Patent application title: Manipulation of Genes of the Mevalonate and Isoprenoid Pathways to Create Novel Traits in Transgenic Organisms
Inventors:
Frederick M. Hahn (Paia, HI, US)
Adelheid R. Kuehnle (Honolulu, HI, US)
Adelheid R. Kuehnle (Honolulu, HI, US)
IPC8 Class: AC12P1706FI
USPC Class:
435125
Class name: Preparing heterocyclic carbon compound having only o, n, s, se, or te as ring hetero atoms oxygen as only ring hetero atom containing six-membered hetero ring (e.g., fluorescein, etc.)
Publication date: 2008-10-23
Patent application number: 20080261280
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Patent application title: Manipulation of Genes of the Mevalonate and Isoprenoid Pathways to Create Novel Traits in Transgenic Organisms
Inventors:
FREDERICK M. HAHN
Adelheid R. Kuehnle
Agents:
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
Assignees:
Origin: GAINESVILLE, FL US
IPC8 Class: AC12P1706FI
USPC Class:
435125
Abstract:
Disclosed are the uses of specific genes of the mevalonate and isoprenoid
biosynthetic pathways, and of inactive gene sites (the pseudogene) to (1)
enhance biosynthesis of isopentenyl diphosphate, dimethylallyl
diphosphate and isoprenoid pathway derived products in the plastids of
transgenic plants and microalgae, (2) create novel antibiotic resistant
transgenic plants and microalgae, and (3) create a novel selection system
and/or targeting sites for mediating the insertion of genetic material
into plant and microalgae plastids. The specific polynucleotides to be
used, solely or in any combination thereof, are publicly available from
GeneBank and contain open reading frames having sequences that upon
expression will produce active proteins with the following enzyme
activities: (a) acetoacetyl CoA thiolase (EC 2.3.1.9), (b)
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC 4.1.3.5),
(c) HMG-CoA reductase (EC 1.1.1.34), (d) mevalonate kinase (EC 2.7.1.36),
(e) phosphomevalonate kinase (EC 2.7.4.2), (f) mevalonate diphosphate
decarboxylase (EC 4.1.1.33), (g) isopentenyl diphosphate (IPP) isomerase
(EC 5.3.3.2), and (h) phytoene synthase (EC 2.5.1.32).Claims:
1. A method for synthesizing an isoprenoid or an isoprenoid precursor via
a mevalonate pathway in a host cell, wherein the method comprises:i)
culturing a transformed host cell in a suitable medium, wherein the
transformed host cell is a prokaryote that does not normally synthesize
isopentenyl pyrophosphate (IPP) through the mevalonate pathway, and
wherein the host cell comprises one or more nucleic acids heterologous to
the host cell, wherein the one or more heterologous nucleic acids
comprises nucleotide sequences that encode two or more mevalonate pathway
enzymes, wherein said two or more mevalonate pathway enzymes comprises an
enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA as
the first step in the synthesis of the isoprenoid or isoprenoid precursor
and one or more additional mevalonate pathway enzymes selected from:(a)
an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form
HMG-CoA;(b) an enzyme that converts HMG-CoA to mevalonate;(c) an enzyme
that phosphorylates mevalonate to mevalonate 5-phosphate;(d) an enzyme
that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
and(e) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl
pyrophosphate, said culturing providing for production of the two or more
enzymes, resulting in synthesis of said isoprenoid or isoprenoid
precursor in a recoverable amount of at least about 1 mg/L; andii)
recovering the produced isoprenoid or isoprenoid precursor.
2. The method of claim 1, wherein the one or more heterologous nucleic acids is integrated into the chromosome of the host cell.
3. The method of claim 1, wherein the one or more heterologous nucleic acids is contained in at least one extrachromosomal expression vector.
4. The method of claim 3, wherein the one or more heterologous nucleic acids is present in a single expression vector.
5. The method of claim 3, wherein each of the one or more heterologous nucleic acids is contained within a separate expression vector.
6. The method of claim 3, wherein at least two of the one or more heterologous nucleic acids are contained in a single expression vector.
7. The method of claim 3, wherein the one or more heterologous nucleic acids is contained in two expression vectors.
8. A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, the method comprising: culturing a transformed host microorganism in a suitable medium, the transformed host microorganism comprising one or more nucleic acids heterologous to the host microorganism, wherein the host microorganism is a prokaryote that does not normally synthesize IPP through the mevalonate pathway, wherein the one or more nucleic acids comprises nucleotide sequences encoding two or more enzymes selected from:a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA, wherein said enzyme is from Ralstonia, Saccharomyces, or Escherichia coli, wherein said enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is present as the first step in the synthesis of the IPP;b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA, wherein said enzyme is from Blattella or Saccharomyces; c) an enzyme that converts HMG-CoA to mevalonate, wherein said enzyme is from Sulfolobus, Haloferax, or Saccharomyces; d) a Saccharomyces enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;e) a Saccharomyces enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; andf) a Saccharomyces enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, said culturing providing for production of the enzymes, wherein said production of said two or more enzymes results in production of IPP.
9. The method of claim 1, wherein the method further comprises reacting isopentenyl pyrophosphate with dimethylallyl pyrophosphate or a polyprenyl pyrophosphate in the presence of at least one enzyme to provide a polyprenyl pyrophosphate isoprenoid precursor.
10. The method of claim 9, wherein the one or more heterologous nucleic acids further comprises:g) a nucleic acid comprising a nucleotide sequence coding for an enzyme that converts isopentenyl pyrophosphate to dimethylallyl pyrophosphate.
11. The method of claim 1 wherein the isoprenoid precursor is IPP, and wherein the IPP is further modified enzymatically by the action of isopentenyl pyrophosphate isomerase and one or more polyprenyl pyrophosphate synthases to provide an isoprenoid selected from the group consisting of a monoterpene, sesquiterpene, diterpene, sesterterpene, triterpene, tetraterpene, and a steroid.
12. The method of claim 11, wherein the isoprenoid is a monoterpene.
13. The method of claim 12, wherein the monoterpene is selected from the group consisting of limonene, citranellol, and geraniol.
14. The method of claim 11, wherein the isoprenoid is a sesquiterpene.
15. The method of claim 14, wherein the sesquiterpene is selected from the group consisting of periplanone B, arteinisinin, ginkgolide B, forskolin, and farnesol.
16. The method of claim 12, wherein the isoprenoid is a diterpene.
17. The method of claim 1, wherein the prokaryote is Escherichia coli.
18. The method of claim 7, wherein the prokaryote is Escherichia coli.
19. The method of claim 8, wherein the prokaryote is Escherichia coli.
20. The method of claim 11, wherein the prokaryote is Escherichia coli.
21. The method of claim 19, wherein the enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is from E. coli.
22. The method of claim 19, wherein the enzyme that condenses acetyl-CoA with acetoacetyl-CoA is from Saccharomyces.
23. A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, wherein the method comprises: culturing a transformed host microorganism in a suitable medium, the transformed host microorganism comprising at least two operons heterologous to the host microorganism, wherein each of said two operons comprises nucleotide sequences encoding enzymes in the mevalonate pathway, and wherein the host microorganism is a prokaryote that does not normally synthesize IPP through the mevalonate pathway; wherein the mevalonate pathway comprises:(a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA, wherein said enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is present as the first step in the synthesis of IPP;(b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;(c) an enzyme that converts HMG-CoA to mevalonate;(d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;(e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; and(f) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, said culturing providing for production of the enzymes, wherein said production of said two or more enzymes results in production of IPP.
24. The method of claim 23, wherein said at least two operons are contained in a single extrachromosomal expression vector.
25. The method of claim 23, wherein at least one of said at least two operons is contained in a different extrachromosomal expression vector from another of said at least two operons.
26. The method of claim 23, wherein at least one of said at least two operons is integrated into a chromosome of said transformed host microorganism.
27. The method of claim 23, wherein said transformed host microorganism also comprises a heterologous nucleic acid comprising a nucleotide sequence encoding an enzyme that converts IPP to dimethylallyl pyrophosphate, and the method further comprises reacting the IPP with dimethylallyl pyrophosphate and a polyprenyl pyrophosphate synthase to provide a polyprenyl pyrophosphate isoprenoid precursor.
28. The method of claim 23, wherein said transformed host microorganism is E. coli.
29. The method of claim 24, wherein said transformed host microorganism is E. coli.
30. The method of claim 25, wherein said transformed host microorganism is E. coli.
31. The method of claim 26, wherein said transformed host microorganism is E. coli.
32. The method of claim 28, wherein said E. coli also produces IPP by a DXP pathway.
33. The method of claim 28, whereina) said enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is from Ralsionia, Saccharomyces, or Escherichia coli; b) said enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA is from Blattella or Saccharomyces; c) said enzyme that converts HMG-CoA to mevalonate is from Sulfolobus, Haloferax, or Saccharomyces; andd) said enzymes that phosphorylate mevalonate to mevalonate 5-phosphate, that convert mevalonate 5-phosphate to mevalonate 5-pyrophosphate, and that convert mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, are from Saccharomyces.
34. The method of claim 33, wherein the enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA is from E. coli.
35. The method of claim 33, wherein the enzyme that condenses acetyl-CoA to acetoacetyl-CoA to form HMG-CoA is Saccharomyces.
36. The method of claim 1, wherein the two or more enzymes are from at least two distinct organisms.
37. The method of claim 1, wherein at least one of the two or more enzymes is from an organism other than Saccharomyces cerevisiae.
38. The method of claim 1, wherein the one or more nucleic acids comprises nucleotide sequences encoding three enzymes in the mevalonate pathway.
39. The method of claim 1, wherein the one or more nucleic acids comprises nucleotide sequences encoding four enzymes in the mevalonate pathway.
40. The method of claim 1, wherein the one or more nucleic acids comprises nucleotide sequences encoding six enzymes in the mevalonate pathway.
41. The method of claim 23, wherein the IPP is further modified enzymatically by the action of isopentenyl pyrophosphate isomerase to provide dimethylallyl pyrophosphate (DMAPP).
42. The method of claim 41, wherein the DMAPP is further modified enzymatically with one or more polyprenyl pyrophosphate synthases to provide an isoprenoid.
43. The method of claim 41, wherein the isoprenoid is a monoterpene.
44. The method of claim 43, wherein the monoterpene is selected from limonene, citranellol, and geraniol.
45. The method of claim 42, wherein the isoprenoid is a sesquiterpene.
46. The method of claim 45, wherein the sesquiterpene is selected from periplanone B, artemisinin, ginkgolide B, forskolin, and farnesol.
47. The method of claim 42, wherein the isoprenoid is a diterpene.
48. The method of claim 42, wherein the isoprenoid is a triterpene.
49. The method of claim 42, wherein the isoprenoid is a tetraterpene.
50. The method of claim 42, wherein the isoprenoid is a steroid.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application is a continuation of U.S. application Ser. No. 11/489,050, filed Jul. 18, 2006, which is a divisional of U.S. application Ser. No. 11/053,541, filed Feb. 8, 2005, which is a divisional of U.S. application Ser. No. 10/835,516, filed Apr. 28, 2004, now allowed, which is a divisional of U.S. application Ser. No. 09/918,740, filed Jul. 31, 2001, now abandoned, and claims the benefit of U.S. Provisional Application No. 60/221,703, filed Jul. 31, 2000, all of which are hereby incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002]This invention relates to the fields of biotechnology and genetic engineering, in particular to agricultural and aquacultural biotechnology. More specifically, the invention relates to transgenic plants and microalgae, in particular to transplastomic plants and microalgae and means for insertion of genetic material into plastids.
BACKGROUND OF THE INVENTION
[0003]The ubiquitous isoprenoid biosynthetic pathway is responsible for the formation of the most chemically diverse family of metabolites found in nature (Hahn et al., J. Bacteriol. 178:619-624, 1996) including sterols (Popjak, Biochemical symposium no. 29 (T. W. Goodwin, ed.) Academic Press, New York, pp 17-37, 1970), carotenoids (Goodwin, Biochem. J. 123:293-329, 1971), dolichols (Matsuoka et al., J. Biol. Chem. 266:3464-3468, 1991), ubiquinones (Ashby and Edwards, J. Biol. Chem. 265:13157-13164, 1990), and prenylated proteins (Clarke, Annu. Rev. Biochem. 61:355-386, 1992). Biosynthesis of isopentenyl diphosphate (IPP), the essential 5-carbon isoprenoid precursor, occurs by two distinct compartmentalized routes in plants (Lange and Croteau, Proc. Natl. Acad. Sci. USA 96:13714-13719, 1999). In the plant cytoplasm, IPP is assembled from three molecules of acetyl coenzyme A by the well-characterized mevalonate pathway (Lange and Croteau, Proc. Natl. Acad. Sci. USA 96:13714-13719, 1999). However, a recently discovered mevalonate-independent pathway is responsible for the synthesis of IPP in plant chloroplasts (Lichtenthaler et al. FEBS Letters 400:271-274, 1997).
[0004]Following the synthesis of IPP via the mevalonate route, the carbon-carbon double bond must be isomerized to create the potent electrophile dimethylallyl diphosphate (DMAPP). This essential activation step, carried out by IPP isomerase, insures the existence of the two 5-carbon isomers, IPP and DMAPP, which must join together in the first of a series of head to tail condensation reactions to create the essential allylic diphosphates of the isoprenoid pathway (Hahn and Poulter, J. Biol. Chem. 270:11298-11303, 1995). Recently, it was reported that PP isomerase activity was not essential in E. coli, one of many eubacteria containing only the non-mevalonate pathway for the synthesis of both 5-carbon isomers, suggesting the existence of two separate mevalonate-independent routes to IPP and DMAPP (Hahn et al., J. Bacteriol. 181:4499-4504, 1999). Thus, it is unclear whether an IPP isomerase is essential for the synthesis of isoprenoids in plant plastids as well. Regardless of whether IPP isomerase activity is present in plant plastids, the separation by compartmentalization of the two different biosynthetic routes, the mevalonate and deoxyxylulose phosphate pathways (or "non-mevalonate"), for IPP and DMAPP biosynthesis in plants is the fundamental tenet upon which the subject inventions are based.
[0005]The synthesis of IPP by the mevalonate pathway (Eisenreich et al., Chemistry and Biology 5:R221-R233, 1998) is cytoplasm based and occurs as follows: The condensation of two acetyl CoA molecules to yield acetoacetyl CoA is catalyzed by acetoacetyl CoA thiolase (EC 2.3.1.9). The addition of another molecule of acetyl CoA to acetoacetyl CoA is catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC 4.1.3.5) to yield HMG-CoA, which is reduced in the subsequent step to mevalonate by HMG-CoA reductase (EC 1.1.1.34). Mevalonate is phosphorylated by mevalonate kinase (EC 2.7.1.36) to yield phosphomevalonate, which is phosphorylated, by phosphomevalonate kinase (EC 2.7.4.2) to form mevalonate diphosphate. The conversion of mevalonate diphosphate to IPP with the concomitant release of CO2 is catalyzed by mevalonate diphosphate decarboxylase (EC 4.1.1.33).
[0006]In organisms utilizing the deoxyxylulose phosphate pathway (aka "non-mevalonate pathway", "methylerydritol phosphate (MEP) pathway", and "Rohmer pathway"), the five carbon atoms in the basic isoprenoid unit are derived from pyruvate and D-glyceraldehyde phosphate (GAP) (Eisenreich et al., 1998). Thus, synthesis of IPP and/or DMAPP by the non-mevalonate route, which occurs in plastids, is as follows: Pyruvate and GAP are condensed to give 1-deoxy-D-xylulose 5-phosphate (DXP) by DXP synthase (Sprenger et al., Proc. Natl. Acad. Sci. USA 94:12857-12862, 1997). The rearrangement and reduction of DXP to form 2-C-methylerythritol 4-phosphate (MEP), the first committed intermediate in the non-mevalonate pathway biosynthesis of isoprenoids is catalyzed by DXP reductoisomerase (Kuzuyama et al., Tetrahedron Lett. 39:4509-4512, 1998). MEP is then appended to CTP to form 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (Rohdich et al., Proc. Natl. Acad. Sci. USA 96:11758-11763, 1999), followed by phosphorylation of the C2 hydroxyl group (Luttgen et al., Proc. Natl. Acad. Sci. USA 97:1062-1067, 2000) and elimination of CMP, to form a 2,4-cyclic diphosphate (Herz et al., Proc. Natl. Acad. Sci. USA 97:2486-2490, 2000). Interestingly, Herz et al. reported the possible existence of bifunctional proteins with both YgbP and YgbB activities. Once the remaining steps to the fundamental five-carbon isoprenoid building blocks, IPP and DMAPP, in the non-mevalonate pathway are discovered, they will serve as additional targets for inhibitors with antibiotic and herbicidal activity.
[0007]Since the non-mevalonate pathway is ultimately responsible for the biosynthesis of compounds critical for photosynthesis such as the prenyl side-chain of chlorophylls, which serve as lipophilic anchors for the photoreceptors and the photoprotective carotenoid pigments, any enzyme, gene, or regulatory sequence involved in the biosynthesis of IPP and/or DMAPP can be a potential target for herbicides. For example, the antibiotic fosmidomycin, a specific inhibitor of the enzyme DXP reductoisomerase (Kuzuyama et al, Tetrahedron Lett. 39:7913-7916, 1998) has been shown to have significant herbicidal activity, especially in combination with other herbicides (Kamuro et al. "Herbicide" U.S. Pat. No. 4,846,872; issued Jul. 11, 1989). The report of an Arabidopsis thaliana albino mutant being characterized as a disruption of the CLA1 gene, later revealed as encoding DXP synthase by Rohmer et al. (Lois et al., Proc. Natl. Acad. Sci. USA 95:2105-2110, 1998), also illustrates the potential of non-mevalonate pathway enzymes as targets for compounds with herbicidal activity. Accordingly, one of ordinary skill in the art can readily understand that as additional compounds are discovered exhibiting herbicidal activity based on their effects on the non-mevalonate pathway, those compounds could be used in accord with the teachings herein.
[0008]The synthesis of carotenoids from IPP and DMAPP takes place in plant plastids by a genetically- and enzymatically-defined pathway (Cunningham and Gantt, Ann. Rev. Plant Mol. Biol. 39:475-502, 1998). Enhanced production of carotenoids such as lycopene and β-carotene in plants is highly desirable due to the reported health benefits of their consumption (Kajiwara et al, Biochem. J. 324:421-426, 1997). Enhanced carotenoid production in plants can also have a dramatic effect on their coloration and be highly desirable to the growers of ornamentals, for example. The IPP isomerase reaction is considered to be a rate-limiting step for isoprenoid biosynthesis (Ramos-Valdivia et al., Nat. Prod. Rep. 6:591-603, 1997). Kajiwara et al. reported that the expression of heterologous IPP isomerase genes in a strain of E. coli specifically engineered to produce carotenoids resulted in over a 2-fold increase in β-carotene formation. Recently, it has been reported that expression of an additional gene for DXP synthase in an E. coli strain specifically engineered to produce carotenoids also increased the level of lycopene substantially (Harker and Bramley, FEBS Letters 448:115-119, 1999). Increased isoprenoid production also has been shown in bacteria by combining carotenogenic genes from bacteria with an orf encoding IPP isomerase; and was even further enhanced when additionally combined with the dxs gene from the MEP pathway to supply the precursors IPP and DMAPP (Albrecht et al. Nature Biotechnology 18: 843-846, 2000).
[0009]Accumulation of one specific isoprenoid, such as beta-carotene (yellow-orange) or astaxanthin (red-orange), can serve to enhance flower color or nutriceutical composition depending if the host is cultivated as an ornamental or as an output crop; and if the product accumulates in the tissue of interest (i.e. flower parts or harvestable tissue). In plants, tissue with intrinsic carotenoid enzymes can accumulate ketocarotenoids such as astaxanthin in chromoplasts of reproductive tissues of tobacco by addition of the biosynthetic enzyme beta-carotene ketolase (Mann et al., Nature Biotechnology 18: 888-892, 2000). Astaxanthin is the main carotenoid pigment found in aquatic animals; in microalgae it accumulates in the Chlorophyta such as in species of Haematococcus and Chlamydomonas. Thus, an increase in the essential 5-carbon precursors, IPP and DMAPP, by expression of orfs encoding IPP isomerase and orfs upstream thereof, can feed into the production output of such valuable isoprenoids in organisms other than bacteria.
[0010]As a further example of utility, Petunia flower color is usually due to the presence of modified cyanidin and delphinidin anthocyanin pigments to produce shades in red to blue groupings. Recently produced yellow seed-propagated multiflora and grandiflora petunias obtain their coloration from the presence of beta-carotene, lutein and zeaxanthin carotenoid pigments in combination with colorless flavonols (Nielsen and Bloor, Scienia Hort. 71: 257-266, 1997). Industry still lacks bright yellow and orange clonally propagated trailing petunias. Metabolic engineering of the carotenoid pathway is desired to introduce these colors in this popular potted and bedding plant.
[0011]Plant genetic engineering has evolved since the 1980s from arbitrarily located monocistronic insertions into a nuclear chromosome, often subject to multiple copies, rearrangements and methylation, to predetermined sites for defined multicistronic or multigenic operon insertions into a plastid chromosome (plastome), which thus far is thought impervious to typical nuclear gene inactivation. While breeding of crop plants by nuclear genome engineering is nevertheless a proven technology for major agronomic crops and for traits such as herbicide resistance, introgression of genes into the plastome is a highly promising breeding approach for several reasons as described by Bock and Hagemann (Bock and Hagemann, Prog. Bot. 61:76-90, 2000). Of note is the containment of transgenes in the transplastomic plant: Plastids are inherited through the maternal parent in most plant species and thus plastid-encoded transgenes are unable to spread in pollen to non-target species. Therefore plastid engineering can minimize negative impacts of genetically engineered plants. A report on potential transfer by pollen of herbicide resistance into weedy relatives of cultivated crops (Keeler et al., Herbicide Resistant Crops: Agricultural, Economic, Environmental, Regulatory and Technological Aspects, pp. 303-330, 1996) underscores the value of using plastid engineering rather than nuclear engineering for critical production traits such as herbicide resistance. Daniell et al. have recently demonstrated herbicide resistance through genetic engineering of the chloroplast genome (Daniell et al., Nat. Biotechnol., 16:345-348, 1998).
