga cagcaagcgc aacgtgggcg ccgtgagctg ggacagcatc
1260gaccagctgc cccccgagac caccgacgag cccctggaga agggctacag ccaccagctg
1320aactacgtga tgtgcttcct gatgcagggc agccgcggca ccatccccgt gctgacctgg
1380acccacaaga gcgtcgactt caacaacatc atccccagca gccagatcac ccagatcccc
1440ctgaccaaga gcaccaacct gggcagcggc accagcgtgg tgaagggccc cggcttcacc
1500ggcggcgaca tcctgcgccg caccagcccc ggccagatca gcaccctgcg cgtgaacatc
1560accgcccccc tgagccagcg ctaccgcgtc cgcatccgct acgccagcac caccaacctg
1620cagttccaca ccagcatcga cggccgcccc atcaaccagg gcaacttcag cgccaccatg
1680agcagcggca gcaacctgca gagcggcagc ttccgcaccg tgggcttcac cacccccttc
1740aacttcagca acggcagcag cgtgttcacc ctgagcgccc acgtgttcaa cagcggcaac
1800gaggtgtaca tcgaccgcat cgagttcgtg cccgccgagg tgacctag
1848153615PRTArtificial Sequencecap8AFdm3 protein 153Met Thr Ser Asn Gly
Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly 1 5
10 15 Arg Gln Gln His Arg Gly Leu Asp Ser Ser
Thr Thr Lys Asp Val Ile 20 25
30 Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly
Phe 35 40 45 Pro
Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr 50
55 60 Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu Gln Val Glu 65 70
75 80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
Lys Asn Lys Ala Leu 85 90
95 Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala
100 105 110 Leu Ser
Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His 115
120 125 Ser Gln Gly Arg Ile Arg Glu
Leu Phe Ser Gln Ala Glu Ser His Phe 130 135
140 Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr
Glu Val Leu Phe 145 150 155
160 Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys
165 170 175 Asp Ala Gln
Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile 180
185 190 Ala Glu Phe Tyr Lys Arg Gln Leu
Lys Leu Thr Gln Glu Tyr Thr Asp 195 200
205 His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
Arg Gly Ser 210 215 220
Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225
230 235 240 Leu Thr Val Leu
Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg 245
250 255 Leu Tyr Pro Lys Glu Val Lys Thr Glu
Leu Thr Arg Asp Val Leu Thr 260 265
270 Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
Thr Phe 275 280 285
Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu 290
295 300 His Arg Ile Gln Phe
His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn 305 310
315 320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr
Val Ser Thr Arg Pro Ser 325 330
335 Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
Ser 340 345 350 Ser
Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg 355
360 365 Ala Val Ala Asn Thr Asn
Leu Ala Val Trp Pro Ser Ala Val Tyr Ser 370 375
380 Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn
Asp Gln Thr Asp Glu 385 390 395
400 Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser
405 410 415 Trp Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu 420
425 430 Glu Lys Gly Tyr Ser His Gln
Leu Asn Tyr Val Met Cys Phe Leu Met 435 440
445 Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp
Thr His Lys Ser 450 455 460
Val Asp Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro 465
470 475 480 Leu Thr Lys
Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly 485
490 495 Pro Gly Phe Thr Gly Gly Asp Ile
Leu Arg Arg Thr Ser Pro Gly Gln 500 505
510 Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser
Gln Arg Tyr 515 520 525
Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr 530
535 540 Ser Ile Asp Gly
Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met 545 550
555 560 Ser Ser Gly Ser Asn Leu Gln Ser Gly
Ser Phe Arg Thr Val Gly Phe 565 570
575 Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr
Leu Ser 580 585 590
Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu
595 600 605 Phe Val Pro Ala
Glu Val Thr 610 615 1541923DNAArtificial
Sequence8AFdm3 T coding sequence 154atgacggccg acaacaacac cgaggccctg
gacagcagca ccaccaagga cgtgatccag 60aagggcatca gcgtggtggg cgacctgctg
ggcgtggtgg gcttcccctt cggcggcgcc 120ctggtgagct tctacaccaa cttcctgaac