[0012]Moreover, plastids are the site of essential biosynthetic activity. Although most associate photosynthesis as the primary function of the chloroplast, studies document that the chloroplast is the center of activity for functions involving carbon metabolism, nitrogen metabolism, sulfur metabolism, biochemical regulation, and various essential biosynthetic pathways including amino acid, vitamin, and phytohormone biosynthesis. Crop traits of interest such as nutritional enhancement require genetic manipulations that impact plastid biosynthetic pathways such as carotenoid production. While nuclear-encoded gene products can be exported from the engineered nucleus into the plastid for such manipulations, the biosynthetic genes themselves can be inserted into the plastid for expression and activity. As we begin to pyramid multiple genes often required for pathway manipulations (such as the aforementioned carotenoid biosynthesis) the repeated use of selection markers is expected to lead to unstable crops through homology-dependent gene silencing (Meyer and Saedler, Ann. Rev. Plant. Physiol. Mol. Biol. 47:23-48, 1996). In addition, the requirement for higher expression levels of transgenes for effective phenotypes such as vitamin levels and herbicide and pest resistance levels often falls short in nuclear transformations. These deficiencies are overcome through plastid transformation or combining plastid with nuclear transformations: The plastid recognizes strings of genes linked together in multicistronic operons and, due to the high copy number of genes within a plastid and within plastids in a cell, can produce a hundred- to thousand-fold the amount of transgene product. Accordingly, there is a continuing need for improved methods of producing plants having transformed plastids (transplastomic plants).
[0013]Golden rice is one example for which plastid engineering can complement nuclear engineering of pathways that reside in the plastid, yet have met with limited success. The metabolic pathway for beta-carotene (pro-vitamin A) was assembled in rice plastids by introduction into the nuclear genome of four separate genes, three encoding plastid-targeted proteins using three distinct promoters, plus a fourth selectable marker gene using a repeated promoter (Ye et al. Science 287:303-305, 2000). The wild-type rice endosperm is free of carotenoids but it does produce geranylgeranyl diphosphate; combining phytoene synthase, phytoene desaturase, and lycopene-beta cyclase resulted in accumulation of beta-carotene to make "golden rice." However, the quantity produced was lower than the minimum desired for addressing vitamin A deficiency. An increased supply of precursors for increasing intermediates, such as geranylgeranyl diphosphate, is predicted to significantly increase isoprenoid production. Insertion of an operon encoding the entire mevalonate pathway into the rice plastome of the "golden rice" genotype, using for example the methods as described in Khan and Maliga, Nature Biotechnology 17: 910-914, 1999, can provide a means for making improvements in metabolic engineering of this important monocot crop.
[0014]Proplastid and chloroplast genetic engineering have been shown to varying degrees of homoplasmy for several major agronomic crops including potato, rice, maize, soybean, grape, sweet potato, and tobacco including starting from non-green tissues. Non-lethal selection on antibiotics is used to proliferate cells containing plastids with antibiotic resistance genes. Plastid transformation methods use two plastid-DNA flanking sequences that recombine with plastid sequences to insert chimeric DNA into the spacer regions between functional genes of the plastome, as is established in the field (see Bock and Hagemann, Prog. Bot. 61:76-90, 2000, and Guda et al., Plant Cell Reports 19:257-262, 2000, and references therein).
[0015]Antibiotics such as spectinomycin, streptomycin, and kanamycin can shut down gene expression in chloroplasts by ribosome inactivation. These antibiotics bleach leaves and form white callus when tissue is put onto regeneration medium in their presence. The bacterial genes aadA and neo encode the enzymes aminoglycoside-3N-adenyltransferase and neomycin phosphotransferase, which inactivate these antibiotics, and can be used for positive selection of plastids engineered to express these genes. Polynucleotides of interest can be linked to the selectable genes and thus can be enriched by selection during the sorting out of engineered and non-engineered plastids. Consequently, cells with plastids engineered to contain genes for these enzymes (and linkages thereto) can overcome the effects of inhibitors in the plant cell culture medium and can proliferate, while cells lacking engineered plastids cannot proliferate. Similarly, plastids engineered with polynucleotides encoding enzymes from the mevalonate pathway to produce IPP from acetyl CoA in the presence of inhibitors of the non-mevalonate pathway can overcome otherwise inhibitory culture conditions. By utilizing the polynucleotides disclosed herein in accord with this invention, an inhibitor targeting the non-mevalonate pathway and its components can be used for selection purposes of transplastomic plants produced through currently available methods, or any future methods which become known for production of transplastomic plants, to contain and express said polynucleotides and any linked coding sequences of interest.
[0016]This selection process of the subject invention is unique in that it is the first selectable trait that acts by pathway complementation to overcome inhibitors. This is distinguished from the state of the art of selection by other antibiotics to which resistance is conferred by inactivation of the antibiotic itself, e.g. compound inactivation as for the aminoglyoside 3'-adenyltransferase gene or neo gene. This method avoids the occurrence of resistant escapes due to random insertion of the resistance gene into the nuclear genome or by spontaneous mutation of the ribosomal target of the antibiotic, as is known to occur in the state of the art. Moreover, this method requires the presence of an entire functioning mevalonate pathway in plastids. For example, if one of the enzyme activities of the mevalonate pathway is not present in the plastid, resistance will not be conferred.
[0017]There is strong evidence indicating that the origin of plastids within the cell occurred via endosymbiosis and that plastids are derived from cyanobacteria. As such, the genetic organization of the plastid is prokaryotic in nature (as opposed to the eukaryotic nuclear genome of the plant cell). The plastid chromosome ranges from roughly 110 to 150 Kb in size (196 for the green alga Chlamydomonas), much smaller than that of most cyanobacteria. However, many of the bacterium genes have either been lost because their function was no longer necessary for survival, or were transferred to the chromosomes of the nuclear genome. Most, but not all, of the genes remaining on the plastid chromosome function in either carbon metabolism or plastid genetics. However, many genes involved in these functions, as well as the many other functions and pathways intrinsic to plastid function, are also nuclear encoded, and the translated products are transported from the cytoplasm to the plastid. Studies have documented nuclear encoded genes with known activity in the plastid that are genetically more similar to homologous genes in bacteria rather than genes of the same organism with the same function but activity in the cytoplasm as reviewed for example in Martin et al. (1998) Nature 393:162-165 and references therein.
[0018]The process whereby genes are transported from the plastid to the nucleus has been addressed. Evidence indicates that copies of many plastid genes are found among nuclear chromosomes. For some of these, promoter regions and transit peptides (small stretches of DNA encoding peptides that direct polypeptides to the plastid) become associated with the gene that allows it to be transcribed, and the translated polypeptide relocated back into the plastid. Once this genetic apparatus has become established, the genes present in the plastid chromosome may begin to degrade until they are no longer functional, i.e., any such gene becomes a pseudogene.
[0019]As is common in prokaryotic systems, many genes that have a common function are organized into an operon. An operon is a cluster of contiguous genes transcribed from one promoter to give rise to a polycistron mRNA. Proteins from each gene in the polycistron are then translated. There are 18 operons in the plastid chromosome of tobacco (Nicotiana tabacum). Although many of these involve as few as two genes, some are large and include many genes. Evolutionary studies indicate that gene loss--as pseudogenes or completely missing sequences--occurs as individuals rather than as blocks of genes or transcriptional units. Thus other genes surrounding a pseudogene in a polycistronic operon remain functional.
[0020]The rpl23 operon consists of genes whose products are involved in protein translation. Most of these genes are ribosomal proteins functioning in either the large or small ribosomal subunit. One particular gene of note, infA, encodes an initiation factor protein that is important in initiating protein translation. Although this gene is functional in many plants, it is a pseudogene in tobacco and all other members of that family (Solanaceae), including the horticulturally valuable tomato, petunia, and potato crops. A recent survey of plant groups has indicated that there have been numerous loses of functionality of infA (Millen et al., Plant Cell 13; 645-658, 2001). This as well as other pseudogenes are identified in species whose chloroplast genomes have not yet been fully sequenced.
[0021]Pseudogenes such as infA become potential target sequences for insertion of intact orfs. Inserted orfs are controlled by regulatory upstream and downstream elements of the polycistron and are promoterless themselves. Pseudogenes are known for a multiplicity of crops and algae with chloroplast genomes that are already fully sequenced. Crops include grains such as rice and trees such as Pinus. Of note in the latter are the eleven ndh genes; all may serve as potential targets for transgene insertion.
[0022]Transplastomic solanaceous crops are highly desirable in order to eliminate the potential for gene transfer from engineered lines to wild species, as demonstrated in Lycopersicon (Dale, P. J. 1992. Spread of engineered genes to wild relatives. Plant Physiol. 100:13-15.). A method for plastid engineering that enables altered pigmentation, for improved nutrition in tomato or improved flower color in Petunia and ornamental tobacco as examples, is desirable for solanaceous crops. The infA gene is widely lost among rosids and some asterids; among the latter, infA is a pseudogene in all solanaceous species examined (representing 16 genera). The solanaceous infA DNA sequences show high similarity, with all nucleotide changes within infA being documented. Thus one set of flanking sequences of reasonable length as known in the art should serve for directed insertion of an individual or multiple orfs into the infA sites of the solanaceous species. It is documented in a solanaceous species that flanking sequences for genes to be inserted into the plastome are not required to be specific for the target species, as incompletely homologous plastid sequences are integrated at comparable frequencies (Kavanagh et al., Genetics 152:1111-1122, 1999).
[0023]The upstream 5' region, often referred to as the 5' UTR, is important on the expression level of a transcript as it is translated Knowing the translation products of surrounding genes in a polycistron allows one to select a pseudogene site that is affiliated with a strong 5' UTR for optimizing plastid expression in a particular tissue. The plastid genome in many plant species can have multiple pseudogenes that are located in different polycistronic sites. So, if one has a choice, one can select a site based on whether it is actively transcribed in green vs non-green plastid; and then if the polycistron has high or low relative expression in that plastid type. Moreover, monocistronic mRNA of ndhD was detected in developed leaves but not in greening or expanding leaves of barley (Hordeum vulgare), despite this gene being part of a polycistronic unit as reported by del Campo et al. (1997) Plant Physiol 114:748. Thus, one can time transgene product production by treating an inactive gene, based on developmental expression, as a pseudogene for targetting and integration purposes using the invention disclosed herein.
[0024]Algal species are becoming increasingly exploited as sources of nutraceuticals, pharmaceuticals, and lend themselves to aquaculture. Mass production of the isoprenoid compound astaxanthin produced by the green microalga Haemotcoccus is one successful example of the above. Metabolic engineering that would increase product yields and composition in microalgae would significantly benefit the industry. The development of organellar transformation for the unicellular green alga Chlamydomonas reinhardtii, with its single large chloroplast, opens the door for conducting studies on genetic manipulation of the isoprenoid pathway. Filamentous or multicellular algae are also of interest as untapped biofactories, as are other nongreen algae whose pathways for producing unique fatty acids, amino acids, and pigments can be ameliorated for commercial benefit.
[0025]The biolistic DNA delivery method is a general means with which to transform the chloroplast of algae (Boynton and Gillham, Methods Enzymol. 217:510-536, 1993). Sequencing of at least six plastomes from algae should facilitate transformation systems by confirming insertion sites, including pseudogene sites, and the regulatory elements directing heterologous gene expression. What is required is a dominant marker for selection of stable transformants to which natural resistance is absent (Stevens and Purton, J. Phycol 33: 713-722, 1997). For Chlamydomonas, chloroplasts can be engineered using markers that confer spectinomycin resistance following their integration into the plastome via homologous recombination. By utilizing the polynucleotides disclosed herein in accord with this invention, an inhibitor targeting the non-mevalonate pathway and its components can be used for selection purposes of transplastomic algae produced through currently available methods, or any future methods which become known for production of transplastomic algae, to contain and express said polynucleotides and any linked coding sequences of interest. This is a novel selection vehicle for transplastomic algae. Moreover, elevating the supply of essential precursors for isoprenoid production in algae as described above is enabled by this invention.
SUMMARY OF THE INVENTION
[0026]This invention relates to the presence of enzymatic activities necessary to form IPP from acetyl CoA, generally known as the mevalonate pathway, within plant and microalgae plastids. This invention may also require the presence of IPP isomerase activity within plastids resulting from the insertion into said plants and microalgae of a polynucleotide encoding a polypeptide with IPP isomerase activity. This invention may be achieved by the use of any polynucleotide, be it a DNA molecule or molecules, or any hybrid DNA/RNA molecule or molecules, containing at least one open reading frame that when expressed provides a polypeptide(s) exhibiting said activities within plastids. These open reading frames may be identical to their wild type progenitors, or alternatively may be altered in any manner (for example, with plastid-optimized codon usage), may be isolated from the host organism to be modified, may originate from another organism or organisms, or may be any combination of origin so long as the encoded proteins are able to provide the desired enzymatic activity within the target plastids. The described open reading frames may be inserted directly into plastids using established methodology or any methodology yet to be discovered. Alternatively, plastid localization of the desired activities may be achieved by modifying genes already residing in the cell nucleus, inserting foreign polynucleotides for nuclear residence, or inserting polynucleotides contained on exogenous, autonomous plasmids into the cell cytoplasm so that in all cases their encoded proteins are transported into the plastid. For example, a chloroplast transit (targeting) peptide can be fused to a protein of interest. Any combination of the above methods for realizing said activities in plant and microalgae plastids can be utilized. By causing the complete mevalonate pathway enzymatic activity to occur in plastids normally possessing only the non-mevalonate pathway, the presence of said activities within the chloroplasts of a specific plant or microalgae will endow it with resistance to a compound, molecule, etc. that targets a component of the non-mevalonate pathway, be it an enzyme, gene, regulatory sequence, etc., thereby also providing a useful selection system based on circumvention of the inhibition of the non-mevalonate pathway in transplastomic plants and microalgae.
[0027]In addition, this invention relates to the use of open reading frames encoding polypeptides with enzymatic activities able to convert acetyl CoA to IPP, generally known as the mevalonate pathway, and a polypeptide with IPP isomerase activity as a method for increasing the production of IPP, DMAPP, and isoprenoid pathway derived products whose level within plant and microalgae plastids is dependent on the level of IPP and/or DMAPP present within the plastids. The presence of exogenous genes encoding 1-deoxy-D-xylulose-5-phosphate synthase and IPP isomerase have been shown to increase the production of carotenoids in eubacteria, presumably due to an increased production of IPP and/or DMAPP. Thus, insertion of the entire mevalonate pathway, solely or coupled with an additional IPP isomerase, into plastids will increase the level of IPP and/or DMAPP, resulting in an increased level of carotenoids and other yet to be determined isoprenoid pathway derived products within plant and microalgae plastids. This invention can utilize an open reading frame encoding the enzymatic activity for IPP isomerase independently or in addition to said open reading frames comprising the entire mevalonate pathway to obtain the increased level of isoprenoid pathway derived products within plant and microalgae plastids. This invention may be achieved by the use of any DNA molecule or molecules, or any hybrid DNA/RNA molecule or molecules, containing open reading frames able to provide said activities within plant and microalgae plastids. These open reading frames may be identical to their wild type progenitors, may be altered in any manner, may be isolated from the plant to be modified, may originate from another organism or organisms, or may be any combination of origin so long as the encoded proteins are able to provide said activities within plastids. The described open reading frames may be inserted directly into plant and microalgae plastids using established methodology or any methodology yet to be discovered. Alternatively, plastid localization of the desired activities may be achieved by modifying genes already residing in the nucleus, inserting foreign genes for nuclear residence, or inserting genes contained on exogenous, autonomous plasmids into the cytoplasm so that in all cases their encoded proteins are transported into the plastid. Any combination of the above methods for realizing said activities in plastids can be utilized.
[0028]Further, this invention also relates to the direct insertion of any foreign gene into a plant or microalgae chloroplast by coupling it to the open reading frames encoding polypeptides with enzymatic activities able to convert acetyl CoA to IPP, thus comprising the entire mevalonate pathway. By utilizing a compound, molecule, etc. that targets a component of the non-mevalonate pathway be it an enzyme, gene, regulatory sequence, etc., a method of selection analogous to the use of kanamycin and spectinomycin resistance for the transformation event is achieved. As inhibition of the non-mevalonate pathway in a plant or microalgae results in the impairment of photosynthesis, the presence of the mevalonate pathway biosynthetic capability is apparent, thus enabling the facile screening of concomitant incorporation into plastids of a foreign gene coupled to the open reading frames comprising the entire mevalonate pathway. The use of a polynucleotide comprising an open reading frame encoding a polypeptide with IPP isomerase activity in addition to the open reading frames encoding the mevalonate pathway is a particularly preferred embodiment, which provides all enzymatic activities necessary to synthesize both IPP and DMAPP and overcome the effect(s) of inhibition of the non-mevalonate pathway.
[0029]Further, this invention is unique and novel in that the transforming DNA, that is integrated by two or more homologous/heterologous recombination events, is purposefully targeted into inactive gene sites selected based on prior knowledge of transcription in plastid type, developmental expression including post-transcriptional editing, and post-transcriptional stability. Additionally, this invention uses the regulatory elements of known inactive genes (pseudogenes) to drive production of a complete transforming gene unrelated to the inserted gene site. Thus, by utilizing the transgene insertion method disclosed herein in accord with this invention, any foreign gene can be targeted to an inactive gene site (the pseudogene) through currently available methods of gene transfer, or any future methods which become known for production of transgenic and transplastomic plants, to contain and express said foreign gene and any linked coding sequences of interest. This gene insertion process of the subject invention is unique in that it is the first method specifically acting by pseudogene insertion to overcome the need for promoters and other regulatory elements normally associated with a transforming DNA vector while permitting site-specific recombination in organellar genomes. The use of the infA pseudogene insertion site in the solanaceous crops in particular is a preferred embodiment for the transformation of plastids using the open reading frames for the mevalonate pathway as well as for providing the necessary precursors for modified output traits in plants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030]FIG. 1 is a map of cloning vector pFCO1 containing S. cerevisiae orfs encoding phosphomevalonate kinase (PMK), mevalonate kinase (MVK), and mevalonate diphosphate decarboxylase (MDD).
[0031]FIG. 2 is a map of expression vector pFCO2 containing S. cerevisiae orfs encoding phosphomevalonate kinase (PMK), mevalonate kinase (MVK), and mevalonate diphosphate decarboxylase (MDD).
[0032]FIG. 3 is a map of cloning vector pHKO1 containing S. cerevisiae orf encoding acetoacetyl thiolase (AACT); A. thaliana orfs encoding HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGRt).
[0033]FIG. 4 is a map of expression vector pHKO2 containing S. cerevisiae orfs encoding phosphomevalonate kinase (PMK), mevalonate kinase (MVK), mevalonate diphosphate decarboxylase (MDD), and acetoacetyl thiolase (AACT); A. thaliana orfs encoding HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGRt) which in their summation are designated Operon A, encoding the entire mevalonate pathway.
[0034]FIG. 5 is a map of cloning vector pHKO3 containing S. cerevisiae orfs encoding phosphomevalonate kinase (PMK), mevalonate kinase (MVK), mevalonate diphosphate decarboxylase (MDD), and acetoacetyl thiolase (AACT); A. thaliana orfs encoding HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGRt) which in their summation are designated Operon B, encoding the entire mevalonate pathway.
[0035]FIG. 6 is an illustration of how the mevalonate (MEV) pathway, by providing an alternative biosynthetic route to IPP circumvents blocks in the MEP pathway due to a mutation in the gene for deoxyxylulose phosphate synthase (dxs) and due to inhibition by fosmidomycin of deoxyxylulose phosphate reductoisomerase (dxr).
[0036]FIG. 7 is a map of vector pBSNT27 containing N. tabcum chloroplast DNA (cpDNA) and the N. tabcum infA pseudogene and pBSNT27 sequence (SEQ ID NO: 17)
[0037]FIG. 8 is a map of plastid transformation vector pHKO4 containing N. tabcum chloroplast DNA (cpDNA) flanking the insertion of Operon B into the infA pseudogene.
[0038]FIG. 9 is a map of cloning vector pHKO5 containing S. cerevisiae orfs encoding phosphomevalonate kinase (PMK), mevalonate kinase (MVK), and mevalonate diphosphate decarboxylase (MDD), and acetoacetyl thiolase (AACT); A. thaliana orfs encoding HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGRt); R. capsulatus orf encoding IPP isomerase (IPPI) which in their summation are designated Operon C, encoding the entire mevalonate pathway and IPP isomerase.
[0039]FIG. 10 is a map of cloning vector pFHO1 containing S. cerevisiae orf encoding acetoacetyl thiolase (AACT); A. thaliana orf encoding HMG-CoA synthase (HMGS); Streptomyces sp CL190 orf encoding HMG-CoA reductase (HMGR).
[0040]FIG. 11 is a map of cloning vector pFHO2 containing S. cerevisiae orfs encoding phosphomevalonate kinase (PMK), mevalonate kinase (MVK), and mevalonate diphosphate decarboxylase (MDD), and acetoacetyl thiolase (AACT); A. thaliana orf encoding HMG-CoA synthase (HMGS); Streptomyces sp CL190 orf encoding HMG-CoA reductase (HMGR) which in their summation are designated Operon D, encoding the entire mevalonate pathway.
[0041]FIG. 12 is a map of cloning vector pFHO3 containing S. cerevisiae orfs encoding phosphomevalonate kinase (PMK), mevalonate kinase (MVK), and mevalonate diphosphate decarboxylase (MDD), and acetoacetyl thiolase (AACT); A. thaliana orf encoding HMG-CoA synthase (HMGS); Streptomyces sp CL190 orf encoding HMG-CoA reductase (HMGR); R. capsulatus orf encoding IPP isomerase (IPPI) which in their summation are designated Operon E, encoding the entire mevalonate pathway and IPP isomerase.
[0042]FIG. 13 is a map of cloning vector pFHO4 containing a S. cerevisiae orf encoding acetoacetyl thiolase (AACT) coupled to the Streptomyces sp CL190 gene cluster which in their summation are designated Operon F, encoding the entire mevalonate pathway and IPP isomerase.
[0043]FIG. 14 is a plastid transformation vector pHKO7 containing N. tabacum chloroplast DNA (cpDNA) flanking the insertion of Operon C into the infA pseudogene.
[0044]FIG. 15 is a map of expression vector pHKO9 containing Operon B.
[0045]FIG. 16 is a map of expression vector pHK10 containing Operon C.
[0046]FIG. 17 is a map of plastid transformation vector pFHO6 containing N. tabacum chloroplast DNA (cpDNA) flanking the insertion of both Operon E and the R. capsulatus orf encoding phytoene synthase (PHS) into the infA pseudogene.
BRIEF DESCRIPTION OF THE SEQUENCES
[0047]SEQ ID NO: 1) is a PCR primer containing Saccharomyces cerevisiae DNA.
[0048]SEQ ID NO: 2) is a PCR primer containing S. cerevisiae DNA.
[0049]SEQ ID NO: 3) is a PCR primer containing S. cerevisiae DNA.
[0050]SEQ ID NO: 4) is a PCR primer containing S. cerevisiae DNA.
[0051]SEQ ID NO: 5) is a PCR primer containing S. cerevisiae DNA.
[0052]SEQ ID NO: 6) is a PCR primer containing S. cerevisiae DNA.
[0053]SEQ ID NO: 7) is a PCR primer containing Arabidopsis thaliana DNA.
[0054]SEQ ID NO: 8) is a PCR primer containing A. thaliana DNA.
[0055]SEQ ID NO: 9) is a PCR primer containing A. thaliana DNA.
[0056]SEQ ID NO: 10) is a PCR primer containing A. thaliana DNA.
[0057]SEQ ID NO: 11) is a PCR primer containing S. cerevisiae DNA.
[0058]SEQ ID NO: 12) is a PCR primer containing S. cerevisiae DNA.
[0059]SEQ ID NO: 13) is an Oligonucleotide containing S. cerevisiae DNA.
[0060]SEQ ID NO: 14) is an Oligonucleotide containing A. thaliana and S. cerevisiae DNA.
[0061]SEQ ID NO: 15) is an Oligonucleotide containing S. cerevisiae DNA.
[0062]SEQ ID NO: 16) is an Oligonucleotide containing S. cerevisiae DNA.
[0063]SEQ ID NO: 17) is a Vector pBSNT27 containing Nicotiana tabacum DNA.
[0064]SEQ ID NO: 18) is an Oligonucleotide containing N. tabacum and S. cerevisiae DNA.
[0065]SEQ ID NO: 19) is an Oligonueleotide containing N. tabacum and A. thaliana DNA.
[0066]SEQ ID NO: 20) is a PCR primer containing Rhodobacter capsulatus DNA.
[0067]SEQ ID NO: 21) is a PCR is a primer containing R. capsulatus DNA.
[0068]SEQ ID NO: 22) is a PCR primer containing Schizosaccharomyces pombe DNA.
[0069]SEQ ID NO: 23) is a PCR primer containing S. pombe DNA.
[0070]SEQ ID NO: 24) is a PCR primer containing Streptomyces sp CL190 DNA.
[0071]SEQ ID NO: 25) is a PCR primer containing Streptomyces sp CL190 DNA.
[0072]SEQ ID NO: 26) is an Oligonucleotide containing S. cerevisiae DNA.
[0073]SEQ ID NO: 27) is an Oligonucleotide containing S. cerevisiae DNA.
[0074]SEQ ID NO: 28) is an Oligonucleotide containing Streptomyces sp CL190 and R. capsulatus DNA.
[0075]SEQ ID NO: 29) is an Oligonucleotide containing R. capsulatus DNA.
[0076]SEQ ID NO: 30) is an Oligonucleotide containing Streptomyces sp CL190 and S. cerevisiae DNA.
[0077]SEQ ID NO: 31) is an Oligonucleotide containing Streptomyces sp CL190 DNA.
[0078]SEQ ID NO: 32) is an Oligonucleotide containing N. tabacum and S. cerevisiae DNA.
[0079]SEQ ID NO: 33) is an Oligonucleotide containing N. tabacum and R. capsulatus DNA.
[0080]SEQ ID NO: 34) is an Oligonucleotide containing N. tabacum and S. cerevisiae DNA.
[0081]SEQ ID NO: 35) is an Oligonucleotide containing N. tabacum and S. pombe DNA.
[0082]SEQ ID NO: 36) is an Oligonucleotide containing NotI restriction site.
[0083]SEQ ID NO: 37) is an Oligonucleotide containing NotI restriction site.
[0084]SEQ ID NO: 38) is an Oligonucleotide containing S. cerevisiae DNA.
[0085]SEQ ID NO: 39) is an Oligonucleotide containing A. thaliana DNA.
[0086]SEQ ID NO: 40) is an Oligonucleotide containing S. cerevisiae DNA.
[0087]SEQ ID NO: 41) is an Oligonucleotide containing R. capsulatus DNA.
[0088]SEQ ID NO: 42) is an Oligonucleotide containing S. cerevisiae DNA.
[0089]SEQ ID NO: 43) is an Oligonucleotide containing S. pombe DNA.
[0090]SEQ ID NO: 44) is an Oligonucleotide containing R. capsulatus DNA.
[0091]SEQ ID NO: 45) is an Oligonucleotide containing R. capsulatus DNA.
[0092]SEQ ID NO: 46) is an Oligonucleotide containing S. pombe DNA.
[0093]SEQ ID NO: 47) is an Oligonucleotide containing S. pombe DNA.
[0094]SEQ ID NO: 48) is Saccharomyces cerevisiae orf for phosphomevalonate kinase (ERG8).
[0095]SEQ ID NO: 49) Saccharomyces cerevisiae orf for mevalonate kinase (ERG12).
[0096]SEQ ID NO: 50) Saccharomyces cerevisiae orf for mevalonate diphosphate decarboxylase (ERG19).
[0097]SEQ ID NO: 51) Saccharomyces cerevisiae orf for acetoacetyl thiolase.
[0098]SEQ ID NO: 52) Arabidopsis thaliana orf for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase.
[0099]SEQ ID NO: 53) Arabidopsis thaliana orf for HMG-CoA reductase.
[0100]SEQ ID NO: 54) Schizosaccharomyces pombe IDI1 (IPP isomerase).
[0101]SEQ ID NO: 55) Rhodobacter capsulatus idiB (IPP isomerase).
[0102]SEQ ID NO: 56) Streptomyces sp CL190 orf encoding HMG-CoA reductase.
[0103]SEQ ID NO: 57) Streptomyces sp CL190 gene cluster containing mevalonate pathway and IPP isomerase orfs.
[0104]SEQ ID NO: 58) is Operon A containing A. thaliana and S. cerevisiae DNA SEQ ID NO: 59) is Operon B containing A. thaliana and S. cerevisiae DNA.
[0105]SEQ ID NO: 60) is Operon C containing A. thaliana, S. cerevisiae, and R. capsulatus DNA.
[0106]SEQ ID NO: 61) is Operon D containing A. thaliana, S. cerevisiae, and Streptomycs sp CL190 DNA.
[0107]SEQ ID NO. 62) is Operon E containing A. thaliana, S. cerevisiae, Streptomycs sp CL190 DNA, and R. capsulatus DNA.
[0108]SEQ ID NO: 63) is Operon F containing S. cerevisiae and Streptomycs sp CL190 DNA.
[0109]SEQ ID NO: 64) is Operon G containing A. thaliana, S. cerevisiae and S. pombe DNA.
[0110]SEQ ID NO, 65) is PCR primer containing R. capsulatus DNA.
[0111]SEQ ID NO: 66) is PCR primer containing R. capsulatus DNA.
[0112]SEQ ID NO: 67) is an Oligonucleotide containing N. tabacum and R. capsulatus DNA.
[0113]SEQ ID NO: 68) is an Oligonucleotide containing N. tabacum and R. capsulatus DNA.
[0114]SEQ ID NO: 69) is an Oligonucleotide containing N. tabacum and S. cerevisiae DNA.
[0115]SEQ ID NO: 70) is an Oligonucleotide containing N. tabacum and R. capsulatus DNA.
[0116]SEQ ID NO: 71) is Rhodobacter capsulatus orf encoding phytoene synthase (crtB).
[0117]SEQ ID NO: 72) is plastid transformation vector pHKO4, containing Operon B, containing A. thaliana and S. cerevisiae DNA.
[0118]SEQ ID NO: 73) is plastid transformation vector pHKO7, containing Operon C, containing A. thaliana, S. cerevisiae, and R. capsulatus DNA.
[0119]SEQ ID NO: 74) is plastid transformation vector pHKO8, containing Operon G, containing A. thaliana, S. cerevisiae, and S. pombe DNA.
[0120]SEQ ID NO: 75) is plastid transformation vector pFHO5 containing R. capsulatus DNA encoding phytoene synthase.
[0121]SEQ ID NO: 76) is plastid transformation vector pFHO6, containing Operon E, containing, A. thaliana, S. cerevisiae, Streptomycs sp CL190 DNA, and R. capsulatus DNA.
DETAILED DESCRIPTION
[0122]In the description that follows, a number of terms used in genetic engineering are utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
[0123]A protein is considered an isolated protein if it is a protein isolated from a host cell in which it is naturally produced. It can be purified or it can simply be free of other proteins and biological materials with which it is associated in nature for example, if it is recombinantly produced.
[0124]An isolated nucleic acid is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule, but is not flanked by both of the coding or noncoding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic or plastomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic or plastomic DNA; (c) a separate molecule such as a cDNA, a genomic or plastomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of (i) DNA molecules, (ii) transfected cells, and (iii) cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
[0125]One DNA portion or sequence is downstream of second DNA portion or sequence when it is located 3' of the second sequence. One DNA portion or sequence is upstream of a second DNA portion or sequence when it is located 5' of that sequence.
[0126]One DNA molecule or sequence and another are heterologous to one another if the two are not derived from the same ultimate natural source, or are not naturally contiguous to each other. The sequences may be natural sequences, or at least one sequence can be derived from two different species or one sequence can be produced by chemical synthesis provided that the nucleotide sequence of the synthesized portion was not derived from the same organism as the other sequence.
[0127]A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.
[0128]A nucleotide sequence is operably linked when it is placed into a fictional relationship with another nucleotide sequence. For instance, a promoter is operably linked to a coding sequence if the promoter effects its transcription or expression. Generally, operably linked means that the sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, it is well known that certain genetic elements, such as enhancers, may be operably linked even at a distance, i.e., even if not contiguous.
[0129]In a plastome, sequences are physically linked by virtue of the chromosome configuration, but they are not necessarily operably linked due to differential expression for example. Transgenes can be physically linked prior to transformation, or can become physically linked once they insert into a plastome. Transgenes can become operably linked if they share regulatory sequences upon insertion into a plastome.
[0130]The term recombinant polynucleotide refers to a polynucleotide which is made by the combination of two otherwise separated segments of sequence accomplished by the artificial manipulation of isolated segments of polynucleotides by genetic engineering techniques or by chemical synthesis. In so doing one may join together polynucleotide segments of desired functions to generate a desired combination of functions.
[0131]The polynucleotides may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Caruthers (1981) Tetra. Letts., 22:1859-1862 or the triester method according to Matteuci et al. (1981) J. Am. Chem. Soc., 103: 3185, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
[0132]Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host will typically, but not always, comprise a replication system (i.e. vector) recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably, but not necessarily, also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (expression vectors) may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides may also be included where appropriate, preferably from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.
[0133]Variants or sequences having substantial identity or homology with the polynucleotides encoding enzymes of the mevalonate pathway may be utilized in the practice of the invention. Such sequences can be referred to as variants or modified sequences. That is, a polynucleotide sequence may be modified yet still retain the ability to encode a polypeptide exhibiting the desired activity. Such variants or modified sequences are thus equivalents. Generally, the variant or modified sequence will comprise at least about 40%-60%, preferably about 60%-80%, more preferably about 80%-90%, and even more preferably about 90%-95% sequence identity with the native sequence.
[0134]Sequence relationships between two or more nucleic acids or polynucleotides are generally defined as sequence identity, percentage of sequence identity, and substantial identity. In determining sequence identity, a "reference sequence" is used as a basis for sequence comparison. The reference may be a subset or the entirety of a specified sequence. That is, the reference sequence may be a full-length gene sequence or a segment of the gene sequence.
[0135]Methods for alignment of sequences for comparison are well known in the art. See, for example, Smith et al. (1981) Adv. Appl. Math. 2:482; Needleman et al. (1970) J. Mol. Blot. 48:443; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; CLUSTAL in the PC/Gene Program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA. Preferred computer alignment methods also include the BLASTP, BLASTN, and BLASTX algorithms. See, Altschul et al. (1990) J. Mol. Biol. 215:403-410.
[0136]Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. "Percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions as compared to the reference window for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
[0137]Polynucleotide sequences having "substantial identity" are those sequences having at least about 50%-60% sequence identity, generally at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described above. Preferably sequence identity is determined using the default parameters determined by the program. Substantial identity of amino acid sequence generally means sequence identity of at least 50%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.
[0138]Nucleotide sequences are generally substantially identical if the two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5EC lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. Nucleic acid molecules that do not hybridize to each other under stringent conditions may still be substantially identical if the polypeptides they encode are substantially identical. This may occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
[0139]As noted, hybridization of sequences may be carried out under stringent conditions. By "stringent conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 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 at least about 30EC for short probes (e.g., 10 to 50 nucleotides) and at least about 60EC for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary stringent conditions include hybridization with a buffer solution of 30 to 35% formamide, 1.0 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37EC, and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55EC. It is recognized that the temperature, salt, and wash conditions may be altered to increase or decrease stringency conditions. For the post-hybridization washes, the critical factors are the ionic strength and temperature of the final wash solution. See, Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284.
[0140]As indicated, fragments and variants of the nucleotide sequences of the invention are encompassed herein. By "fragment" is intended a portion of the nucleotide sequence. Fragments of the polynucleotide sequence will generally encode polypeptides which retain the biological/enzymatic activity of the native protein. Those of skill in the art routinely generate fragments of polynucleotides of interest through use of commercially available restriction enzymes; synthetic construction of desired polynucleotides based on known sequences; or use of "erase-a-base" technologies such as Bal 31 exonuclease, by which the skilled artisan can generate hundreds of fragments of a known polynucleotide sequence from along the entire length of the molecule by time-controlled, limited digestion. Fragments that retain at least one biological or enzymatic activity of the native protein are equivalents of the native protein for that activity.
[0141]By "variants" is intended substantially similar sequences. For example, for nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of an enzyme of the mevalonate pathway. Variant nucleotide sequences include synthetically derived sequences, such as those generated for example, using site-directed mutagenesis. Generally, nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, 70%, generally 80%, preferably 85%, 90%, up to 95% sequence identity to its respective native nucleotide sequence. Activity of polypeptides encoded by fragments or variants of polynucleotides can be confirmed by assays disclosed herein.
[0142]Variant" in the context of proteins is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or human manipulation. Conservative amino acid substitutions will generally result in variants that retain biological function. Such variants are equivalents of the native protein. Variant proteins that retain a desired biological activity are encompassed within the subject invention. Variant proteins of the invention may include those that are altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulation are generally known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods and Enzymol; 154:367-382; and the references cited therein.
[0143]An expression cassette may contain at least one polynucleotide of interest to be cotransformed into the organism. Such an expression cassette is preferably provided with a plurality of restriction sites for insertion of the sequences of the invention to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
[0144]The cassette may include 5' and 3' regulatory sequences operably linked to a polynucleotide of interest. By "operably linked" is intended, for example, a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. When a polynucleotide comprises a plurality of coding regions that are operably linked such that they are under the control of a single promoter, the polynucleotide may be referred to as an "operon".
[0145]The expression cassette will optionally include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a polynucleotide sequence of interest and a transcriptional and translational termination region functional in plants or microalgae. The transcriptional initiation region, the promoter, is optional, but may be native or analogous, or foreign or heterologous, to the intended host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By "foreign" is intended that the transcriptional initiation region is not found in the native organism into which the transcriptional initiation region is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcriptional initiation region that is heterologous to the coding sequence.
[0146]The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[0147]Where appropriate, the polynucleotides of interest may be optimized for expression in the transformed organism. That is, the genes can be synthesized using plant or algae plastid-preferred codons corresponding to the plastids of the plant or algae of interest. Methods are available in the art for synthesizing such codon optimized polynucleotides. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Of course, the skilled artisan will appreciate that for the transplastomic purposes described herein, sequence optimization should be conducted with plastid codon usage frequency in mind, rather than the plant or algae genome codon usage exemplified in these references.
[0148]It is now well known in the art that when synthesizing a polynucleotide of interest for improved expression in a host cell it is desirable to design the gene such that its frequency of codon usage approaches the frequency of codon usage of the host cell. It is also well known that plastome codon usage may vary from that of the host plant or microalgae genome. For purposes of the subject invention, "frequency of preferred codon usage" refers to the preference exhibited by a specific host cell plastid in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a plastid can be calculated by averaging frequency of preferred codon usage in a number of genes expressed by the plastid. It usually is preferable that this analysis be limited to genes that are among those more highly expressed by the plastid. Alternatively, the polynucleotide of interest may be synthesized to have a greater number of the host plastid's most preferred codon for each amino acid, or to reduce the number of codons that are rarely used by the host.
[0149]The expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region), Elroy-Stein et al. (1989) PNAS USA 86:6126-6130; potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allison et al. (1986); MDMV Leader (Maize Dwarf Mosaic Virus) Virology 154:9-20; and human immunoglobulin heavy-chain binding protein (BiP), Macejak et al. (1991) Nature 353:90-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al. (1987) Nature 325:622-625; tobacco mosaic virus leader (TMV), Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256; and maize chlorotic mottle virus leader (MCMV), Lommel et al. (1991) Virology 81:382-385. See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.
[0150]In preparing an expression cassette, the various polynucleotide fragments may be manipulated, so as to provide for the polynucleotide sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the polynucleotide fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous nucleotides, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
[0151]In addition, expressed gene products may be localized to specific organelles in the target cell by ligating DNA or RNA coded for peptide leader sequences to the polynucleotide of interest. Such leader sequences can be obtained from several genes of either plant or other sources. These genes encode cytoplasmically-synthesized proteins directed to, for example, mitochondria (the F1-ATPase beta subunit from yeast or tobacco, cytochrome c1 from yeast), chloroplasts (cytochrome oxidase subunit Va from yeast, small subunit of rubisco from pea), endoplasmic reticulum lumen (protein disulfide isomerase), vacuole (carboxypeptidase Y and proteinase A from yeast, phytohaemagglutinin from French bean), peroxisomes (D-aminoacid oxidase, uricase) and lysosomes (hydrolases).
[0152]Following transformation, a plant may be regenerated, e.g., from single cells, callus tissue, or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues, and organs of the plant. Available techniques are reviewed in Vasil et al. (1984) in Cell Culture and Somatic Cell Genetics of Plants, Vols. I, II and III, Laboratory Procedures and Their Applications (Academic press); and Weissbach et al. (1989) Methods for Plant Mol. Biol.
[0153]The transformed plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
[0154]The particular choice of a transformation technology will be determined by its efficiency to transform certain target species, as well as the experience and preference of the person practicing the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant or microalgae plastids is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.
[0155]Also according to the invention, there is provided a plant or microalgae cell having the constructs of the invention. A further aspect of the present invention provides a method of making such a plant cell involving introduction of a vector including the construct into a plant cell. For integration of the construct into the plastid genome (the plastome), such introduction will be followed by recombination between the vector and the plastome genome to introduce the operon sequence of nucleotides into the plastome. RNA encoded by the introduced nucleic acid construct (operon) may then be transcribed in the cell and descendants thereof, including cells in plants regenerated from transformed material. A gene stably incorporated into the plastome of a plant or microalgae is passed from generation to generation to descendants of the plant or microalgae, so such descendants should show the desired phenotype.
[0156]The present invention also provides a plant or microalgae culture comprising a plant cell as disclosed. Transformed seeds and plant parts are also encompassed. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny, meaning descendants, not limited to the immediate generation of descendants but including all generations of descendants. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to naturally occurring, deliberate, or inadvertent caused mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
[0157]In addition to a plant or microalgae, the present invention provides any clone of such a plant or microalgae, seed, selfed or hybrid or mated descendants, and any part of any of these, such as cuttings or seed for plants. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed, and so on. Also encompassed by the invention is a plant or microalgae which is a sexually or asexually propagated off-spring, clone, or descendant of such a plant or microalgae, or any part or propagule of said plant, off-spring, clone, or descendant. Plant or microalgae extracts and derivatives are also provided.
[0158]The present invention may be used for transformation of any plant species, including, but not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), nice (Oryza saliva), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea ssp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumn occidental), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables, ornamentals, and conifers.
[0159]Preferably, plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea, and other root, tuber, or seed crops. Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, and sorghum. Horticultural plants to which the present invention may be applied may include lettuce; endive; and vegetable brassicas including cabbage, broccoli, and cauliflower; and carnations and geraniums. The present invention may be applied to tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum, petunia, rose, poplar, eucalyptus, and pine.
[0160]Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans including guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
[0161]Microalgae include but are not limited to the Chlorophyta and the Rhodophyta and may be such organisms as Chlamydomonas, Haematococcus, and Ouneliella.
[0162]Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Unless indicated otherwise, the respective contents of the documents cited herein are hereby incorporated by reference to the extent they are not inconsistent with the teachings of this specification.