accatctggc ccagcgagga cccctggaag 180gccttcatgg agcaggtgga ggccctgatg
gaccagaaga tcgccgacta cgccaagaac 240aaggcactgg ccgagctaca gggcctccag
aacaacgtgg aggactatgt gagcgccctg 300agcagctggc agaagaaccc cgctgcaccg
ttccgcaacc cccacagcca gggccgcatc 360cgcgagctgt tcagccaggc cgagagccac
ttccgcaaca gcatgcccag cttcgccatc 420agcggctacg aggtgctgtt cctgaccacc
tacgcccagg ccgccaacac ccacctgttc 480ctgctgaagg acgcccaaat ctacggagag
gagtggggct acgagaagga ggacatcgcc 540gagttctaca agcgccagct gaagctgacc
caggagtaca ccgaccactg cgtgaagtgg 600tacaacgtgg gtctagacaa gctccgcggc
agcagctacg agagctgggt gaacttcaac 660cgctaccgcc gcgagatgac cctgaccgtg
ctggacctga tcgccctgtt ccccctgtac 720gacgtgcgcc tgtaccccaa ggaggtgaag
accgagctga cccgcgacgt gctgaccgac 780cccatcgtgg gcgtgaacaa cctgcgcggc
tacggcacca ccttcagcaa catcgagaac 840tacatccgca agccccacct gttcgactac
ctgcaccgca tccagttcca cacgcgtttc 900cagcccggct actacggcaa cgacagcttc
aactactgga gcggcaacta cgtgagcacc 960cgccccagca tcggcagcaa cgacatcatc
accagcccct tctacggcaa caagagcagc 1020gagcccgtgc agaaccttga gttcaacggc
gagaaggtgt accgcgccgt ggctaacacc 1080aacctggccg tgtggccctc tgcagtgtac
agcggcgtga ccaaggtgga gttcagccag 1140tacaacgacc agaccgacga ggccagcacc
cagacctacg acagcaagcg caacgtgggc 1200gccgtgagct gggacagcat cgaccagctg
ccccccgaga ccaccgacga gcccctggag 1260aagggctaca gccaccagct gaactacgtg
atgtgcttcc tgatgcaggg cagccgcggc 1320accatccccg tgctgacctg gacccacaag
agcgtcgact tcaacaacat catccccagc 1380agccagatca cccagatccc cctgaccaag
agcaccaacc tgggcagcgg caccagcgtg 1440gtgaagggcc ccggcttcac cggcggcgac
atcctgcgcc gcaccagccc cggccagatc 1500agcaccctgc gcgtgaacat caccgccccc
ctgagccagc gctaccgcgt ccgcatccgc 1560tacgccagca ccaccaacct gcagttccac
accagcatcg acggccgccc catcaaccag 1620ggcaacttca gcgccaccat gagcagcggc
agcaacctgc agagcggcag cttccgcacc 1680gtgggcttca ccaccccctt caacttcagc
aacggcagca gcgtgttcac cctgagcgcc 1740cacgtgttca acagcggcaa cgaggtgtac
atcgaccgca tcgagttcgt gcccgccgag 1800gtgaccttcg aggccgagta cgacctggag
agggctcaga aggccgtgaa cgagctgttc 1860accagcagca accagatcgg cctgaagacc
gacgtgaccg actaccacat cgatcaggtg 1920tag
1923155640PRTArtificial Sequence8AFdm3 T
protein 155Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr
Lys 1 5 10 15 Asp
Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30 Val Gly Phe Pro Phe
Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe 35
40 45 Leu Asn Thr Ile Trp Pro Ser Glu Asp
Pro Trp Lys Ala Phe Met Glu 50 55
60 Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr
Ala Lys Asn 65 70 75
80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95 Val Ser Ala Leu
Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg 100
105 110 Asn Pro His Ser Gln Gly Arg Ile Arg
Glu Leu Phe Ser Gln Ala Glu 115 120
125 Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly
Tyr Glu 130 135 140
Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe 145
150 155 160 Leu Leu Lys Asp Ala
Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys 165
170 175 Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln
Leu Lys Leu Thr Gln Glu 180 185
190 Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys
Leu 195 200 205 Arg
Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg 210
215 220 Glu Met Thr Leu Thr Val
Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr 225 230
235 240 Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr
Glu Leu Thr Arg Asp 245 250
255 Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly
260 265 270 Thr Thr
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe 275
280 285 Asp Tyr Leu His Arg Ile Gln
Phe His Thr Arg Phe Gln Pro Gly Tyr 290 295
300 Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn
Tyr Val Ser Thr 305 310 315
320 Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly
325 330 335 Asn Lys Ser
Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys 340
345 350 Val Tyr Arg Ala Val Ala Asn Thr
Asn Leu Ala Val Trp Pro Ser Ala 355 360
365 Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr
Asn Asp Gln 370 375 380
Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly 385
390 395 400 Ala Val Ser Trp
Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp 405
410 415 Glu Pro Leu Glu Lys Gly Tyr Ser His
Gln Leu Asn Tyr Val Met Cys 420 425
430 Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr
Trp Thr 435 440 445
His Lys Ser Val Asp Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr 450
455 460 Gln Ile Pro Leu Thr
Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val 465 470
475 480 Val Lys Gly Pro Gly Phe Thr Gly Gly Asp
Ile Leu Arg Arg Thr Ser 485 490
495 Pro Gly Gln Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu
Ser 500 505 510 Gln
Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln 515
520 525 Phe His Thr Ser Ile Asp
Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser 530 535
540 Ala Thr Met Ser Ser Gly Ser Asn Leu Gln Ser
Gly Ser Phe Arg Thr 545 550 555
560 Val Gly Phe Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe
565 570 575 Thr Leu
Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp 580
585 590 Arg Ile Glu Phe Val Pro Ala
Glu Val Thr Phe Glu Ala Glu Tyr Asp 595 600
605 Leu Glu Arg Ala Gln Lys Ala Val Asn Glu Leu Phe
Thr Ser Ser Asn 610 615 620
Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val 625
630 635 640
1561923DNAArtificial Sequence8AFlongdm3T coding sequence 156atgacggccg
acaacaacac cgaggccctg gacagcagca ccaccaagga cgtgatccag 60aagggcatca
gcgtggtggg cgacctgctg ggcgtggtgg gcttcccctt cggcggcgcc 120ctggtgagct
tctacaccaa cttcctgaac accatctggc ccagcgagga cccctggaag 180gccttcatgg
agcaggtgga ggccctgatg gaccagaaga tcgccgacta cgccaagaac 240aaggcactgg
ccgagctaca gggcctccag aacaacgtgg aggactatgt gagcgccctg 300agcagctggc
agaagaaccc cgctgcaccg ttccgcaacc cccacagcca gggccgcatc 360cgcgagctgt
tcagccaggc cgagagccac ttccgcaaca gcatgcccag cttcgccatc 420agcggctacg
aggtgctgtt cctgaccacc tacgcccagg ccgccaacac ccacctgttc 480ctgctgaagg
acgcccaaat ctacggagag gagtggggct acgagaagga ggacatcgcc 540gagttctaca
agcgccagct gaagctgacc caggagtaca ccgaccactg cgtgaagtgg 600tacaacgtgg
gtctagacaa gctccgcggc agcagctacg agagctgggt gaacttcaac 660cgctaccgcc
gcgagatgac cctgaccgtg ctggacctga tcgccctgtt ccccctgtac 720gacgtgcgcc
tgtaccccaa ggaggtgaag accgagctga cccgcgacgt gctgaccgac 780cccatcgtgg
gcgtgaacaa cctgcgcggc tacggcacca ccttcagcaa catcgagaac 840tacatccgca
agccccacct gttcgactac ctgcaccgca tccagttcca cacgcgtttc 900cagcccggct
actacggcaa cgacagcttc aactactgga gcggcaacta cgtgagcacc 960cgccccagca
tcggcagcaa cgacatcatc accagcccct tctacggcaa caagagcagc 1020gagcccgtgc
agaaccttga gttcaacggc gagaaggtgt accgcgccgt ggctaacacc 1080aacctggccg
tgtggccctc tgcagtgtac agcggcgtga ccaaggtgga gttcagccag 1140tacaacgacc
agaccgacga ggccagcacc cagacctacg acagcaagcg caacgtgggc 1200gccgtgagct
gggacagcat cgaccagctg ccccccgaga ccaccgacga gcccctggag 1260aagggctaca
gccaccagct gaactacgtg atgtgcttcc tgatgcaggg cagccgcggc 1320accatccccg
tgctgacctg gacccacaag agcgtcgact tcttcaacat gatcgacagc 1380aagaagatca
cccagctgcc cctggtgaag gcctacaagc tccagagcgg cgccagcgtg 1440gtggcaggcc
cccgcttcac cggcggcgac atcatccagt gcaccgagaa cggcagcgcc 1500gccaccatct
acgtgacccc cgacgtgagc tacagccaga agtaccgcgc ccgcatccgc 1560tacgccagca
ccaccaacct gcagttccac accagcatcg acggccgccc catcaaccag 1620ggcaacttca
gcgccaccat gagcagcggc agcaacctgc agagcggcag cttccgcacc 1680gtgggcttca
ccaccccctt caacttcagc aacggcagca gcgtgttcac cctgagcgcc 1740cacgtgttca
acagcggcaa cgaggtgtac atcgaccgca tcgagttcgt gcccgccgag 1800gtgaccttcg
aggccgagta cgacctggag agggctcaga aggccgtgaa cgagctgttc 1860accagcagca
accagatcgg cctgaagacc gacgtgaccg actaccacat cgatcaggtg 1920tag
1923157640PRTArtificial Sequence8AFlongdm3T protein 157Met Thr Ala Asp
Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys 1 5
10 15 Asp Val Ile Gln Lys Gly Ile Ser Val
Val Gly Asp Leu Leu Gly Val 20 25
30 Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr
Asn Phe 35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu 50
55 60 Gln Val Glu Ala Leu
Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn 65 70
75 80 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln
Asn Asn Val Glu Asp Tyr 85 90
95 Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe
Arg 100 105 110 Asn
Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 115
120 125 Ser His Phe Arg Asn Ser
Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu 130 135
140 Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala
Asn Thr His Leu Phe 145 150 155
160 Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175 Glu Asp
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu 180
185 190 Tyr Thr Asp His Cys Val Lys
Trp Tyr Asn Val Gly Leu Asp Lys Leu 195 200
205 Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn
Arg Tyr Arg Arg 210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr 225
230 235 240 Asp Val Arg
Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp 245
250 255 Val Leu Thr Asp Pro Ile Val Gly
Val Asn Asn Leu Arg Gly Tyr Gly 260 265
270 Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro
His Leu Phe 275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr 290
295 300 Tyr Gly Asn Asp
Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr 305 310
315 320 Arg Pro Ser Ile Gly Ser Asn Asp Ile
Ile Thr Ser Pro Phe Tyr Gly 325 330
335 Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly
Glu Lys 340 345 350
Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
355 360 365 Val Tyr Ser Gly
Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln 370
375 380 Thr Asp Glu Ala Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Asn Val Gly 385 390
395 400 Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro
Glu Thr Thr Asp 405 410
415 Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys
420 425 430 Phe Leu Met
Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr 435
440 445 His Lys Ser Val Asp Phe Phe Asn
Met Ile Asp Ser Lys Lys Ile Thr 450 455
460 Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly
Ala Ser Val 465 470 475
480 Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu
485 490 495 Asn Gly Ser Ala
Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser 500
505 510 Gln Lys Tyr Arg Ala Arg Ile Arg Tyr
Ala Ser Thr Thr Asn Leu Gln 515 520
525 Phe His Thr Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn
Phe Ser 530 535 540
Ala Thr Met Ser Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr 545
550 555 560 Val Gly Phe Thr Thr
Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe 565
570 575 Thr Leu Ser Ala His Val Phe Asn Ser Gly
Asn Glu Val Tyr Ile Asp 580 585
590 Arg Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr
Asp 595 600 605 Leu
Glu Arg Ala Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn 610
615 620 Gln Ile Gly Leu Lys Thr
Asp Val Thr Asp Tyr His Ile Asp Gln Val 625 630
635 640 1581962DNAArtificial Sequencecap8AFdm3T
coding serquence 158atgactagta acggccgcca gtgtgctggt attcgccctt
atgacggccg acaacaacac 60cgaggcctgg acagcagcac caccaaggac gtgatccaga
agggcatcag cgtggtgggc 120gacctgctgg gcgtggtggg cttccccttc ggcggcgccc
tggtgagctt ctacaccaac 180ttcctgaaca ccatctggcc cagcgaggac ccctggaagg
ccttcatgga gcaggtggag 240gccctgatgg accagaagat cgccgactac gccaagaaca
aggcactggc cgagctacag 300ggcctccaga acaacgtgga ggactatgtg agcgccctga
gcagctggca gaagaacccc 360gctgcaccgt tccgcaaccc ccacagccag ggccgcatcc
gcgagctgtt cagccaggcc 420gagagccact tccgcaacag catgcccagc ttcgccatca
gcggctacga ggtgctgttc 480ctgaccacct acgcccaggc cgccaacacc cacctgttcc
tgctgaagga cgcccaaatc 540tacggagagg agtggggcta cgagaaggag gacatcgccg
agttctacaa gcgccagctg 600aagctgaccc aggagtacac cgaccactgc gtgaagtggt
acaacgtggg tctagacaag 660ctccgcggca gcagctacga gagctgggtg aacttcaacc
gctaccgccg cgagatgacc 720ctgaccgtgc tggacctgat cgccctgttc cccctgtacg
acgtgcgcct gtaccccaag 780gaggtgaaga ccgagctgac ccgcgacgtg ctgaccgacc
ccatcgtggg cgtgaacaac 840ctgcgcggct acggcaccac cttcagcaac atcgagaact
acatccgcaa gccccacctg 900ttcgactacc tgcaccgcat ccagttccac acgcgtttcc