[0163]Percentages and ratios given herein are by weight, and temperatures are in degrees Celsius unless otherwise indicated. The references cited within this application are herein incorporated by reference to the extent applicable. Where necessary to better exemplify the invention, percentages and ratios may be cross-combined.
EXAMPLE 1
Isolation of Orfs Encoding Enzymes of the Mevalonate Pathway for the Construction of Vectors pFCO1 and pFCO2
[0164]In an exemplified embodiment, vectors containing open reading frames (orfs) encoding enzymes of the mevalonate pathway are constructed. Polynucleotides derived from the yeast Saccharomyces cerevisiae, the plant Arabidopsis thaliana, and the eubacterium Streptomyces sp CL190 are used for the construction of vectors, including plastid delivery vehicles, containing orfs for biosynthesis of the mevalonate pathway enzymes. Construction of the vectors is not limited to the methods described. It is routine for one skilled in the art to choose alternative restriction sites, PCR primers, etc. to create analogous plasmids containing the same orfs or other orfs encoding the enzymes of the mevalonate pathway. Many of the steps in the construction of the plasmids of the subject invention can utilize the joining of blunt-end DNA fragments by ligation. As orientation with respect to the promoter upstream (5') of the described orfs can be critical for biosynthesis of the encoded polypeptides, restriction analysis is used to determine the orientation in all instances involving blunt-end ligations. A novel directional ligation methodology, chain reaction cloning (Pachuk et al., Gene 243:19-25, 2000), can also be used as an alternative to standard ligations in which the resultant orientation of the insert is not fixed. All PCR products are evaluated by sequence analysis as is well known in the art.
[0165]The construction of a synthetic operon comprising three yeast orfs encoding phosphomevalonate kinase, mevalonate kinase, and mevalonate diphosphate decarboxylase is described by Hahn et al. (Hahn et al., J. Bacteriol. 183:1-11, 2001). This same synthetic operon, contained within plasmid pFCO2, is able to synthesize, in vivo, polypeptides with enzymatic activities able to convert exogenously supplied mevalonate to IPP as demonstrated by the ability of the mevalonate pathway orfs to complement the temperature sensitive dxs::kanr lethal mutation in E. coli strain FH11 (Hahn et al., 2001).
[0166]Plasmids pFCO1 and pFCO2 containing a synthetic operon for the biosynthesis of IPP from mevalonate are constructed as follows. Three yeast orfs encoding mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase are isolated from S. cerevisiae genomic DNA by PCR using the respective primer sets
TABLE-US-00001 FH0129-2: (SEQ ID NO: 1) 5' GGACTAGTCTGCAGGAGGAGTTTTAATGTCATTACCGTTCTTAAC TTCTGCACCGGG-3' (sense) and FH0129-1: (SEQ ID NO: 2) 5' TTCTCGAGCTTAAGAGTAGCAATATTTACCGGAGCAGTTACACTA GCAGTATATACAGTCATTAAAACTCCTCCTGTGAAGTCCATGGTAAATTC G 3' (antisense); FH0211-1: (SEQ ID NO: 3) 5' TAGCGGCCGCAGGAGGAGTTCATATGTCAGAGTTGAGAGCCTTC AGTGCCCCAGGG 3' (sense) and FH0211-2: (SEQ ID NO: 4) 5' TTTCTGCAGTTTATCAAGATAAGTTTCCGGATCTTT 3' (antisense); CT0419-1: (SEQ ID NO: 5) 5' GGAATTCATGACCGTTTACACAGCATCCGTTACCGCACCCG 3' (sense) and CT0419-2: (SEQ ID NO: 6) 5' GGCTCGAGTTAAAACTCCTCTTCCTTTGGTAGACCAGTCTTTGCG 3' (antisense).
Primer FH0129-2 includes a SpeI site (underlined). Primer FH0129-1 contains an XhoI site (underlined), an AflII site (double-underlined), and 54 nucleotides (bold italics) corresponding to the 5' end of the yeast orf for mevalonate diphosphate decarboxylase. Following PCk using primers FH01029-1 and FH0129-2, a product containing the orf encoding yeast mevalonate kinase is isolated by agarose gel electrophoresis and GeneClean purified. Following restriction with SpeI-XhoI, the PCR product is inserted into the SpeI-XhoI sites of pBluescript(SK+) (Stratagene, LaJolla, Calif.) by ligation to create pBRG12. Primers FH0211-1 and FH0211-2 contain a NotI site (underlined) and a PstI site (underlined), respectively. Following PCR using primers FH0211-1 and FH0211-2, a product containing the orf encoding yeast phosphomevalonate kinase is restricted with NotI-PstI, purified by GeneClean, and inserted into pGEM-T Easy (Promega Corp, Madison, Wis.) by ligation to create pERG8. An orf encoding yeast mevalonate diphosphate decarboxylase is isolated by PCR using primers CT0419-1 and CT0419-2 and inserted directly into pGEM-T Easy by ligation to create pERG19. Restriction of pERG8 with NotI-PstI yields a 1.4 Kb DNA fragment containing the orf for phosphomevalonate kinase. Restriction of pBRG12 with NotI-PstI is followed by the insertion of the 1.4 Kb NotI-PstI DNA fragment by ligation to create pBRG812 containing the orfs for both phosphomevalonate kinase and mevalonate kinase and the 5' end of the orf for yeast mevalonate diphosphate decarboxylase. Restriction of pERG19 with AflII-XhoI yields a 1.2 Kb DNA fragment containing the 3' end of the orf for yeast mevalonate diphosphate decarboxylase missing in pBRG812. Insertion of the 1.2 Kb AflII-XhoI DNA fragment into pBRG812/AflII-XhoI by ligation yields pFCO1 containing the three yeast mevalonate pathway orfs (FIG. 1). Restriction of pFCO1 with XhoI is followed by treatment with the Klenow fragment of T7 DNA polymerase and dNTPs to create blunt ends. Subsequent restriction of pFCO1/XhoI/Klenow with Sac yields a 3.9 Kb DNA fragment containing the three yeast mevalonate pathway orfs. Following agarose gel electrophoresis and GeneClean purification of the 3.9 Kb DNA fragment, it is inserted into the SmaI-SacI sites of pNGH1-amp (Garrett et al., J. Biol. Chem. 273:12457-12465, 1998) by ligation to create pFCO2 (FIG. 2).
EXAMPLE 2
Construction of E. coli Strain FH11 (JM101/dxs::kanr/pDX4)
[0167]A mutant E. coli strain containing a disruption of the chromosomal dxs gene is constructed as described by Hamilton et al. (Hamilton et al., J. Bacteriol. 171:4617-4622, 1989). The strains are grown at 30EC or 44EC in Luria-Bertani (LB) supplemented with the following antibiotics as necessary; ampicillin (Amp) (50 (g/ml), chloramphenicol (Cam) (30 (g/nl), and kanamycin (Kan) (25 (g/ml). Within phagemid DD92 (F. R. Blattner, University of Wisconsin, Madison, Wis.) is a 19.8 Kb EcoRI fragment of E. coli genomic DNA containing dxs, the gene for DXP synthase. Following the isolation of the phage from E. coli strain LE392, DD92 is restricted with SphI, and the resultant 6.3 Kb fragment is isolated by agarose gel electrophoresis. GeneClean purification of the SphI fragment and restriction with SmaI yields a 2.0 Kb SphI-SmaI fragment containing E. coli dxs. The 2.0 Kb fragment is purified by GeneClean and inserted by ligation into the SphI-HindIII sites of pMAK705, a plasmid containing a temperature-sensitive origin of replication (Hamilton et al., J. Bacteriol. 171:4617-4622, 1989). The resulting plasmid containing wt dxs, pDX4, is restricted with SapI, a unique site located in the middle of the dxs gene, and the 5'-overhangs are filled in with Klenow and dNTPs. The blunt-ended DNA fragment is purified by GeneClean and treated with shrimp alkaline phosphatase (SAP, USB Corp., Cleveland, Ohio) according to the manufacturers instructions. pUC4K (Amersham Pharmacia Biotech, Piscataway, N.J.) is restricted with EcoRI, Klenow-treated, and the resulting 1.3 Kb blunt-ended DNA fragment containing the gene for Kan resistance is inserted into the filled-in SapI site of pDX4 by blunt-end ligation to create pDX5 with a disruption in E. coli dxs. Competent E. coli JM101 cells are transformed with pDX5, a pMAK705 derivative containing dxs::kanr, and grown to an optical density (A600) of 0.6 at 30EC. Approximately 10,000 cells are plated out on LB/Cam medium prewarmed to 44EC. The plates were incubated at 44EC, and several of the resulting colonies are grown at 44EC in 4 ml of LB/Cam medium. Four 50 ml LB/Cam cultures are started with 0.5 ml from four of the 4 ml cultures and grown overnight at 30EC. Four fresh 50 ml LB/Cam cultures are started with 100 μl of the previous cultures and grown overnight at 30EC. An aliquot of one of the 50 ml cultures is serially diluted 5×105 fold, and 5 μl is plated on LB/Cam medium. Following incubation at 30EC, the resulting colonies are used to individually inoculate 3 ml of LB medium containing Cam and Kan. Twelve LB/Cain/Kan cultures are grown overnight at 30EC and used for plasmid DNA isolation. E. coli cells where the disrupted copy of dxs is incorporated into the genome are identified by restriction analysis of the isolated plasmid DNA and verified by sequence analysis of the DNA contained in the plasmids. The E. coli JM101 derivative containing the dxs::kanr mutation is designated FH11 (Hahn et al. 2001).
EXAMPLE 3
Assay Demonstrating Synthesis of IPP from Mevalonic Acid in E. coli
[0168]The episomal copy of dxs contained on pDX4 in E. coli strain FH11 is "turned off" at 44EC due to a temperature sensitive origin of replication on the pMAK705 derivative (Hamilton et al., J. Bacteriol. 171:4617-4622, 1989). The inability of FH11 to grow at the restrictive temperature demonstrates that dxs is an essential single copy gene in E. coli (Hahn et al., 2001). A cassette containing three yeast mevalonate pathway orfs is removed from pFCO1 and inserted into pNGH1-Amp to form pFCO2 for testing the ability of the mevalonate pathway orfs to complement the dxs::kanr disruption when FH11 is grown at 44EC on medium containing mevalonate. The utility of strain FH11 as a component of an assay for testing the ability of mevalonate pathway orfs to direct the synthesis of IPP is demonstrated as follows;
[0169]Colonies of E. coli strain FH11 transformed with pFCO2 or pNGH1-Amp, the expression vector without an insert, are isolated by incubation at 30EC on LB plates containing Kan and Amp. Four ml LB/Kan/Amp cultures containing either FH11/pFCO2 or FH11/pNGH1-Amp are grown overnight at 30EC. Following a 10.000-fold dilution, 10 μl portions from the cultures are spread on LB/Kan/Amp plates that are prewarmed to 44E C or are at rt. Approximately 1.3 mg of mevalonic acid is spread on each plate used for FH11/pFCO2. The prewarmed plates are incubated at 44EC, and the rt plates are incubated at 30EC overnight.
[0170]FH11/pNGH1-amp cells will not grow at the restrictive temperature of 44EC and FH11/pFCO2 cells are unable to grow at of 44EC unless mevalonic acid (50 mg/L) is added to the growth medium thus establishing the ability of the polypeptides encoded by the mevalonate pathway orfs contained in the synthetic operon within pFCO2 to form IPP from mevalonate in vivo (Hahn et al., 2001).
EXAMPLE 4
Isolation of Mevalonate Pathway Orfs
[0171]In a specific, exemplified embodiment, the isolation of orfs, each encoding a polypeptide with either HMG-CoA synthase enzyme activity, HMG-CoA reductase enzyme activity, or acetoacetyl-CoA thiolase enzyme activity, and construction of vectors containing these orfs is as follows: Synthesis of A. thaliana first strand cDNAs is performed utilizing PowerScriptJ (reverse transcriptase (Clontech Laboratories, Inc., Palo Alto, Calif.) according to the manufacturer's instructions. Specifically, a microfuge tube containing 5 μl of A. thaliana RNA (Arabidopsis Biological Resource Center, Ohio State University, Columbus, Ohio), 1.8 μl poly(dT)15 primer (0.28 μg/μl, Integrated DNA Technologies, Inc. Coralville, Iowa), and 6.2 μl DEPC-treated H2O is heated at 70EC for 10 min and then immediately cooled on ice. The mixture is spun down by centrifugation and 4 μl of 5× First-Strand Buffer (Clontech), 2μ(l Advantage UltraPure PCR dNTP mix (10 mM each, Clontech) and 2μ(l 100 mM DTT are added and the entire contents mixed by pipetting. Following the addition of 1μ(l reverse transcriptase (Clontech) and mixing by pipetting, the contents are incubated at 42E C for 90 min and then heated at 70EC for 15 min to terminate the reaction.
[0172]The resulting A. thaliana first strand cDNAs are used as templates for the synthesis of an orf encoding HMG-CoA synthase and a truncated HMG-CoA reductase by PCR in a Perkin-Elmer GeneAmp PCR System 2400 thermal cycler utilizing the Advantage7-HF 2 PCR Kit (Clontech) according to the manufacturer's instructions. An A. thaliana HMG-CoA synthase orf is isolated using the following PCR primers:
TABLE-US-00002 (SEQ ID NO: 7) 1) 5' GCTCTAGATGCGCAGGACGCACATATGGCGAAGAACGTTGGGAT TTTGGCTATGGATATCTATTTCCC 3' (sense); and (SEQ ID NO: 8) 2) 5' CGCTCGAGTCGACGGATCCTCAGTGTCCATTGGCTACAGATCCA TCTTCACCTTTCTTGCC 3' (antisense);
containing the restriction site XbaI shown underlined, the restriction site XhoI shown in bold italic and the restriction site SalI shown double underlined. Specifically, 2 (l cDNA, 5 μl 10×HF 2 PCR Buffer (Clontech), 5 μl 10× HF 2 dNTP Mix (Clontech), 1 μl each of the primers described above, 1 μl 50× Advantage-HF 2 Polymerase Mix (Clontech), and 35 μl PCR-Grade H2O (Clontech) are combined in a 0.5 ml PCR tube. The mixture is heated at 94EC for 15 sec then subjected to 40 PCR cycles consisting of 15 sec at 94EC and 4 min at 68EC. After a final incubation at 68EC for 3 min, the reaction is cooled to 4EC. Agarose gel electrophoresis is performed on a 10 μl aliquot to confirm the presence of a DNA fragment of the predicted size of 1.4 Kb. The PCR is repeated in triplicate to generate enough product for its isolation by gel excision and purification by GeneClean (Qbiogene, Inc., Carlsbad Calif.). Following restriction with XbaI-XhoI and purification by GeneClean, the 1.4 Kb PCR product is inserted into the XbaI-XhoI sites of pBluescript(SK+) by ligation to form putative pBSHMGS constructs. Sequence analysis of several of the candidate constructs is performed to identify inserts with DNA identical to the published A. thaliana orf for HMG-CoA synthase and are used for the construction of pBSHMGSR as described below.
[0173]An A. thaliana orf encoding a polypeptide with HMG-CoA reductase enzyme activity is synthesized by PCR essentially as described above using the following primers:
3) 5' CCGCTCGAGCACGTGGAGGCACATATGCAATGCTGTGAGATGCCT GTTGGATACATTCAGATTCCTGTTGGG 3' (sense) (SEQ ID NO: 9); and4) 5' GGGTACCTGCGGCCGGATCCCGGGTCATGTTGTTGTTGTTGTCGT TGTCGTTGCTCCAGAGATGTCTCGG 3' (antisense) (SEQ ID NO: 10); containing the restriction site XhoI shown underlined, the restriction site KpnI shown in italic, the restriction site EagI shown in bold, and the restriction site SmaI shown double underlined. The 1.1 Kb PCR product is isolated by agarose gel electrophoresis, purified by GeneClean and inserted into the pT7Blue-3 vector (Novagen, Inc., Madison, Wis.) using the Perfectly BluntJ Cloning Kit (Novagen) according to the manufacturer's instructions. Sequence analysis is performed to identify constructs containing A. thaliana DNA encoding the desired C-terminal portion of the published HMG-CoA reductase amino acid sequence and are designated pHMGR.
[0174]PCR is performed on S. cerevisiae genomic DNA (Invitrogen, Corp., Carlsbad, Calif.) by using the Advantage®-HF 2 PCR Kit (Clontech) according to the manufacturer's instructions and the following primers;
TABLE-US-00003 (SEQ ID NO: 11) 5) 5' ACAACACCGCGGCGGCCGCGTCGACTACGTAGGAGGCACATATGTC TCAGAACGTTTACATTGTATCGACTGCC 3' (sense); and (SEQ ID NO: 12) 6) 5' GCTCTAGAGGATCCTCATATCTTTTCAATGACAATAGAGGAAGCACC ACCACC 3' (antisense);
containing the restriction site NotI shown underlined, the restriction site SacII shown in italic, the restriction site Sail shown in bold, the restriction site SnaBI shown double underlined, and the restriction site XbaI in bold italic. The 1.2 Kb PCR product is isolated by agarose gel electrophoresis, purified by GeneClean and inserted into the vector pT7Blue-3 (Novagen,) using the Perfectly BluntJ Cloning Kit (Novagen) according to the manufacturer's instructions. Sequence analysis is performed to identify constructs containing S. cerevisiae DNA identical to the published orf encoding acetoacetyl-CoA thiolase and they are designated pAACT.
EXAMPLE 5
Construction of pHKO1
[0175]In an exemplified embodiment, a pBluescript(SK+) derivative containing an operon with orfs encoding polypeptides with enzymatic activities for HMG-CoA synthase, HMG-CoA reductase, and acetoacetyl-CoA thiolase is constructed as follows: Following restriction of pHMGR with XhoI-KpnI, isolation of the 1.1 Kb DNA fragment by agarose gel electrophoresis, and purification by GeneClean, the 1.1 Kb XhoI-KpnI DNA fragment containing the orf encoding the C-terminal portion of A. thaliana HMG-CoA reductase is inserted into the SalI-KpnI sites of pBSHMGS by ligation to create pBSHMGSR. Following restriction of pAACT with SacII-XbaI, isolation of the 1.2 Kb DNA fragment containing the orf encoding yeast acetoacetyl-CoA thiolase by agarose gel electrophoresis, and purification by GeneClean, the 1.2 Kb SacII-XbaI DNA fragment is inserted into the SacII-XbaI sites of pBSHMGSR by ligation to create pHKO1 (FIG. 3).
EXAMPLE 6
Construction of pHKO2
[0176]In a specific, exemplified embodiment, a vector containing a synthetic operon consisting of six orfs encoding polypeptides with acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase enzymatic activities, thus comprising the entire mevalonate pathway, is constructed as follows: Restriction of pHKO1 with EagI yields a 3.7 Kb DNA fragment containing orfs encoding yeast acetoacetyl-CoA thiolase, A. thaliana HMG-CoA synthase, and a truncated A. thaliana HMG-CoA reductase. Following isolation of the 3.7 Kb EagI DNA fragment by agarose gel electrophoresis and purification by GeneClean, it is directionally inserted into the NotI site of pFCO2 (Hahn et al., 2001) utilizing the methodology of chain reaction cloning (Pachuk et al., 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the following bridge oligonucleotide primers:
TABLE-US-00004 (SEQ ID NO: 13) 1) 5' TGGAATTCGAGCTCCACCGCGGTGGCGGCCGCGTCGACGCCGGC GGAGGCACATATGTCT 3'; and (SEQ ID NO: 14) 2) 5' AACAACAACAACATGACCCGGGATCCGGCCGCAGGAGGAGTTCA TATGTCAGAGTTGAGA 3';
as follows. Agarose gel electrophoresis is performed on the 8.1 Kb pFCO2/NotI DNA fragment and the 3.7 Kb EagI DNA fragment isolated from pHKO1 to visually estimate their relative concentrations. Approximately equivalent amounts of each fragment totaling 4.5 μl, 1 μl of each bridge oligo at a concentration of 200 nM, 5 μl Ampligase® 10× Reaction Buffer (Epicentre), 3 μl Ampligase® (5 U/(l) (Epicentre), and 35.5 μl PCR grade H2O are added to a 0.5 ml PCR tube. The mixture is heated at 94EC for 2 min then subjected to 50 PCR cycles consisting of 30 sec at 94EC, 30 sec at 60EC, and 1 min at 66EC. After a final incubation at 66E C for 5 min, the reaction is cooled to 4EC. Colonies resulting from the transformation of E. coli strain NovaBlue (Novagen) with 1 μl of the directional ligation reaction are grown in LB medium supplemented with ampicillin at a final concentration of 50 μg/ml. Restriction analysis with NaeI-KpnI of mini-prep plasmid DNA from the liquid cultures is performed to identify candidate pHKO2 constructs by the presence of both a 5.7 and a 6.2 Kb DNA fragment. Further analysis by restriction with SmaI-XhoI to generate both a 3.9 and 7.9 Kb DNA fragment confirms the successful construction of pHKO2 (FIG. 4).
EXAMPLE 7
Assay Demonstrating the Synthesis of IPP from Acetyl-CoA in E. coli
[0177]In a specific, exemplified embodiment, a derivative of pNGH1-amp (Hahn et al., 2001), containing the entire mevalonate pathway, is assayed (FIG. 5) for its ability to synthesize IPP from endogenous acetyl-CoA in E. coli strain FH11, containing the temperature sensitive dxs::kanrr knockout (Hahn et al., 2001), as follows: Colonies resulting from the transformation of FH11, by pHKO2, containing orfs encoding polypeptides with enzymatic activities for acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, are isolated by incubation at 30EC on LB plates containing Kan and Amp. Several 4 ml LB/Kan/amp samples are individually inoculated with single colonies from the FH11/pHKO2 transformation. Following growth at 30EC overnight, the FH11/pHKO2 cultures are diluted 100,000-fold, and 5 μl aliquots are spread on LB/Kan/amp plates at room temperature (rt) or that are prewarmed to 44EC. The prewarmed plates are incubated at 44EC, and the rt plates are incubated at 30EC overnight. FH11 and FH11/pNGH1 amp cells will not grow at the restrictive temperature of 44EC (Hahn et al., 2001). FH11/pHKO2 cells are able to grow at 44EC, thus establishing the ability, of a synthetic operon comprising the entire mevalonate pathway, to form IPP from acetyl-CoA and thereby overcome the dxs::kanr block to MEP pathway biosynthesis of IPP in E. coli strain FH11.
EXAMPLE 8
Construction of pHKO3
[0178]In another exemplified embodiment, a derivative of pBluescript(SK+) containing an operon comprising orfs, which in their summation is the entire mevalonate pathway, is constructed as follows: pHKO1, containing orfs encoding acetoacetyl-CoA thiolase, HMG-CoA synthase, and an N-terminal truncated HMG-CoA reductase, is restricted with SalI-NotI and purified by GeneClean. The pBluescript(SK+) derivative pFCO1, containing the orfs encoding mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, has been described above in Example 1. Following restriction of pFCO1 with XhoI-NotI, isolation by agarose gel electrophoresis, and purification by GeneClean, the 3.9 Kb DNA fragment containing the mevalonate pathway orfs is inserted into pHKO1/SalI-NotI by directional ligation (Pachuk et al., 2000) utilizing thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the following bridging oligonucleotides:
TABLE-US-00005 (SEQ ID NO: 15) 1) 5' CTCAACTCTGACATATGAACTCCTCCTGCGGCCGCCGCGGTGGA GCTCCAGCTTTTGTTCCC 3'; and (SEQ ID NO: 16) 2) 5' GGTCTACCAAAGGAAGAGGAGTTTTAACTCGACGCCGGCGGAGG CACATATGTCTCAGAACG 3';
essentially as described for the construction of pHKO2. Restriction analysis is performed with KpnI to confirm the successful construction of pHKO3 (FIG. 6).
EXAMPLE 9
Construction of Tobacco Plastid Transformation Vector pHKO4
[0179]In an exemplified embodiment, a vector containing a Nicotiana tabacum plastid pseudogene is utilized to create a plastid transformation vector as follows: The pBluescript(SK+) derivative designated as pBSNT27 (FIG. 7, SEQ ID NO: 17) contains a 3.3 Kb BglI-BamHI DNA fragment of the N. tabacum chloroplast genome corresponding approximately to base-pairs 80553-83810 of the published nucleotide sequence (Sugiura, M., 1986, and Tsudsuki, T., 1998.). A unique restriction site contained within the tobacco infA pseudogene located on pBSNT27 is cleaved with BglII and the resulting 5' overhangs are filled in with Klenow and dNTPs. The resulting 6.2 Kb blunt-ended DNA fragment is GeneClean purified. Following restriction of pHKO3 with EagI, filling in of the resulting 5' overhangs with Klenow and dNTPs, isolation by agarose gel electrophoresis, and purification by GeneClean, the resulting 7.7 Kb blunt-ended DNA fragment, containing orfs encoding the entire mevalonate pathway, is directionally inserted into the blunt-ended BglII site of pBSNT27 utilizing chain reaction cloning (Pachuk et al., 2000.), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the following bridging oligonucleotides:
TABLE-US-00006 (SEQ ID NO: 18) 1) 5' GATCTTTCCTGAAACATAATTTATAATCAGATCGGCCGCAGGAG GAGTTCATATGTCAGAGTTGAG 3'; and (SEQ ID NO: 19) 2) GACAACAACAACAACATGACCCGGGATCCGGCCGATCTAAACAAACC CGGAACAGACCGTTGGGAA 3';
to form the tobacco plastid-specific transformation vector pHKO4 (FIG. 8).
[0180]Alternatively, other derivatives of pBSNT27 can be constructed, using skills as known in the art, that are not reliant upon an available restriction site(s) in the pseudogene. For example, although the infA pseudogene comprises basepairs 3861-4150 in pBSNT27, there are unique restriction sites in close proximity, upstream and downstream, that can be utilized to excise the entire pseudogene followed by its replacement with an orf or gene cluster comprising multiple orfs, e.g., the complete mevalonate pathway described above. Specifically, there is a unique BsrGI site at 3708 base pairs and a unique SexAI restriction site at 4433 base pairs within pBSNT27. Thus, as will be readily apparent to those skilled in the art, one can replace the infA pseudogene entirely by inserting a BsrGI-SexAI DNA fragment containing DNA, comprising orfs encoding the entire mevalonate pathway, that is flanked by the excised DNA originally flanking the infA pseudogene, i.e. DNA corresponding to 3708-3860 and 4151-4433 base pairs in pBSNT27. The resultant construct will be missing the pseudogene, but will contain the excised flanking DNA restored to its original position and now surrounding the mevalonate pathway orfs. Also, a similar strategy, that will also be apparent to those skilled in the art in view of this disclosure can be employed that restores the intact pseudogene to a location between the DNA originally flanking it, yet linked to an orf or orfs located upstream and/or downstream of the pseudogene and adjacent to the original flanking DNA.
EXAMPLE 10
Construction of Vectors Containing Orfs Encoding IPP Isomerase (pHKO5 and pHKO6)
[0181]In a specific, exemplified embodiment, orfs encoding IPP isomerase are isolated and vectors containing an operon comprising orfs for the entire mevalonate pathway and an additional orf for IPP isomerase are constructed as follows: A Rhodobacter capsulatus orf encoding a polypeptide with IPP isomerase activity is isolated by PCR from genomic DNA (J. E. Hearst, Lawrence Berkeley Laboratories, Berkeley, Calif.) using the following primers:
TABLE-US-00007 (SEQ ID NO: 20) 1) 5' CGCTCGAGTACGTAAGGAGGCACATATGAGTGAGCTTATACCCG CCTGGGTTGG 3' (sense); and (SEQ ID NO: 21) 2) 5' GCTCTAGAGATATCGGATCCGCGGCCGCTCAGCCGCGCAGGATC GATCCGAAAATCC 3' (antisense);
containing the restriction sites XhoI shown underlined, BsaAI shown in bold, XbaI shown in italic, EcoRV shown double underlined, and NotI shown in bold italic. The PCR product is restricted with XhoI-XbaI, isolated by agarose gel electrophoresis, purified by GeneClean, and inserted into the XhoI-XbaI sites of pBluescript(SK+) by ligation to form pBSIDI. Sequence analysis is performed to identify the plasmids containing R. capsulatus DNA identical to the complementary sequence of base pairs 34678-34148, located on contig rc04 (Rhodobacter Capsulapedia, University of Chicago, Chicago, Ill.). Following restriction of pBSIDI with BsaAI-EcoRV, agarose gel electrophoresis and GeneClean purification, the 0.5 Kb BsaAI-EcoRV DNA fragment containing the R. capsulatus orf is inserted into the dephosphorylated SmaI site of pHKO3 by blunt-end ligation to create pHKO5 (FIG. 9). This establishes the isolation of a previously unknown and unique orf encoding R. capsulatus IPP isomerase.
[0182]A Schizosaccharomyces pombe orf encoding a polypeptide with IPP isomerase activity is isolated from plasmid pBSF19 (Hahn and Poulter, J. Biol. Chem. 270:11298-11303, 1995) by PCR using the following primers
TABLE-US-00008 (SEQ ID NO: 22) 3) 5' GCTCTAGATACGTAGGAGGCACATATGAGTTCCCAACAAGAGAA AAAGGATTATGATGAAGAACAATTAAGG 3' (sense); and (SEQ ID NO: 23) 4) 5' CGCTCGAGCCCGGGGGATCCTTAGCAACGATGAATTAAGGTATC TTGGAATTTTGACGC 3' (antisense);
containing the restriction site BsaAI shown in bold and the restriction site SmaI shown double underlined. The 0.7 Kb PCR product is isolated by agarose gel electrophoresis, purified by GeneClean and inserted into the pT7Blue-3 vector (Novagen, Inc., Madison, Wis.) using the Perfectly BluntJ Cloning Kit (Novagen) according to the manufacturer's instructions. Sequence analysis is performed to identify constructs containing S. pombe DNA identical to the published DNA sequence (Hahn and Poulter, 1995) and are designated pIDI. Following restriction of pIDI with BsaAI-SmaI, isolation by agarose gel electrophoresis, and purification by GeneClean, the 0.7 Kb BsaAI-SmaI DNA fragment containing the orf encoding S. pombe IPP isomerase is inserted into the dephosphorylated SmaI site of pHKO3 by blunt-end ligation to create pHKO6.
EXAMPLE 11
Construction of Vectors Containing Alternative Orfs for Mevalonate Pathway Enzymes and IPP Isomerase
[0183]In another exemplified embodiment, vectors containing open reading frames (orfs) encoding enzymes of the mevalonate pathway and IPP isomerase other than those described above are constructed. Polynucleotides derived from the yeast Saccharomyces cerevisiae, the plant Arabidopsis thaliana, and the bacteria Rhodobacter capsulatus and Streptomyces sp strain CL190 are used for the construction of vectors, including plastid delivery vehicles, containing orfs for biosynthesis of the encoded enzymes. Construction of the vectors is not limited to the methods described. One skilled in the art may choose alternative restriction sites, PCR primers, etc. to create analogous plasmids containing the same orfs or other orfs encoding the enzymes of the mevalonate pathway and IPP isomerase.
[0184]Specifically, by way of example, genomic DNA is isolated from Streptomyces sp strain CL190 (American Type Culture Collection, Manassas, Va.) using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's instructions. An off encoding a polypeptide with HMG-CoA reductase activity (Takahashi et al., J. Bacteriol. 181:1256-1263, 1999) is isolated from the Streptomyces DNA by PCR using the following primers:
TABLE-US-00009 (SEQ ID NO: 24) 1) 5' CCGCTCGAGCACGTGAGGAGGCACATATGACGGAAACGCACGCC ATAGCCGGGGTCCCGATGAGG 3' (sense); and (SEQ ID NO: 25) 2) 5' GGGGTACCGCGGCCGCACGCGTCTATGCACCAACCTTTGCGGTC TTGTTGTCGCGTTCCAGCTGG 3' (antisense);
containing the restriction site XhoI shown underlined, the restriction site KpnI shown in italics, the restriction site NotI shown in bold, and the restriction site MluI shown double underlined. The 1.1 Kb PCR product is isolated by agarose gel electrophoresis, purified by GeneClean and inserted into the pT7Blue-3 vector (Novagen, Inc., Madison, Wis.) using the Perfectly BluntJ Cloning Kit (Novagen) according to the manufacturer's instructions. Sequence analysis is performed to identify constructs containing Streptomyces sp CL190 DNA identical to the published sequence and are designated pHMGR2.
[0185]Alternatively, using skills as known in the art, an orf encoding a truncated S. cerevisiae HMG-CoA reductase (Chappel et al., U.S. Pat. No. 5,349,126 1994) can be isolated by PCR and inserted into pT7Blue-3 (Novagen, Inc., Madison, Wis.) to construct a vector for use in building a gene cluster comprising the entire mevalonate pathway, in an analgous fashion to the use of the Streptomyces sp CL190 orf encoding HMG-CoA reductase, as described herein.
[0186]Following restriction of pAACT (see Example 4) with SacII-XbaI, isolation of the 1.2 Kb DNA fragment containing the orf encoding yeast acetoacetyl-CoA thiolase by agarose gel electrophoresis, and purification by GeneClean, the 1.2 Kb SacII-XbaI DNA fragment is inserted into the SacII-XbaI sites of pBSHMGS (see Example 4) by ligation to create pBSCTGS. Following restriction of pHMGR2 with Mol-KpnI, isolation of the 1.1 Kb DNA fragment by agarose gel electrophoresis, and purification by GeneClean, the 11.1 Kb XhoI-KpnI DNA fragment containing the orf encoding Streptomyces sp CL190 HMG-CoA reductase is inserted into the Mol-KpnI sites of pBSCTGS by ligation to create the pBluescript(SK+) derivative, pFHO1 (FIG. 10).
[0187]A derivative of pFHO1 containing an operon with orfs, which in their summation comprise the entire mevalonate pathway, is constructed as follows: pFHO1 is restricted with SnaBI and the resulting 6.6 Kb blunt-ended DNA fragment is purified by GeneClean. Following the restriction of pFCO1 (see Example 1) with NotI-XhoI, the resulting 3.9 Kb DNA fragment is isolated by agarose gel electrophoresis and purified by GeneClean. The 5' overhangs of the 3.9 Kb DNA fragment are filled in with Klenow and dNTPs. Following purification by GeneClean, the blunt-ended DNA fragment containing three mevalonate pathway orfs (Hahn et al., 2001) is inserted into the SnaBI site of pFHO1 utilizing directional ligation methodology (Pachuk et al., 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the bridging oligonucleotides:
TABLE-US-00010 (SEQ ID NO: 26) 3) 5' GAGCTCCACCGCGGCGGCCGCGTCGACTACGGCCGCAGGAGGAG TTCATATGTCAGAGTT 3'; and (SEQ ID NO: 27) 4) 5' TCTACCAAAGGAAGAGGAGTTTTAACTCGAGTAGGAGGCACATA TGTCTCAGAACGTTTA 3'; to form pFHO2 (FIG. 11).
[0188]A derivative of pFHO2 containing an operon with orfs, which in their summation comprise the entire mevalonate pathway and an orf encoding IPP isomerase is constructed as follows: pFHO2 is restricted with MluI and the resulting 5' overhangs are filled in with Klenow and dNTPs. The 10.6 Kb blunt-ended DNA fragment is purified by GeneClean. Following restriction of pBSIDI with BsaAI-EcoRV, agarose gel electrophoresis and GeneClean purification, the resulting blunt-ended 0.5 Kb DNA fragment containing the R. capsulatus IPP isomerase orf is inserted into the filled in MluI site of pFHO2 utilizing directional ligation methodology (Pachuk et al., 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the following bridging oligonucleotides:
TABLE-US-00011 (SEQ ID NO: 28) 5) 5' CAAGACCGCAAAGGTTGGTGCATAGACGCGGTAAGGAGGCACAT ATGAGTGAGCTTATAC 3'; and (SEQ ID NO: 29) 6) 5' CCTGCGCGGCTGAGCGGCCGCGGATCCGATCGCGTGCGGCCGCG GTACCCAATTCGCCCT 3'; to form pFHO3 (FIG. 12).
[0189]Following the restriction of pBluescript(SK+) with SacII-XbaI and purification by GeneClean, a 1.3 Kb SacII-XbaI DNA fragment containing the orf encoding S. cerevisiae acetoacetyl-CoA thiolase, isolated from pAACT (see Example 4) by restriction and agarose gel electrophoresis, is inserted into pBluescript(SK+)/SacII-XbaI by ligation. The resulting plasmid, pBSAACT, is restricted with XbaI, treated with Klenow and dNTPs, and purified by GeneClean. Following restriction of Streptomyces sp CL190 genomic DNA with SnaBI, a blunt-ended 6.8 Kb DNA fragment, containing five (5) orfs encoding polypeptides with HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase and IPP isomerase enzymatic activities (Takagi et al., J. Bacteriol. 182:4153-4157, 2000 and Kuzuyama et al., Proc. Natl. Acad. Sci. USA 98:932-7, 2001), is isolated by agarose gel electrophoresis, purified by GeneClean and inserted into the filled in XbaI site of pBSAACT utilizing directional ligation methodology (Pachuk et al., 2000), thermostable Ampligasequadrature (Epicentre Technologies, Madison, Wis.), and the bridging oligonucleotides:
TABLE-US-00012 (SEQ ID NO: 30) 7) 5' TGTCATTGAAAAGATATGAGGATCCTCTAGGTACTTCCCTGGCG TGTGCAGCGGTTGACG 3'; and (SEQ ID NO: 31) 8) 5' CGATTCCGCATTATCGGTACGGGTGCCTACCTAGAACTAGTGGA TCCCCCGGGCTGCAGG 3';
to form pFHO4 (FIG. 13). Transformation experiments to isolate pFHO4 constructs are performed with E. coli competent cells utilizing media containing ampicillin. Alternatively, media containing only fosmidomycin (20 μg/ml) as the selection agent is used for the direct isolation of pFHO4 constructs containing the Streptomyces sp CL190 gene cluster.The construction of vectors pHKO2, pHKO3, pHKO5, pHKO6, pFHO2, pFHO3, and pFHO4, illustrates the many ways of combining orfs isolated from a variety of organisms to encode polypeptides such that in their summation they comprise the entire mevalonate pathway or comprise the entire mevalonate pathway and IPP isomerase.
EXAMPLE 12
Construction of Tobacco Plastid Transformation Vectors pHKO7 and pHKO8
[0190]In a specific, exemplified embodiment, tobacco plastid-specific transformation vectors containing orfs, which in their summation comprise the mevalonate pathway, and an additional off encoding IPP isomerase are constructed as follows: Restriction of pHKO5 with NotI generates a DNA fragment containing six orfs comprising the entire mevalonate pathway and an additional orf encoding R. capsulatus IPP isomerase. Restriction of pHKO6 with EagI generates a DNA fragment containing the six orfs comprising the complete mevalonate pathway and an additional orf encoding S. pombe IPP isomerase. Following isolation by agarose gel electrophoresis and purification by GeneClean, the 8.2 Kb NotI DNA fragment from pHKO5 is blunt-ended with Klenow and dNTPs and inserted into the blunt-ended BglII site of pBSNT27 utilizing chain reaction cloning (Pachuk et al., 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the following bridging oligonucleotides:
TABLE-US-00013 (SEQ ID NO: 32) 1) 5' CTTTCCTGAAACATAATTTATAATCAGATCGGCCGCAGGAGGAG TTCATATGTCAGAGTT 3'; and (SEQ ID NO: 33) 2) 5'TTCGGATCGATCCTGCGCGGCTGAGCGGCCGATCTAAACAAACCC GGAACAGACCGTTGG 3';
to create the plastid delivery vehicle pHKO7 (FIG. 14) containing orfs encoding the entire mevalonate pathway and an orf encoding R. capsulatus IPP isomerase. Following isolation by agarose gel electrophoresis and purification by GeneClean, the 8.4 Kb EagI DNA fragment from pHKO6 is blunt-ended with Klenow and dNTPs and inserted into the blunt-ended BglII site of pBSNT27 utilizing chain reaction cloning (Pachuk et al, 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the following bridging oligonucleotides:
TABLE-US-00014 (SEQ ID NO: 34) 3) 5' CTTTCCTGAAACATAATTTATAATCAGATCGGCCGCAGGAGGAG TTCATATGTCAGAGT 3'; and (SEQ ID NO: 35) 4) 5' TCGTTGCTAAGGATCCCCCGGGATCCGGCCGATCTAAACAAACC CGGAACAGACCGTTGG 3';
to create the plastid delivery vehicle pHKO8 containing orfs encoding the entire mevalonate pathway plus the S. pombe IPP isomerase orf.
[0191]Alternatively, either of the IPP isomerase orfs described above can be solely inserted, without orfs for the mevalonate pathway, directly into pBSNT27 (or into any suitable plant transformation vector, known in the art), using skills known in the art.
EXAMPLE 13
Construction of Vectors Used for Increasing Carotenoid Production (pHKO9, pHK10, pHK11, pHK12, and pHK13)
[0192]In yet another exemplified embodiment, a derivative of pTrcHisB (Invitrogen) containing a synthetic operon comprising orfs, which in their summation is the entire mevalonate pathway, is constructed as follows: A unique NotI site was inserted into pTrcHisB utilizing the following oligonucleotides:
TABLE-US-00015 1) 5' CATGGCGGCCGCG 3'; (SEQ ID NO: 36) and 2) 5' GATCCGCGOCCGC 3'; (SEQ ID NO: 37)
that upon annealing, form a double-stranded DNA linker containing NotI with 5' overhangs compatible with StyI and BamHI. Following restriction of pTrcHisB with StyI-BamHI, isolation of the resulting 4.3 Kb DNA fragment by agarose gel electrophoresis, and its purification by GeneClean, the NotI linker was inserted into pTrcHisB/StyI-BamHI by ligation. Restriction analysis with BsaAI-NotI confirms the successful construction of pTrcHisB-NotI (pTHBN1) by the presence of both 2.5 and 1.8 Kb DNA fragments. Following restriction of pHKO3 with EagI, the 7.7 Kb DNA fragment, containing the six mevalonate pathway orfs, is isolated by agarose gel electrophoresis, purified by GeneClean, and inserted into the NotI site of pTHBN1 utilizing directional ligation methodology (Pachuk et al., 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the bridging oligonucleotides:
TABLE-US-00016 (SEQ ID NO: 38) 3) 5' TTAAATAAGGAGGAATAAACCATGGCGGCCGCAGGAGGAGTTCA TATGTCAGAGTTGAGA 3'; and (SEQ ID NO: 39) 4) 5' AACAACAACAACATGACCCGGGATCCGGCCGCGATCCGAGCTCG AGATCTGCAGCTGGTA 3';
to form pHKO9 (FIG. 15).
[0193]Derivatives of pTHBN1 containing the entire mevalonate pathway plus an additional orf encoding IPP isomerase are constructed as follows: Following restriction of pHKO5 with NotI, the 8.2 Kb DNA fragment, containing the six mevalonate pathway orfs plus an orf encoding R. capsulatus IPP isomerase, is isolated by agarose gel electrophoresis, purified by GeneClean, and inserted into the NotI site of pTHBN1 utilizing directional ligation methodology (Pachuk et al., 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the bridging oligonucleotides:
TABLE-US-00017 (SEQ ID NO: 40) 5) 5' TCGATTAAATAAGGAGGAATAAACCATGGCGGCCGCAGGAGGAG TTCATATGTCAGAGTT 3'; and (SEQ ID NO: 41) 6) 5' GATTTTCGGATCGATCCTGCGCGGCTGAGCGGCCGCGATCCGAG CTCGAGATCTGCAGCT 3';
to form pHK10 (FIG. 16). Following restriction of pHKO6 with EagI, the 8.4 Kb DNA fragment, containing the six mevalonate pathway orfs plus an orf encoding S. pombe IPP isomerase, is isolated by agarose gel electrophoresis, purified by GeneClean, and inserted into the NotI site of pTHBN1 utilizing directional ligation methodology (Pachuk et al., 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the following bridging oligonucleotides:
TABLE-US-00018 (SEQ ID NO: 42) 7) 5' TCGATTAAATAAGGAGGAATAAACCATGGCGGCCGCAGGAGGAG TTCATATGTCAGAGTT 3'; and (SEQ ID NO: 43) 8) 5' TTCATCGTTGCTAAGGATCCCCCGGGATCCGGCCGCGATCCGAG CTCGAGATCTGCAGCT 3'; to form pHK11.