agcccggcta ctacggcaac 960gacagcttca actactggag cggcaactac gtgagcaccc
gccccagcat cggcagcaac 1020gacatcatca ccagcccctt ctacggcaac aagagcagcg
agcccgtgca gaaccttgag 1080ttcaacggcg agaaggtgta ccgcgccgtg gctaacacca
acctggccgt gtggccctct 1140gcagtgtaca gcggcgtgac caaggtggag ttcagccagt
acaacgacca gaccgacgag 1200gccagcaccc agacctacga cagcaagcgc aacgtgggcg
ccgtgagctg ggacagcatc 1260gaccagctgc cccccgagac caccgacgag cccctggaga
agggctacag ccaccagctg 1320aactacgtga tgtgcttcct gatgcagggc agccgcggca
ccatccccgt gctgacctgg 1380acccacaaga gcgtcgactt caacaacatc atccccagca
gccagatcac ccagatcccc 1440ctgaccaaga gcaccaacct gggcagcggc accagcgtgg
tgaagggccc cggcttcacc 1500ggcggcgaca tcctgcgccg caccagcccc ggccagatca
gcaccctgcg cgtgaacatc 1560accgcccccc tgagccagcg ctaccgcgtc cgcatccgct
acgccagcac caccaacctg 1620cagttccaca ccagcatcga cggccgcccc atcaaccagg
gcaacttcag cgccaccatg 1680agcagcggca gcaacctgca gagcggcagc ttccgcaccg
tgggcttcac cacccccttc 1740aacttcagca acggcagcag cgtgttcacc ctgagcgccc
acgtgttcaa cagcggcaac 1800gaggtgtaca tcgaccgcat cgagttcgtg cccgccgagg
tgaccttcga ggccgagtac 1860gacctggaga gggctcagaa ggccgtgaac gagctgttca
ccagcagcaa ccagatcggc 1920ctgaagaccg acgtgaccga ctaccacatc gatcaggtgt
ag 1962159653PRTArtificial Sequencecap8AFdm3T
protein 159Met Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp
Gly 1 5 10 15 Arg
Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp Val Ile
20 25 30 Gln Lys Gly Ile Ser
Val Val Gly Asp Leu Leu Gly Val Val Gly Phe 35
40 45 Pro Phe Gly Gly Ala Leu Val Ser Phe
Tyr Thr Asn Phe Leu Asn Thr 50 55
60 Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
Gln Val Glu 65 70 75
80 Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu
85 90 95 Ala Glu Leu Gln
Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala 100
105 110 Leu Ser Ser Trp Gln Lys Asn Pro Ala
Ala Pro Phe Arg Asn Pro His 115 120
125 Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
His Phe 130 135 140
Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe 145
150 155 160 Leu Thr Thr Tyr Ala
Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys 165
170 175 Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly
Tyr Glu Lys Glu Asp Ile 180 185
190 Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
Asp 195 200 205 His
Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser 210
215 220 Ser Tyr Glu Ser Trp Val
Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr 225 230
235 240 Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro
Leu Tyr Asp Val Arg 245 250
255 Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr
260 265 270 Asp Pro
Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe 275
280 285 Ser Asn Ile Glu Asn Tyr Ile
Arg Lys Pro His Leu Phe Asp Tyr Leu 290 295
300 His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly
Tyr Tyr Gly Asn 305 310 315
320 Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser
325 330 335 Ile Gly Ser
Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser 340
345 350 Ser Glu Pro Val Gln Asn Leu Glu
Phe Asn Gly Glu Lys Val Tyr Arg 355 360
365 Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
Val Tyr Ser 370 375 380
Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu 385
390 395 400 Ala Ser Thr Gln
Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser 405
410 415 Trp Asp Ser Ile Asp Gln Leu Pro Pro
Glu Thr Thr Asp Glu Pro Leu 420 425
430 Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
Leu Met 435 440 445
Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser 450
455 460 Val Asp Phe Asn Asn
Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro 465 470
475 480 Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly
Thr Ser Val Val Lys Gly 485 490
495 Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly
Gln 500 505 510 Ile
Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr 515
520 525 Arg Val Arg Ile Arg Tyr
Ala Ser Thr Thr Asn Leu Gln Phe His Thr 530 535
540 Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn
Phe Ser Ala Thr Met 545 550 555
560 Ser Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe
565 570 575 Thr Thr
Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser 580
585 590 Ala His Val Phe Asn Ser Gly
Asn Glu Val Tyr Ile Asp Arg Ile Glu 595 600
605 Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr
Asp Leu Glu Arg 610 615 620
Ala Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly 625
630 635 640 Leu Lys Thr
Asp Val Thr Asp Tyr His Ile Asp Gln Val 645
650 160687PRTArtificial SequenceFR8a+34 protein 160Met Lys
Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser 1 5
10 15 Pro Asp Leu Gly Thr Leu Val
Pro Arg Gly Ser Met Ala Asp Ile Gly 20 25
30 Ser Thr Met Thr Ser Asn Gly Arg Gln Cys Ala Gly
Ile Arg Pro Tyr 35 40 45
Asp Gly Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp
50 55 60 Val Ile Gln
Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val 65
70 75 80 Gly Phe Pro Phe Gly Gly Ala
Leu Val Ser Phe Tyr Thr Asn Phe Leu 85
90 95 Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys
Ala Phe Met Glu Gln 100 105
110 Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
Lys 115 120 125 Ala
Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val 130
135 140 Ser Ala Leu Ser Ser Trp
Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn 145 150
155 160 Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe
Ser Gln Ala Glu Ser 165 170
175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val
180 185 190 Leu Phe
Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu 195
200 205 Leu Lys Asp Ala Gln Ile Tyr
Gly Glu Glu Trp Gly Tyr Glu Lys Glu 210 215
220 Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu
Thr Gln Glu Tyr 225 230 235
240 Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg
245 250 255 Gly Ser Ser
Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu 260
265 270 Met Thr Leu Thr Val Leu Asp Leu
Ile Ala Leu Phe Pro Leu Tyr Asp 275 280
285 Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr
Arg Asp Val 290 295 300
Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr 305
310 315 320 Thr Phe Ser Asn
Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp 325
330 335 Tyr Leu His Arg Ile Gln Phe His Thr
Arg Phe Gln Pro Gly Tyr Tyr 340 345
350 Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser
Thr Arg 355 360 365
Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn 370
375 380 Lys Ser Ser Glu Pro
Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val 385 390
395 400 Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala
Val Trp Pro Ser Ala Val 405 410
415 Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln
Thr 420 425 430 Asp
Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala 435
440 445 Val Ser Trp Asp Ser Ile
Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu 450 455
460 Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn
Tyr Val Met Cys Phe 465 470 475
480 Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His
485 490 495 Lys Ser
Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln 500
505 510 Leu Pro Leu Thr Lys Ser Thr
Asn Leu Gly Ser Gly Thr Ser Val Val 515 520
525 Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg
Arg Thr Ser Pro 530 535 540
Gly Gln Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln 545
550 555 560 Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe 565
570 575 His Thr Ser Ile Asp Gly Arg Pro
Ile Asn Gln Gly Asn Phe Ser Ala 580 585
590 Thr Met Ser Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe
Arg Thr Val 595 600 605
Gly Phe Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr 610
615 620 Leu Ser Ala His
Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg 625 630
635 640 Ile Glu Phe Val Pro Ala Glu Val Thr
Phe Glu Ala Glu Tyr Asp Leu 645 650
655 Glu Arg Ala Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser
Asn Gln 660 665 670
Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val 675
680 685
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