[0194]Derivatives of pTHBN1 containing only an orf encoding IPP isomerase are constructed as follows: pTHBN1 is restricted with NotI and the resulting 5' overhangs are filled in with Klenow and dNTPs. The 4.3 Kb pTHBN1/NotI blunt-ended DNA fragment is GeneClean purified. Following restriction of pBSIDI with BsaAI-EcoRV, agarose gel electrophoresis and GeneClean purification, the resulting blunt-ended 0.5 Kb DNA fragment containing the R. capsulatus IPP isomerase orf is inserted into the filled in NotI site of pTHBN1 utilizing chain reaction cloning (Pachuk et al., 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the following bridging oligonucleotides:
TABLE-US-00019 (SEQ ID NO: 44) 9) 5' TTAAATAAGGAGGAATAAACCATGGCGGCCGTAAGGAGGCACAT ATGAGTGAGCTTATAC T 3'; and (SEQ ID NO: 45) 10) 5' GCCTGCGCGGCTGAGCGGCCGCGGATCCGATGGCCGCGATCCGA GCTCGAGATCTGCAGCT 3';
to form pHK12. Following restriction of pIDI with BsaAI-SmaI, agarose gel electrophoresis and GeneClean purification, the resulting blunt-ended 0.7 Kb DNA fragment containing the S. pombe IPP isomerase orf is inserted into the filled in NotI site of pTHBN1 utilizing chain reaction cloning (Pachuk et al., 2000), thermostable Ampligase® (Epicentre Technologies, Madison, Wis.), and the bridging oligonucleotides:
TABLE-US-00020 (SEQ ID NO: 46) 11) 5' TTAAATAAGGAGGAATAAACCATGGCGGCCGTAGGAGGCACATA TGAGTTCCCAACAAGA 3'; and (SEQ ID NO: 47) 12) 5' ACCTTAATTCATCGTTGCTAAGGATCCCCCGGCCGCGATCCGAG CTCGAGATCTGCAGCT 3'; to form pHK13.
EXAMPLE 14
Increased Isoprenoid Production in Cells Containing the MEP Pathway
[0195]In another exemplified embodiment, a carotenoid producing E. coli strain is utilized to demonstrate the effect of the insertion of orfs encoding the entire mevalonate pathway, or orfs encoding the entire mevalonate pathway and IPP isomerase, or an orf encoding just IPP isomerase, on production of lycopene as follows: Following the transformation of E. coli TOP10 F'(Invitrogen) with pAC-LYC (Cunningham et al, J. Bacteriol. 182:5841-5848, 2000), transformed cells are isolated on LB/Cam (30 Mg/ml) plates grown at 30EC. TOP10 F'/pAC-LYC competent cells are prepared by the CaCl2 method (Sambrook et al., 1989) following growth in LB/Cain in darkness at 28EC and 225 rpm to an optical density (A600) of 0.6. Competent TOP10 F'/pAC-LYC cells are transformed with one of the following plasmids; pTrcHisB; pHKO9, a pTrcHisB derivative containing the entire mevalonate pathway; pHK10, a pTrcHisB derivative containing the entire mevalonate pathway plus the orf encoding R. capsulatus IPP isomerase; pHK11, a pTrcHisB derivative containing the entire mevalonate pathway plus the orf encoding S. pombe IPP isomerase; pHK12, a pTrcHisB derivative containing the orf encoding R. capsulatus IPP isomerase; and pHK13, a pTrcHisB derivative containing the orf encoding S. pombe IPP isomerase. The bacterial strains described above, comprising pTHBN1 derivatives containing the mevalonate pathway orfs and/or an orf encoding IPP isomerase, are designated HK1, HK2, HK3, HK4, and HK5 respectively. The resulting transformants are isolated as colonies from LB/Cam/amp plates grown at 30EC. Single colonies of TOP10 F'/pAC-LYC/pTrcHisB and HK1 (TOP10 F'/pAC-LYC/pHKO9) are used to individually inoculate 4 ml LB/Cam/amp cultures and grown overnight in the dark at 28EC and 225 rpm. The cultures are serially diluted 10,000 to 100,000-fold, plated on LB/Cam/amp medium containing IPTG, and grown in the dark at rt for 2 to 10 days. The plates are visually examined for an increase in lycopene production as evident by a "darkening" of the light pink colored colonies that are present on the control plates corresponding to TOP10 F/pAC-LYC/pTrcHisB. The same experiments are performed with strains HK2, HK3, HK4, and HK5 to determine, visually, the effect of the orfs contained within pHK10, pHK11, pHK12, and pHK13 on lycopene production in TOP10 F'/pAC-LYC cells. The quantification of the carotenoid lycopene in cells, identified as potential overproducers due to their darker color when compared to the color of TOP10 F'/pAC-LYC/pTHBN1 cells, is performed utilizing a spectrophotometric assay as described by Cunningham et al. (Cunningham et al., 2000). Increased production of lycopene in E. coli cells containing the entire mevalonate pathway or the entire mevalonate pathway plus an additional orf for IPP isomerase establishes that the presence in cells of an additional biosynthetic pathway for the formation of IPP or IPP and DMAPP enhances the production of isoprenoid compounds, such as carotenoids, that are derived from IPP and DMAPP.
EXAMPLE 15
Demonstration of Antibiotic Resistance Due to the Mevalonate Pathway in MEP Pathway Dependent Cells
[0196]In still another exemplified embodiment, E. coli cells are transformed with DNA containing orfs, which in their summation comprise the entire mevalonate pathway, and the resulting cells are tested for resistance to the antibiotic fosmidomycin as follows. Following the separate transformation of E. coli TOP10 F' (Invitrogen) with pHKO2, pHKO3 and pHKO9, transformed cells are isolated on LB/Amp (50 μg/ml) plates grown at 30EC. Single colonies of TOP10 F'/pHKO2 (designated strain HK6), TOP10 F'/pHKO3 (designated strain HK7), and TOP10 F'/pHKO9 (designated strain HK8), are used to individually inoculate 4 ml LB/amp cultures and grown overnight at 30EC, 225 rpm. The HK6 and HK7 cultures are serially diluted 10,000 to 100,000-fold and plated on LB containing fosmidomycin (20 μg/ml). The HK8 cultures are serially diluted 10,000 to 100,000-fold and plated on LB/IPTG containing fosmidomycin (20 μg/ml) Controls are performed with cells comprising TOP10 FT transformed with the parent vectors of pHKO2, pHKO3 and pHKO9, by plating on the appropriate medium containing fosmidomycin establishing that E. coli control cells are unable to grow on medium containing fosmidomycin. The ability of transformed E. coli cells to grow in the presence of the antibiotic fosmidomycin establishes that the inserted DNA, comprising the entire mevalonate pathway and thus an alternative biosynthetic route to IPP, is functional and can circumvent the inhibition of an enzyme in the trunk line of the MEP pathway.
EXAMPLE 16
Construction of Plastid Transformation Vectors
[0197]In a specific, exemplified embodiment, a plant plastid transformation vector containing a synthetic operon comprising orfs, which in their summation is the entire mevalonate pathway, is constructed as follows: Plasmid pHKO3, a pBluescript derivative containing all six mevalonate pathway orfs, is assembled by restriction of pFCO1 to yield a 3.9 Kb NotI-XhoI DNA fragments containing three mevalonate orfs and its subsequent insertion into the SalI-NotI sites of pHKO1 by directional ligation as described above in Example 8. The plastid transformation vehicle, pHK14 containing the entire mevalonate pathway is constructed as follows: Plastid vector pGS104 (Serino and Maliga, Plant J. 12:687-701, 1997) is restricted with NcoI-XbaI and the two resulting DNA fragment are separated by agarose gel electrophoresis. Following isolation of the larger DNA fragment by gel excision and its purification by GeneClean, the NcoI-XbaI 5' overhangs are dephosphorylated using SAP and filled in with Klenow and dNTPs. The resulting blunt-ended, dephosphorylated DNA fragment derived from pGS104 is GeneClean purified. Following restriction of pHKO3 with EagI, isolation by agarose gel electrophoresis, and purification by GeneClean, the 7.7 Kb DNA fragment is treated with Klenow and dNTPs to fill in the 5' overhangs. The resulting blunt-ended DNA fragment containing the mevalonate pathway is purified by GeneClean and inserted into the dephosphorylated, Klenow-treated NcoI-XbaI sites of pGS104 by blunt-end ligation to yield pHK14.
[0198]Derivatives of pGS104 containing the entire mevalonate pathway plus an additional orf encoding IPP isomerase are constructed as follows: Following restriction of pHKO5 with NotI and treatment with Klenow and dNTPs, the resulting 8.2 Kb blunt-ended DNA fragment, containing the six mevalonate pathway orfs plus an orf encoding R. capsulatus IPP isomerase, is isolated by agarose gel electrophoresis, purified by GeneClean, and inserted into the dephosphorylated, filled in NcoI-XbaI sites of pGS104 by blunt-end ligation to yield pHK15. Following restriction of pHKO6 with EagI and treatment with Klenow and dNTPs, the resulting 8.4 Kb blunt-ended DNA fragment, containing the six mevalonate pathway orfs plus an orf encoding S. pombe IPP isomerase, is isolated by agarose gel electrophoresis, purified by GeneClean, and inserted into the dephosphorylated, filled in NcoI-XbaI sites of pGS104 by blunt-end ligation to yield pHK16.
[0199]Derivatives of pGS104 containing only an orf encoding IPP isomerase are constructed as follows: Following restriction of pBSIDI with BsaAI-EcoRV, agarose gel electrophoresis and GeneClean purification, the resulting blunt-ended 0.5 Kb DNA fragment containing the R. capsulatus IPP isomerase orf is inserted into the dephosphorylated, filled in NcoI-XbaI sites of pGS104 by blunt-end ligation to yield pHK17. Following restriction of pIDI with BsaAI-SmaI, agarose gel electrophoresis and GeneClean purification, the resulting blunt-ended 0.7 Kb DNA fragment containing the S. pombe IPP isomerase orf is inserted into the dephosphorylated, filled in NcoI-XbaI sites of pGS104 by blunt-end ligation to yield pHK18.
EXAMPLE 17
Construction of Transplastomic Plants Containing Orfs Encoding the Mevalonate Pathway or Orfs Encoding the Mevalonate Pathway Coupled with IPP Isomerase
[0200]In another exemplified embodiment, tobacco is engineered at the plastid level by using any of the plastid transformation vectors described above, or their equivalents, such as variants of those plastid transformation vectors as can be routinely constructed by means known in the art and containing the orfs as taught and described above. Specifically, Nicotiana tabacum var. `Xanthi NC` leaf sections (1×0.5 cm strips from in vitro plants with 3 to 5 cm long leaves) are centered in the dish, top side up and bombarded with 1 μm gold micro particles (Kota et al., 1999) coated with DNA containing orfs, which in their summation comprise the entire mevalonate pathway, using a PDS 1000 He device, at 1100 psi. Toxicity is evident in tobacco after three weeks of growth on medium containing the antibiotic fosmidomycin at a concentration of at least 500 micromolar. Transplastomic plants are recovered from leaf sections cultured under lights on standard RMOP shoot regeneration medium or on a Murashige-Skoog salts shoot regeneration medium with 3% sucrose, Gamborg's B5 vitamins, 2 mg/L 6-benzylamino-purine and Phytagel (2.7 g/L), containing 500 μM fosmidomycin for the direct selection of insertion of the entire mevalonate pathway into plastids. Alternatively, the regeneration medium contains an antibiotic, e.g. spectinomycin, for selection based on antibiotic resistance due to any co-transformed gene on the transforming DNA vector, as would be readily apparent to the skilled artisan. De novo green leaf tissue is visible after three weeks. Tissue is removed to undergo a second round of selection on shoot regeneration medium with 500 μM fosmidomycin to encourage homoplasmy and plants are rooted. Genomic DNA is isolated from T0 leaf tissue or T1 leaf tissue derived from in vitro germinated transplastomic seeds utilizing the DNeasy Plant Mini Kit (Qiagen Inc, Valencia, Calif.) according to the manufacturer's instructions and is subjected to analysis as is known in the art to confirm homoplasmy. The ability to select directly for a transformation event corresponding to the successful insertion of the mevalonate pathway orfs into plastids establishes the use of orfs, which in their summation comprise the entire mevalonate pathway, as a selectable marker for plastid transformation. The construction of fosmidomycin resistant plants establishes the ability of the mevalonate pathway, when functioning in plant plastids, to provide an alternate biosynthetic route to IPP, thus overcoming the effect of an inhibitor targeting an enzyme in the trunk line of the MEP pathway.
EXAMPLE 18
Metabolic Engineering in Transplastomic Solanaceae Plants
[0201]In another exemplified embodiment, Solanaceae species are engineered at the plastid level using infA pseudogene insertion of a selectable marker and orfs for expression. Specifically, leaf sections of a genetically defined white petunia (or other petunia), are engineered, as for the Solanaceous species tobacco (see Example 16), using vectors pHKO4 or pHKO7, or their equivalents, for insertion of orfs encoding the entire mevalonate pathway or orfs encoding the entire mevalonate pathway and IPP isomerase. Transplastomic Solanaceae plants containing orfs encoding the entire mevalonate pathway and IPP isomerase, and containing an additional orf encoding phytoene synthase, are created by insertion of a pBSNT27 (see Example 9) derived vector, constructed as follows;
[0202]A Rhodobacter capsulatus orf encoding a polypeptide with phytoene synthase activity is isolated by PCR from genomic DNA using the primers
1) 5' GCGATATCGGATCCAGGAGGACCATATGATC(GCCGAAGCGGATATGGA GGTCTGC 3' (sense) (SEQ ID NO: 65)2) 5' GCGATATCAAGCTTGGATCCTCAATCCATCGCCAGGCCGCGGTCGCGC GC 3' (antisense) (SEQ ID NO: 66) containing the restriction site BamHI shown underlined. The 1.1 Kb PCR product is isolated by agarose gel electrophoresis, purified by GeneClean and inserted into the pT7Blue-3 vector (Novagen) using the Perfectly Blunt (Cloning Kit (Novagen) according to the mnanufacturer's instructions. Sequence analysis is performed to identify constructs containing R. capsulatus DNA identical to the published DNA sequence (SEQ ID NO: 71) and are designated pPHS. Following restriction of pPHS with BamHI, isolation by agarose gel electrophoresis, and purification by GeneClean, the 1.1 Kb BamHI DNA fragment containing the orf encoding R. capsulatus phytoene synthase is inserted into the BglII site of pBSNT27 utilizing chain reaction cloning (Pachuk et al., 2000), thermostable Ampligase ((Epicentre Technologies, Madison, Wis.), and the bridging oligonucleotides
TABLE-US-00021 (SEQ ID NO: 67) 3) 5' CTTTCCTGAAACATAATTTATAATCAGATCCAGGAGGACCATAT GATCGCCGAAGCGGAT 3'; and (SEQ ID NO: 68) 4) 5' CGACCGCGGCCTGGCGATGGATTGAGGATCTAAACAAACCCGGA ACAGACCGTTGGGAAG 3';
to create plastid transformation vector pFHO5. Following restriction of pFHO5 with XcmI, a unique site in the infA pseudogene, and purification by GeneClean, the resulting 3' overhangs are removed by treatment with Mung Bean nuclease and the resulting blunt-ended DNA fragment is purified by GeneClean. Vector pFHO3 is restricted with NotI and the resulting 8.3 Kb DNA fragment, containing Operon E, is isolated by agarose gel electrophoresis and purified by GeneClean. The 5' overhangs of the isolated DNA fragment are filled in with Klenow and dNTPs and the resulting blunt end DNA fragment, containing Operon E, is inserted into the Mung Bean nuclease treated XcmI site of pFHO5 utilizing chain reaction cloning (Pachuk et al., 2000), thermostable Ampligase ((Epicentre Technologies, Madison, Wis.), and the bridging oligonucleotides
TABLE-US-00022 (SEQ ID NO: 69) 5) 5' ATTTTTCATCTCGAATTGTATTCCCACGAAGGCCGCGTCGACTA CGGCCGCAGGAGGAGT3'; and (SEQ ID NO: 70) 6) 5' TTCGGATCGATCCTGCGCGGCTGAGCGGCCGGAATGGTGAAGTT GAAAAACGAATCCTTC3';
to create the plastid transformation vector pFHO6 (FIG. 17).
[0203]Alternatively, an orf encoding IPP isomerase can be inserted into the XcmI site of pFHO5, utilizing skills as known in the art, to create a plastid transformation vector containing both an orf encoding phytoene synthase and an off encoding IPP isomerase. Another alternative uses the infA pseudogene as an insertion site for orfs, encoding phytoene synthase, and/or IPP isomerase, and/or the entire mevalonate pathway, linked with the aadA gene as is known in the art for selection of transplastomic plastids on 500 microgram per liter spectinomycin.
[0204]The BioRad PDS 1000 He gene gun is used to deliver BioRad tungsten M10 (0.7 micron approx.) microspheres into petunia (Petunia hybrida `Mitchell`) leaves positioned top-side up. Intact leaves, or equivalent tissues of about 6-8 cm2 per sample are plated onto shoot regeneration medium consisting of Murashige and Skoog basal medium, B5 vitamins, 3% sucrose, 0.7% (w/v) agar and 3 mg/l BA (6-benzylamino-purine), 0.1 mg/l IAA (Deroles and Gardner, Plant Molec. Biol. 11: 355-364, 1988) in 100×10 mm plastic Petri dishes. Leaves are centered in the target zone of the gene gun for bombardment at 1100 psi, third shelf from bottom, ˜5.6 cm gap, 28 mgHg vacuum. M10 microspheres are coated with DNA using standard procedures of CaCl2 and spermidine precipitation, 1.5 to 2 ug DNA/bombardment. After bombardment, tissues are cultured in light in the presence of antibiotic (500 micromolar fosmidomycin). Each leaf sample is then cut into about 6 pieces and cultured on petunia shooting medium containing 500 micromolar fosmidomycin for 3 to 8 weeks, with subculture onto fresh medium every three weeks. Any green shoots are removed and leaves plated onto the same medium containing 500 micromolar fosmidomycin. Plantlets with at least four leaves and of solid green color (no bleaching on petioles or whorls) are transferred for rooting onto solidified hormone-free Murashige and Skoog salts with B5 vitamins and 2% sucrose and are grown to flowering. The dependency of increased carotenoid production in Solanacae on the combination of the orfs inserted, be it an orf encoding phytoene synthase alone; or orfs encoding the entire mevalonate pathway and phytoene synthase; or orfs encoding phytoene synthase, the entire mevalonate pathway and IPP isomerase; or orfs for phytoene synthase and IPP isomerase, establishes that the addition of the mevalonate pathway and/or IPP isomerase to plant plastids enhances the production of isoprenoid compounds that are derived from IPP and DMAPP; and the suitability of a pseudogene insertion site for creating transplastomic Petunia.
EXAMPLE 19
Transformation of Microalgae
[0205]In a specific exemplified embodiment, chloroplast transformants are obtained by microprojectile bombardment of Chlamydomonas reinhardtii cells and subsequent selection on fosmidomycin. Specifically, a genecluster containing the complete mevalonate pathway is substituted, as a selectable marker, for the coding sequence of the aadA gene in the pUC18 derived vector containing 5-atpAaadA:rbcL-3 (Goldschmidt-Clermont M., Nucleic Acids Res. 19:4083-4089, 1991) as follows: Plasmid pUC-atpX-AAD is restricted with NcoI, purified by GeneClean and treated with Mung Bean nuclease to remove the resulting 5' overhangs. Following GeneClean purification, the blunt ended DNA fragment is restricted with HindIII to remove the aadA orf and the remaining DNA fragment, containing approximately 653 base pairs of the C. reinhardtii atpA gene and approximately 437 base pairs of the C. reinhardtii rbcL gene (Goldschmidt-Clermont M., 1991), is isolated by agarose gel electrophoresis and purified by GeneClean. Plasmid pFHO4 is restricted with NdeI, purified by GeneClean, and the resulting 5 overhangs are filled in with Klenow and dNTPs. Following GeneClean purification, the blunt ended DNA fragment is restricted with HindIII and the resulting DNA fragment, containing Operon F (see FIG. 13), is isolated by agarose gel electrophoresis and purified by GeneClean. The blunt end-HindIII fragment is inserted into the blunt end HindIII sites of the DNA fragment isolated from pUC-atpX-AAD by ligation resulting in the orf encoding S. cerevisiae acetoacetyl CoA thiolase, located at the beginning of Operon F, to be in frame with the ATG start codon of the 5atpA DNA in pUC-atpX-AAD (Goldschmidt-Clermont M., 1991). The resulting modified yeast orf only encodes 2 extra amino acids, Met and Ser, appended to the N-terminal Met of the acetoacetyl CoA thiolase polypeptide encoded by Operon F. The resulting chlamydomonas plastid transformation vector is designated pHK19. About 10,000 cells are spread on TAP plates containing 200 micromolar fosmidomycin, plates are dried, and then cells are immediately bombarded with M10 or 1 micron gold particles coated with about 2 micrograms of plasmid DNA using the PDS-1000 He gene gun, 1100 psi, fourth shelf from bottom, ˜2 cm gap, ˜28 mgHg vacuum (alternatively cells are spread over a Nytran nylon 0.45 micron membrane placed on top of TAP agar and bombarded without a drying phase). Plates are incubated in low light for two to three weeks before colonies are counted. Fosmidomycin-resistant colonies are green (vs yellowish for susceptible cells) and transformants are characterized using skills as known in the art. This demonstrates use of orfs encoding the entire mevalonate pathway as a selectable marker for green algae and by virtue of its functioning demonstrates its utility for overproduction of isoprenoid metabolites in microalgae.
EXAMPLE 20
Metabolic Engineering in Transplastomic Gain Crop (Rice)
[0206]In another exemplified embodiment, an operon comprising orfs encoding the entire mevalonate pathway are inserted into the plastids of rice as follows: A DNA fragment isolated from pHKO3, containing the complete mevalonate pathway, or from pFHO2, containing orfs encoding the entire mevalonate pathway and IPP isomerase, is inserted into the NcoI-XbaI sites of plasmid pMSK49 to replace the gfp coding region adjacent to the coding region for streptomycin resistance, aadA; or inserted into the BstXI-NcoI digested DNA of plasmid pMSK48 using skills as is known in the art for direct selection on fosmidomycin. The resulting plasmids contain rice-specific insertion sequences of pMSK35 as described in Khan and Maliga, Nature Biotechnology 17:910-914, 1999. Embryonic suspensions, induced as previously described (Khan and Maliga 1999), of japonica rice Oryza sativa Taipei 309' engineered with the beta-carotene pathway (Ye et al. Science 287:303-305) are plated into filter paper and bombarded with the PDS1000 He device as described in Example 17. After two days on non-selective medium and then one to two weeks in selective AA medium (Toriyama and Hinata, Plant Science 41: 179-183, 1985) tissue is transferred to agar solidified medium of MS salts, and vitamins, 100 mg/L myo-inositol, 4 mg/L 6-benzylaminopurine, 0.5 mg/L indoleacetic acid, 0.5 mg/L1-napthaleneacetic acide, 3% sucrose, 4% maltose and 100 mg/L streptomycin sulfate or 500 μM fosmidomycin. Transplastomic shoots appear following cultivation in the light after three weeks and leaf samples are analyzed for the operon by PCR.
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Sequence CWU
1
76157DNAArtificial SequencePCR primer containing Saccharomyces
cerevisiae DNA 1ggactagtct gcaggaggag ttttaatgtc attaccgttc ttaacttctg
caccggg 57296DNAArtificial SequencePCR primer containing S.
cerevisiae DNA 2ttctcgagct taagagtagc aatatttacc ggagcagtta cactagcagt
atatacagtc 60attaaaactc ctcctgtgaa gtccatggta aattcg
96356DNAArtificial SequencePCR primer containing S.
cerevisiae DNA 3tagcggccgc aggaggagtt catatgtcag agttgagagc cttcagtgcc
ccaggg 56436DNAArtificial SequencePCR primer containing S.
cerevisiae DNA 4tttctgcagt ttatcaagat aagtttccgg atcttt
36541DNAArtificial SequencePCR primer containing S.
cerevisiae DNA 5ggaattcatg accgtttaca cagcatccgt taccgcaccc g
41645DNAArtificial SequencePCR primer containing S.
cerevisiae DNA 6ggctcgagtt aaaactcctc ttcctttggt agaccagtct ttgcg
45768DNAArtificial SequencePCR primer containing Arabidopsis
thaliana DNA 7gctctagatg cgcaggaggc acatatggcg aagaacgttg ggattttggc
tatggatatc 60tatttccc
68861DNAArtificial SequencePCR primer containing A. thaliana
DNA 8cgctcgagtc gacggatcct cagtgtccat tggctacaga tccatcttca cctttcttgc
60c
61972DNAArtificial SequencePCR primer containing A. thaliana DNA
9ccgctcgagc acgtggaggc acatatgcaa tgctgtgaga tgcctgttgg atacattcag
60attcctgttg gg
721071DNAArtificial SequencePCR primer containing A. thaliana DNA
10ggggtacctg cggccggatc ccgggtcatg ttgttgttgt tgtcgttgtc gttgctccag
60agatgtctcg g
711174DNAArtificial SequencePCR primer containing S. cerevisiae DNA
11acaacaccgc ggcggccgcg tcgacgccgg cggaggcaca tatgtctcag aacgtttaca
60ttgtatcgac tgcc
741253DNAArtificial SequencePCR primer containing S. cerevisiae DNA
12gctctagagg atcctcatat cttttcaatg acaatagagg aagcaccacc acc
531365DNAArtificial SequenceOligonucleotide containing S. cerevisiae DNA
13gctctagata cgtaggaggc acatatgagt gagcttatac ccgcctgggt tggtgacaga
60ctggc
651461DNAArtificial SequenceOligonucleotide containing A. thaliana and S.
cerevisiae DNA 14cgctcgagcc cgggggatcc tcagccgcgc aggatcgatc
cgaaaatccg gtcaagatgg 60c
611572DNAArtificial SequenceOligonucleotide
containing S. cerevisiae DNA 15gctctagata cgtaggaggc acatatgagt
tcccaacaag agaaaaagga ttatgatgaa 60gaacaattaa gg
721659DNAArtificial
SequenceOligonucleotide containing S. cerevisiae DNA 16cgctcgagcc
cgggggatcc ttagcaacga tgaattaagg tatcttggaa ttttgacgc
59176215DNAArtificial Sequencemisc_feature()..()Vector pBSNT27 containing
Nicotiana tabacum DNA 17gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
atttttctaa atacattcaa 60atatgtatcc gctcatgaga caataaccct gataaatgct
tcaataatat tgaaaaagga 120agagtatgag tattcaacat ttccgtgtcg cccttattcc
cttttttgcg gcattttgcc 180ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
agatgctgaa gatcagttgg 240gtgcacgagt gggttacatc gaactggatc tcaacagcgg
taagatcctt gagagttttc 300gccccgaaga acgttttcca atgatgagca cttttaaagt
tctgctatgt ggcgcggtat 360tatcccgtat tgacgccggg caagagcaac tcggtcgccg
catacactat tctcagaatg 420acttggttga gtactcacca gtcacagaaa agcatcttac
ggatggcatg acagtaagag 480aattatgcag tgctgccata accatgagtg ataacactgc
ggccaactta cttctgacaa 540cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
catgggggat catgtaactc 600gccttgatcg ttgggaaccg gagctgaatg aagccatacc
aaacgacgag cgtgacacca 660cgatgcctgt agcaatggca acaacgttgc gcaaactatt
aactggcgaa ctacttactc 720tagcttcccg gcaacaatta atagactgga tggaggcgga
taaagttgca ggaccacttc 780tgcgctcggc ccttccggct ggctggttta ttgctgataa
atctggagcc ggtgagcgtg 840ggtctcgcgg tatcattgca gcactggggc cagatggtaa
gccctcccgt atcgtagtta 900tctacacgac ggggagtcag gcaactatgg atgaacgaaa
tagacagatc gctgagatag 960gtgcctcact gattaagcat tggtaactgt cagaccaagt
ttactcatat atactttaga 1020ttgatttaaa acttcatttt taatttaaaa ggatctaggt
gaagatcctt tttgataatc 1080tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
agcgtcagac cccgtagaaa 1140agatcaaagg atcttcttga gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa 1200aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
agagctacca actctttttc 1260cgaaggtaac tggcttcagc agagcgcaga taccaaatac
tgtccttcta gtgtagccgt 1320agttaggcca ccacttcaag aactctgtag caccgcctac
atacctcgct ctgctaatcc 1380tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
taccgggttg gactcaagac 1440gatagttacc ggataaggcg cagcggtcgg gctgaacggg
gggttcgtgc acacagccca 1500gcttggagcg aacgacctac accgaactga gatacctaca
gcgtgagcta tgagaaagcg 1560ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
aagcggcagg gtcggaacag 1620gagagcgcac gagggagctt ccagggggaa acgcctggta
tctttatagt cctgtcgggt 1680ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
gtcagggggg cggagcctat 1740ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
cttttgctgg ccttttgctc 1800acatgttctt tcctgcgtta tcccctgatt ctgtggataa
ccgtattacc gcctttgagt 1860gagctgatac cgctcgccgc agccgaacga ccgagcgcag
cgagtcagtg agcgaggaag 1920cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
ttggccgatt cattaatgca 1980gctggcacga caggtttccc gactggaaag cgggcagtga
gcgcaacgca attaatgtga 2040gttagctcac tcattaggca ccccaggctt tacactttat
gcttccggct cgtatgttgt 2100gtggaattgt gagcggataa caatttcaca caggaaacag
ctatgaccat gattacgcca 2160agctcgaaat taaccctcac taaagggaac aaaagctgga
gctccaccgc ggtggcggcc 2220gctctagaac tagtggatct tcttggctgt tattcaaaag
gtccaacaat gtatatatat 2280tggacatttt gaggcaatta tagatcctgg aaggcaattc
tgattggtca ataaaaatcg 2340atttcaatgc tatttttttt ttgtttttta tgagtttagc
caatttatca tgaaaggtaa 2400aaggggataa aggaaccgtg tgttgattgt cctgtaaata
taagttgtct tcctccatat 2460gtaaaaaggg aataaataaa tcaattaaat ttcgggatgc
ttcatgaagt gcttctttcg 2520gagttaaact tccgtttgtc catatttcga gaaaaagtat
ctcttgtttt tcattcccat 2580tcccataaga atgaatacta tgattcgcgt ttcgaacagg
catgaataca gcatctatag 2640gataacttcc atcttgaaag ttatgtggcg tttttataag
atatccacga tttctctcta 2700tttgtaatcc aatacaaaaa tcaattggtt ccgttaaact
ggctatatgt tgtgtattat 2760caacgatttc tacataaggc ggcaagatga tatcttgggc
agttacagat ccaggaccct 2820tgacacaaat agatgcgtca gaagttccat atagattact
tcttaatata atttctttca 2880aattcattaa aatttcatgt accgattctt gaatgcccgt
tatggtagaa tattcatgtg 2940ggactttctc agattttaca cgtgtgatac atgttccttc
tatttctcca agtaaagctc 3000ttcgcatcgc aatgcctatt gtgtcggctt ggcctttcat
aagtggagac agaataaagc 3060gtccataata aaggcgttta ctgtctgttc ttgattcaac
acacttccac tgtagtgtcc 3120gagtagatac tgttactttc tctcgaacca tagtactatt
atttgattag atcatcgaat 3180cttttatttc tcttgagatt tcttcaatgt tcagttctac
acacgtcttt ttttcggagg 3240tctacagcca ttatgtggca taggagttac atcccgtacg
aaagttaata gtataccact 3300tcgacgaata gctcgtaatg ctgcatctct tccgagaccg
ggacctttta tcatgacttc 3360tgctcgttgc ataccttgat ccactactgt acggatagcg
tttgctgctg cggtttgagc 3420agcaaacggt gttcctcttc tcgtaccttt gaatccagaa
gtaccggcgg aggaccaaga 3480aactactcga ccccgtacat ctgtaacagt gacaatggta
ttattgaaac ttgcttgaac 3540atgaataact ccctttggta ttctacgtgc acccttacgt
gaaccaatac gtccattcct 3600acgcgaacta attttcggta tagcttttgc catattttat
catctcgtaa atatgagtca 3660gagatatatg gatatatcca tttcatgtca aaacagattc
tttatttgta catcggctct 3720tctggcaagt ctgattatcc ctgtctttgt ttatgtctcg
ggttggaaca aattactata 3780attcgtcccc gcctacggat tagtcgacat ttttcacaaa
ttttacgaac ggaagctctt 3840attttcatat ttctcattcc ttaccttaat tctgaatcta
tttcttggaa gaaaataagt 3900ttcttgaaat ttttcatctc gaattgtatt cccacgaaag
gaatggtgaa gttgaaaaac 3960gaatccttca aatctttgtt gtggagtcga taaattatac
gccctttggt tgaatcataa 4020ggacttactt caattttgac tctatctcct ggcagtatcc
gtataaaact atgccggatc 4080tttcctgaaa cataatttat aatcagatct aaacaaaccc
ggaacagacc gttgggaagc 4140gattcagtaa ttaaagcttc atgactcctt tttggttctt
aaagtccctt tgaggtatca 4200actaataaga aagatattag acaacccccc ttttttcttt
ttcacaaata ggaagtttcg 4260aatccaattt ggatattaaa aggattacca gatataacac
aaaatctctc cacctattcc 4320ttctagtcga gcctctcggt ctgtcattat acctcgagaa
gtagaaagaa ttacaatccc 4380cattccacct aaaattcgcg gaattcgttg ataattagaa
tagattcgta gaccaggtcg 4440actgattcgt tttaaattta aaatatttct atagggtctt
ttcctattcc ttctatgtcg 4500cagggttaaa accaaaaaat atttgttttt ttctcgatgt
tttctcacgt tttcgataaa 4560accttctcgt aaaagtattt gaacaatatt ttcggtaata
ttagtagatg ctattcgaac 4620cacccttttt cgatccatat cagcatttcg tatagaagtt
attatctcag caatagtgtc 4680cctacccatg atgaactaaa attattgggg cctccaaatt
tgatataatc aacgtgtttt 4740ttacttattt tttttttgaa tatgatatga attattaaag
atatatgcgt gagacacaat 4800ctactaatta atctatttct ttcaaatacc ccactagaaa
cagatcacaa tttcatttta 4860taatacctcg ggagctaatg aaactatttt agtaaaattt
aattctctca attcccgggc 4920gattgcacca aaaattcgag ttccttttga tttccttcct
tcttgatcaa taacaactgc 4980agcattgtca tcatatcgta ttatcatccc gttgtcacgt
ttgagttctt tacaggtccg 5040cacaattaca gctctgacta cttctgatct ttctaggggc
atatttggta cggcttcttt 5100gatcacagca acaataacgt caccaatatg agcatatcga
cgattgctag ctcctatgat 5160tcgaatacac atcaattctc gagccccgct gttatccgct
acatttaaat gggtctgagg 5220ttgaatcatt tttttaatcc gttctttgaa tgcaaagggc
gaagaaaaaa aagaaatatt 5280tttgtccaaa aaaaaagaaa catgcggttt cgtttcatat
ctaagagccc tttccgcatt 5340tttttctatt acattacgaa ataatgaatt gagttcgtat
aggcatttta gatgctgcta 5400gtgaaatagc ccttctggct atattttctg ttactccacc
catttcataa agtattcgac 5460ccggtttaac aacagctacc caatattcag gggatccccc
gggctgcagg aattcgatat 5520caagcttatc gataccgtcg acctcgaggg ggggcccggt
acccaattcg ccctatagtg 5580agtcgtatta caattcactg gccgtcgttt tacaacgtcg
tgactgggaa aaccctggcg 5640ttacccaact taatcgcctt gcagcacatc cccctttcgc
cagctggcgt aatagcgaag 5700aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
gaatggcgaa tgggacgcgc 5760cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
gcgcagcgtg accgctacac 5820ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
ttcctttctc gccacgttcg 5880ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
agggttccga tttagtgctt 5940tacggcacct cgaccccaaa aaacttgatt agggtgatgg
ttcacgtagt gggccatcgc 6000cctgatagac ggtttttcgc cctttgacgt tggagtccac
gttctttaat agtggactct 6060tgttccaaac tggaacaaca ctcaacccta tctcggtcta
ttcttttgat ttataaggga 6120ttttgccgat ttcggcctat tggttaaaaa atgagctgat
ttaacaaaaa tttaacgcga 6180attttaacaa aatattaacg cttacaattt aggtg
6215181332DNAArtificial SequenceOligonucleotide
containing N. tabacum and S. cerevisiae DNA 18atgtcattac cgttcttaac
ttctgcaccg ggaaaggtta ttatttttgg tgaacactct 60gctgtgtaca acaagcctgc
cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta 120ataagcgagt catctgcacc
agatactatt gaattggact tcccggacat tagctttaat 180cataagtggt ccatcaatga
tttcaatgcc atcaccgagg atcaagtaaa ctcccaaaaa 240ttggccaagg ctcaacaagc
caccgatggc ttgtctcagg aactcgttag tcttttggat 300ccgttgttag ctcaactatc
cgaatccttc cactaccatg cagcgttttg tttcctgtat 360atgtttgttt gcctatgccc
ccatgccaag aatattaagt tttctttaaa gtctacttta 420cccatcggtg ctgggttggg
ctcaagcgcc tctatttctg tatcactggc cttagctatg 480gcctacttgg gggggttaat
aggatctaat gacttggaaa agctgtcaga aaacgataag 540catatagtga atcaatgggc
cttcataggt gaaaagtgta ttcacggtac cccttcagga 600atagataacg ctgtggccac
ttatggtaat gccctgctat ttgaaaaaga ctcacataat 660ggaacaataa acacaaacaa
ttttaagttc ttagatgatt tcccagccat tccaatgatc 720ctaacctata ctagaattcc
aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg 780gtcaccgaga aatttcctga
agttatgaag ccaattctag atgccatggg tgaatgtgcc 840ctacaaggct tagagatcat
gactaagtta agtaaatgta aaggcaccga tgacgaggct 900gtagaaacta ataatgaact
gtatgaacaa ctattggaat tgataagaat aaatcatgga 960ctgcttgtct caatcggtgt
ttctcatcct ggattagaac ttattaaaaa tctgagcgat 1020gatttgagaa ttggctccac
aaaacttacc ggtgctggtg gcggcggttg ctctttgact 1080ttgttacgaa gagacattac
tcaagagcaa attgacagct tcaaaaagaa attgcaagat 1140gattttagtt acgagacatt
tgaaacagac ttgggtggga ctggctgctg tttgttaagc 1200gcaaaaaatt tgaataaaga
tcttaaaatc aaatccctag tattccaatt atttgaaaat 1260aaaactacca caaagcaaca
aattgacgat ctattattgc caggaaacac gaatttacca 1320tggacttcat aa
1332191191DNAArtificial
SequenceOligonucleotide containing N. tabacum and A. thaliana DNA
19atgaccgttt acacagcatc cgttaccgca cccgtcaaca tcgcaaccct taagtattgg
60gggaaaaggg acacgaagtt gaatctgccc accaattcgt ccatatcagt gactttatcg
120caagatgacc tcagaacgtt gacctctgcg gctactgcac ctgagtttga acgcgacact
180ttgtggttaa atggagaacc acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc
240gacctacgcc aattaagaaa ggaaatggaa tcgaaggacg cctcattgcc cacattatct
300caatggaaac tccacattgt ctccgaaaat aactttccta cagcagctgg tttagcttcc
360tccgctgctg gctttgctgc attggtctct gcaattgcta agttatacca attaccacag
420tcaacttcag aaatatctag aatagcaaga aaggggtctg gttcagcttg tagatcgttg
480tttggcggat acgtggcctg ggaaatggga aaagctgaag atggtcatga ttccatggca
540gtacaaatcg cagacagctc tgactggcct cagatgaaag cttgtgtcct agttgtcagc
600gatattaaaa aggatgtgag ttccactcag ggtatgcaat tgaccgtggc aacctccgaa
660ctatttaaag aaagaattga acatgtcgta ccaaagagat ttgaagtcat gcgtaaagcc
720attgttgaaa aagatttcgc cacctttgca aaggaaacaa tgatggattc caactctttc
780catgccacat gtttggactc tttccctcca atattctaca tgaatgacac ttccaagcgt
840atcatcagtt ggtgccacac cattaatcag ttttacggag aaacaatcgt tgcatacacg
900tttgatgcag gtccaaatgc tgtgttgtac tacttagctg aaaatgagtc gaaactcttt
960gcatttatct ataaattgtt tggctctgtt cctggatggg acaagaaatt tactactgag
1020cagcttgagg ctttcaacca tcaatttgaa tcatctaact ttactgcacg tgaattggat
1080cttgagttgc aaaaggatgt tgccagagtg attttaactc aagtcggttc aggcccacaa
1140gaaacaaacg aatctttgat tgacgcaaag actggtctac caaaggaata a
1191201197DNAArtificial SequencePCR primer containing Rhodobacter
capsulatus DNA 20atgtctcaga acgtttacat tgtatcgact gccagaaccc
caattggttc attccagggt 60tctctatcct ccaagacagc agtggaattg ggtgctgttg
ctttaaaagg cgccttggct 120aaggttccag aattggatgc atccaaggat tttgacgaaa
ttatttttgg taacgttctt 180tctgccaatt tgggccaagc tccggccaga caagttgctt
tggctgccgg tttgagtaat 240catatcgttg caagcacagt taacaaggtc tgtgcatccg
ctatgaaggc aatcattttg 300ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg
tagctggtgg ttgtgaatct 360atgactaacg caccatacta catgccagca gcccgtgcgg
gtgccaaatt tggccaaact 420gttcttgttg atggtgtcga aagagatggg ttgaacgatg
cgtacgatgg tctagccatg 480ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta
ctagagaaca acaagacaat 540tttgccatcg aatcctacca aaaatctcaa aaatctcaaa
aggaaggtaa attcgacaat 600gaaattgtac ctgttaccat taagggattt agaggtaagc
ctgatactca agtcacgaag 660gacgaggaac ctgctagatt acacgttgaa aaattgagat
ctgcaaggac tgttttccaa 720aaagaaaacg gtactgttac tgccgctaac gcttctccaa
tcaacgatgg tgctgcagcc 780gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt
tgaagccttt ggctattatc 840aaaggttggg gtgaggccgc tcatcaacca gctgatttta
catgggctcc atctcttgca 900gttccaaagg ctttgaaaca tgctggcatc gaagacatca
attctgttga ttactttgaa 960ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta
agattttgaa gctagaccca 1020tctaaggtta atgtatatgg tggtgctgtt gctctaggtc
acccattggg ttgttctggt 1080gctagagtgg ttgttacact gctatccatc ttacagcaag
aaggaggtaa gatcggtgtt 1140gccgccattt gtaatggtgg tggtggtgct tcctctattg
tcattgaaaa gatatga 1197211386DNAArtificial SequencePCR primer
containing R. capsulatus DNA 21atggcgaaga acgttgggat tttggctatg
gatatctatt tccctcccac ctgtgttcaa 60caggaagctt tggaagcaca tgatggagca
agtaaaggga aatacactat tggacttggc 120caagattgtt tagctttttg cactgagctt
gaagatgtta tctctatgag tttcaatgcg 180gtgacatcac tttttgagaa gtataagatt
gaccctaacc aaatcgggcg tcttgaagta 240ggaagtgaga ctgttattga caaaagcaag
tccatcaaga ccttcttgat gcagctcttt 300gagaaatgtg gaaacactga tgtcgaaggt
gttgactcga ccaatgcttg ctatggtgga 360actgcagctt tgttaaactg tgtcaattgg
gttgagagta actcttggga tggacgttat 420ggcctcgtca tttgtactga cagcgcggtt
tatgcagaag gacccgcaag gcccactgga 480ggagctgcag cgattgctat gttgatagga
cctgatgctc ctatcgtttt cgaaagcaaa 540ttgagagcaa gccacatggc tcatgtctat
gacttttaca agcccaatct tgctagcgag 600tacccggttg ttgatggtaa gctttcacag
acttgctacc tcatggctct tgactcctgc 660tataaacatt tatgcaacaa gttcgagaag
atcgagggca aagagttctc cataaatgat 720gctgattaca ttgttttcca ttctccatac
aataaacttg tacagaaaag ctttgctcgt 780ctcttgtaca acgacttctt gagaaacgca
agctccattg acgaggctgc caaagaaaag 840ttcacccctt attcatcttt gacccttgac
gagagttacc aaagccgtga tcttgaaaag 900gtgtcacaac aaatttcgaa accgttttat
gatgctaaag tgcaaccaac gactttaata 960ccaaaggaag tcggtaacat gtacactgct
tctctctacg ctgcatttgc ttccctcatc 1020cacaataaac acaatgattt ggcgggaaag
cgggtggtta tgttctctta tggaagtggc 1080tccaccgcaa caatgttctc attacgcctc
aacgacaata agcctccttt cagcatttca 1140aacattgcat ctgtaatgga tgttggcggt
aaattgaaag ctagacatga gtatgcacct 1200gagaagtttg tggagacaat gaagctaatg
gaacataggt atggagcaaa ggactttgtg 1260acaaccaagg agggtattat agatcttttg
gcaccgggaa cttattatct gaaagaggtt 1320gattccttgt accggagatt ctatggcaag
aaaggtgaag atggatctgt agccaatgga 1380cactga
1386221779DNAArtificial SequencePCR
primer containing Schizosaccharomyces pombe DNA 22atggatctcc
gtcggaggcc tcctaaacca ccggttacca acaacaacaa ctccaacgga 60tctttccgtt
cttatcagcc tcgcacttcc gatgacgatc atcgtcgccg ggctacaaca 120attgctcctc
caccgaaagc atccgacgcg cttcctcttc cgttatatct cacaaacgcc 180gttttcttca
cgctcttctt ctccgtcgcg tattacctcc tccaccggtg gcgtgacaag 240atccgttaca
atacgcctct tcacgtcgtc actatcacag aactcggcgc cattattgct 300ctcatcgctt
cgtttatcta tctcctaggg ttttttggta ttgactttgt tcagtcattt 360atctcacgtg
cctctggtga tgcttgggat ctcgccgata cgatcgatga tgatgaccac 420cgccttgtca
cgtgctctcc accgactccg atcgtttccg ttgctaaatt acctaatccg 480gaacctattg
ttaccgaatc gcttcctgag gaagacgagg agattgtgaa atcggttatc 540gacggagtta
ttccatcgta ctcgcttgaa tctcgtctcg gtgattgcaa aagagcggcg 600tcgattcgtc
gtgaggcgtt gcagagagtc accgggagat cgattgaagg gttaccgttg 660gatggatttg
attatgaatc gattttgggg caatgctgtg agatgcctgt tggatacatt 720cagattcctg
ttgggattgc tggtccattg ttgcttgatg gttatgagta ctctgttcct 780atggctacaa
ccgaaggttg tttggttgct agcactaaca gaggctgcaa ggctatgttt 840atctctggtg
gcgccaccag taccgttctt aaggacggta tgacccgagc acctgttgtt 900cggttcgctt
cggcgagacg agcttcggag cttaagtttt tcttggagaa tccagagaac 960tttgatactt
tggcagtagt cttcaacagg tcgagtagat ttgcaagact gcaaagtgtt 1020aaatgcacaa
tcgcggggaa gaatgcttat gtaaggttct gttgtagtac tggtgatgct 1080atggggatga
atatggtttc taaaggtgtg cagaatgttc ttgagtatct taccgatgat 1140ttccctgaca
tggatgtgat tggaatctct ggtaacttct gttcggacaa gaaacctgct 1200gctgtgaact
ggattgaggg acgtggtaaa tcagttgttt gcgaggctgt aatcagagga 1260gagatcgtga
acaaggtctt gaaaacgagc gtggctgctt tagtcgagct caacatgctc 1320aagaacctag
ctggctctgc tgttgcaggc tctctaggtg gattcaacgc tcatgccagt 1380aacatagtgt
ctgctgtatt catagctact ggccaagatc cagctcaaaa cgtggagagt 1440tctcaatgca
tcaccatgat ggaagctatt aatgacggca aagatatcca tatctcagtc 1500actatgccat
ctatcgaggt ggggacagtg ggaggaggaa cacagcttgc atctcaatca 1560gcgtgtttaa
acctgctcgg agttaaagga gcaagcacag agtcgccggg aatgaacgca 1620aggaggctag
cgacgatcgt agccggagca gttttagctg gagagttatc tttaatgtca 1680gcaattgcag
ctggacagct tgtgagaagt cacatgaaat acaatagatc cagccgagac 1740atctctggag
caacgacaac gacaacaaca acaacatga
177923684DNAArtificial SequencePCR primer containing S. pombe DNA
23atgagttccc aacaagagaa aaaggattat gatgaagaac aattaaggtt gatggaagaa
60gtttgtatcg ttgtagatga aaatgatgtc cctttaagat atggaacgaa aaaggagtgt
120catttgatgg aaaatataaa taaaggtctt ttgcatagag cattctctat gttcatcttt
180gatgagcaaa atcgcctttt acttcagcag cgtgcagaag agaaaattac atttccatcc
240ttatggacga atacatgttg ctcccaccca ttggatgttg ctggtgaacg tggtaatact
300ttacctgaag ctgttgaagg tgttaagaat gcagctcaac gcaagctgtt ccatgaattg
360ggtattcaag ccaagtatat tcccaaagac aaatttcagt ttcttacacg aatccattac
420cttgctccta gtactggtgc ttggggagag catgaaattg actacattct tttcttcaaa
480ggtaaagttg agctggatat caatcccaat gaagttcaag cctataagta tgttactatg
540gaagagttaa aagagatgtt ttccgatcct caatatggat tcacaccatg gttcaaactt
600atttgtgagc attttatgtt taaatggtgg caggatgtag atcatgcgtc aaaattccaa
660gataccttaa ttcatcgttg ctaa
68424531DNAArtificial SequencePCR primer containing Streptomyces sp CL190
DNA 24atgagtgagc ttatacccgc ctgggttggt gacagactgg ctccggtgga caagttggag
60gtgcatttga aagggctccg ccacaaggcg gtgtctgttt tcgtcatgga tggcgaaaac
120gtgctgatcc agcgccgctc ggaggagaaa tatcactctc ccgggctttg ggcgaacacc
180tgctgcaccc atccgggctg gaccgaacgc cccgaggaat gcgcggtgcg gcggctgcgc
240gaggagctgg ggatcaccgg gctttatccc gcccatgccg accggctgga atatcgcgcc
300gatgtcggcg gcggcatgat cgagcatgag gtggtcgaca tctatctggc ctatgccaaa
360ccgcatatgc ggatcacccc cgatccgcgc gaagtggccg aggtgcgctg gatcggcctt
420tacgatctgg cggccgaggc cggtcggcat cccgagcggt tctcgaaatg gctcaacatc
480tatctgtcga gccatcttga ccggattttc ggatcgatcc tgcgcggctg a
5312565DNAArtificial SequencePCR primer containing Streptomyces sp CL190
DNA 25ggggtaccgc ggccgcacgc gtctatgcac caacctttgc ggtcttgttg tcgcgttcca
60gctgg
652660DNAArtificial SequenceOligonucleotide containing S. cerevisiae DNA
26gagctccacc gcggcggccg cgtcgactac ggccgcagga ggagttcata tgtcagagtt
602760DNAArtificial SequenceOligonucleotide containing S. cerevisiae DNA
27tctaccaaag gaagaggagt tttaactcga gtaggaggca catatgtctc agaacgttta
602860DNAArtificial SequenceOligonucleotide containing Streptomyces
sp CL190 and R. capsulatus DNA 28caagaccgca aaggttggtg catagacgcg
gtaaggaggc acatatgagt gagcttatac 602960DNAArtificial
SequenceOligonucleotide containing R. capsulatus DNA 29cctgcgcggc
tgagcggccg cggatccgat cgcgtgcggc cgcggtaccc aattcgccct
603060DNAArtificial SequenceOligonucleotide containing Streptomyces sp
CL190and S. cerevisiae DNA 30tgtcattgaa aagatatgag gatcctctag
gtacttccct ggcgtgtgca gcggttgacg 603160DNAArtificial
SequenceOligonucleotide containing Streptomyces sp CL190 DNA
31cgattccgca ttatcggtac gggtgcctac ctagaactag tggatccccc gggctgcagg
603260DNAArtificial SequenceOligonucleotide containing N. tabacum and S.
cerevisiae DNA 32ctttcctgaa acataattta taatcagatc ggccgcagga
ggagttcata tgtcagagtt 603360DNAArtificial SequenceOligonucleotide
containing N. tabacum and R. capsulatus DNA 33ttcggatcga tcctgcgcgg
ctgagcggcc gatctaaaca aacccggaac agaccgttgg 603459DNAArtificial
SequenceOligonucleotide containing N. tabacum and S. cerevisiae DNA
34ctttcctgaa acataattta taatcagatc ggccgcagga ggagttcata tgtcagagt
593560DNAArtificial SequenceOligonucleotide containing N. tabacum and
S. pombe DNA 35tcgttgctaa ggatcccccg ggatccggcc gatctaaaca aacccggaac
agaccgttgg 603613DNAArtificial SequenceOligonucleotide containing
NotI restriction site 36catggcggcc gcg
133713DNAArtificial SequenceOligonucleotide
containing NotI restriction site 37gatccgcggc cgc
133860DNAArtificial
SequenceOligonucleotide containing S. cerevisiae DNA 38ttaaataagg
aggaataaac catggcggcc gcaggaggag ttcatatgtc agagttgaga
603960DNAArtificial SequenceOligonucleotide containing A. thaliana DNA
39aacaacaaca acatgacccg ggatccggcc gcgatccgag ctcgagatct gcagctggta
604060DNAArtificial SequenceOligonucleotide containing S. cerevisiae DNA
40tcgattaaat aaggaggaat aaaccatggc ggccgcagga ggagttcata tgtcagagtt
604160DNAArtificial SequenceOligonucleotide containing R. capsulatus DNA
41gattttcgga tcgatcctgc gcggctgagc ggccgcgatc cgagctcgag atctgcagct
604260DNAArtificial SequenceOligonucleotide containing S. cerevisiae DNA
42tcgattaaat aaggaggaat aaaccatggc ggccgcagga ggagttcata tgtcagagtt
604360DNAArtificial SequenceOligonucleotide containing S. pombe DNA
43ttcatcgttg ctaaggatcc cccgggatcc ggccgcgatc cgagctcgag atctgcagct
604461DNAArtificial SequenceOligonucleotide containing R. capsulatus DNA
44ttaaataagg aggaataaac catggcggcc gtaaggaggc acatatgagt gagcttatac
60t
614561DNAArtificial SequenceOligonucleotide containing R. capsulatus DNA
45gcctgcgcgg ctgagcggcc gcggatccga tggccgcgat ccgagctcga gatctgcagc
60t
614660DNAArtificial SequenceOligonucleotide containing S. pombe DNA
46ttaaataagg aggaataaac catggcggcc gtaggaggca catatgagtt cccaacaaga
604760DNAArtificial SequenceOligonucleotide containing S. pombe DNA
47accttaattc atcgttgcta aggatccccc ggccgcgatc cgagctcgag atctgcagct
60481356DNASaccharomyces cerevisiae 48atgtcagagt tgagagcctt cagtgcccca
gggaaagcgt tactagctgg tggatattta 60gttttagata caaaatatga agcatttgta
gtcggattat cggcaagaat gcatgctgta 120gcccatcctt acggttcatt gcaagggtct
gataagtttg aagtgcgtgt gaaaagtaaa 180caatttaaag atggggagtg gctgtaccat
ataagtccta aaagtggctt cattcctgtt 240tcgataggcg gatctaagaa ccctttcatt
gaaaaagtta tcgctaacgt atttagctac 300tttaaaccta acatggacga ctactgcaat
agaaacttgt tcgttattga tattttctct 360gatgatgcct accattctca ggaggatagc
gttaccgaac atcgtggcaa cagaagattg 420agttttcatt cgcacagaat tgaagaagtt
cccaaaacag ggctgggctc ctcggcaggt 480ttagtcacag ttttaactac agctttggcc
tccttttttg tatcggacct ggaaaataat 540gtagacaaat atagagaagt tattcataat
ttagcacaag ttgctcattg tcaagctcag 600ggtaaaattg gaagcgggtt tgatgtagcg
gcggcagcat atggatctat cagatataga 660agattcccac ccgcattaat ctctaatttg
ccagatattg gaagtgctac ttacggcagt 720aaactggcgc atttggttga tgaagaagac
tggaatatta cgattaaaag taaccattta 780ccttcgggat taactttatg gatgggcgat
attaagaatg gttcagaaac agtaaaactg 840gtccagaagg taaaaaattg gtatgattcg
catatgccag aaagcttgaa aatatataca 900gaactcgatc atgcaaattc tagatttatg
gatggactat ctaaactaga tcgcttacac 960gagactcatg acgattacag cgatcagata
tttgagtctc ttgagaggaa tgactgtacc 1020tgtcaaaagt atcctgaaat cacagaagtt
agagatgcag ttgccacaat tagacgttcc 1080tttagaaaaa taactaaaga atctggtgcc
gatatcgaac ctcccgtaca aactagctta 1140ttggatgatt gccagacctt aaaaggagtt
cttacttgct taatacctgg tgctggtggt 1200tatgacgcca ttgcagtgat tactaagcaa
gatgttgatc ttagggctca aaccgctaat 1260gacaaaagat tttctaaggt tcaatggctg
gatgtaactc aggctgactg gggtgttagg 1320aaagaaaaag atccggaaac ttatcttgat
aaataa 1356491332DNASaccharomyces cerevisiae
49atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg tgaacactct
60gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta
120ataagcgagt catctgcacc agatactatt gaattggact tcccggacat tagctttaat
180cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa ctcccaaaaa
240ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag tcttttggat
300ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg tttcctgtat
360atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa gtctacttta
420cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc cttagctatg
480gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga aaacgataag
540catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac cccttcagga
600atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga ctcacataat
660ggaacaataa acacaaacaa ttttaagttc ttagatgatt tcccagccat tccaatgatc
720ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg
780gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg tgaatgtgcc
840ctacaaggct tagagatcat gactaagtta agtaaatgta aaggcaccga tgacgaggct
900gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat aaatcatgga
960ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa tctgagcgat
1020gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg ctctttgact
1080ttgttacgaa gagacattac tcaagagcaa attgacagct tcaaaaagaa attgcaagat
1140gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg tttgttaagc
1200gcaaaaaatt tgaataaaga tcttaaaatc aaatccctag tattccaatt atttgaaaat
1260aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac gaatttacca
1320tggacttcat aa
1332501191DNASaccharomyces cerevisiae 50atgaccgttt acacagcatc cgttaccgca
cccgtcaaca tcgcaaccct taagtattgg 60gggaaaaggg acacgaagtt gaatctgccc
accaattcgt ccatatcagt gactttatcg 120caagatgacc tcagaacgtt gacctctgcg
gctactgcac ctgagtttga acgcgacact 180ttgtggttaa atggagaacc acacagcatc
gacaatgaaa gaactcaaaa ttgtctgcgc 240gacctacgcc aattaagaaa ggaaatggaa
tcgaaggacg cctcattgcc cacattatct 300caatggaaac tccacattgt ctccgaaaat
aactttccta cagcagctgg tttagcttcc 360tccgctgctg gctttgctgc attggtctct
gcaattgcta agttatacca attaccacag 420tcaacttcag aaatatctag aatagcaaga
aaggggtctg gttcagcttg tagatcgttg 480tttggcggat acgtggcctg ggaaatggga
aaagctgaag atggtcatga ttccatggca 540gtacaaatcg cagacagctc tgactggcct
cagatgaaag cttgtgtcct agttgtcagc 600gatattaaaa aggatgtgag ttccactcag
ggtatgcaat tgaccgtggc aacctccgaa 660ctatttaaag aaagaattga acatgtcgta
ccaaagagat ttgaagtcat gcgtaaagcc 720attgttgaaa aagatttcgc cacctttgca
aaggaaacaa tgatggattc caactctttc 780catgccacat gtttggactc tttccctcca
atattctaca tgaatgacac ttccaagcgt 840atcatcagtt ggtgccacac cattaatcag
ttttacggag aaacaatcgt tgcatacacg 900tttgatgcag gtccaaatgc tgtgttgtac
tacttagctg aaaatgagtc gaaactcttt 960gcatttatct ataaattgtt tggctctgtt
cctggatggg acaagaaatt tactactgag 1020cagcttgagg ctttcaacca tcaatttgaa
tcatctaact ttactgcacg tgaattggat 1080cttgagttgc aaaaggatgt tgccagagtg
attttaactc aagtcggttc aggcccacaa 1140gaaacaaacg aatctttgat tgacgcaaag
actggtctac caaaggaata a 1191511197DNASaccharomyces cerevisiae
51atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc attccagggt
60tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg cgccttggct
120aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg taacgttctt
180tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg tttgagtaat
240catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc aatcattttg
300ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg ttgtgaatct
360atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt tggccaaact
420gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg tctagccatg
480ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca acaagacaat
540tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa attcgacaat
600gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca agtcacgaag
660gacgaggaac ctgctagatt acacgttgaa aaattgagat ctgcaaggac tgttttccaa
720aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg tgctgcagcc
780gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt tgaagccttt ggctattatc
840aaaggttggg gtgaggccgc tcatcaacca gctgatttta catgggctcc atctcttgca
900gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga ttactttgaa
960ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa gctagaccca
1020tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg ttgttctggt
1080gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa gatcggtgtt
1140gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa gatatga
1197521386DNAArabidopsis thaliana 52atggcgaaga acgttgggat tttggctatg
gatatctatt tccctcccac ctgtgttcaa 60caggaagctt tggaagcaca tgatggagca
agtaaaggga aatacactat tggacttggc 120caagattgtt tagctttttg cactgagctt
gaagatgtta tctctatgag tttcaatgcg 180gtgacatcac tttttgagaa gtataagatt
gaccctaacc aaatcgggcg tcttgaagta 240ggaagtgaga ctgttattga caaaagcaag
tccatcaaga ccttcttgat gcagctcttt 300gagaaatgtg gaaacactga tgtcgaaggt
gttgactcga ccaatgcttg ctatggtgga 360actgcagctt tgttaaactg tgtcaattgg
gttgagagta actcttggga tggacgttat 420ggcctcgtca tttgtactga cagcgcggtt
tatgcagaag gacccgcaag gcccactgga 480ggagctgcag cgattgctat gttgatagga
cctgatgctc ctatcgtttt cgaaagcaaa 540ttgagagcaa gccacatggc tcatgtctat
gacttttaca agcccaatct tgctagcgag 600tacccggttg ttgatggtaa gctttcacag
acttgctacc tcatggctct tgactcctgc 660tataaacatt tatgcaacaa gttcgagaag
atcgagggca aagagttctc cataaatgat 720gctgattaca ttgttttcca ttctccatac
aataaacttg tacagaaaag ctttgctcgt 780ctcttgtaca acgacttctt gagaaacgca
agctccattg acgaggctgc caaagaaaag 840ttcacccctt attcatcttt gacccttgac
gagagttacc aaagccgtga tcttgaaaag 900gtgtcacaac aaatttcgaa accgttttat
gatgctaaag tgcaaccaac gactttaata 960ccaaaggaag tcggtaacat gtacactgct
tctctctacg ctgcatttgc ttccctcatc 1020cacaataaac acaatgattt ggcgggaaag
cgggtggtta tgttctctta tggaagtggc 1080tccaccgcaa caatgttctc attacgcctc
aacgacaata agcctccttt cagcatttca 1140aacattgcat ctgtaatgga tgttggcggt
aaattgaaag ctagacatga gtatgcacct 1200gagaagtttg tggagacaat gaagctaatg
gaacataggt atggagcaaa ggactttgtg 1260acaaccaagg agggtattat agatcttttg
gcaccgggaa cttattatct gaaagaggtt 1320gattccttgt accggagatt ctatggcaag
aaaggtgaag atggatctgt agccaatgga 1380cactga
1386531779DNAArabidopsis thaliana
53atggatctcc gtcggaggcc tcctaaacca ccggttacca acaacaacaa ctccaacgga
60tctttccgtt cttatcagcc tcgcacttcc gatgacgatc atcgtcgccg ggctacaaca
120attgctcctc caccgaaagc atccgacgcg cttcctcttc cgttatatct cacaaacgcc
180gttttcttca cgctcttctt ctccgtcgcg tattacctcc tccaccggtg gcgtgacaag
240atccgttaca atacgcctct tcacgtcgtc actatcacag aactcggcgc cattattgct
300ctcatcgctt cgtttatcta tctcctaggg ttttttggta ttgactttgt tcagtcattt
360atctcacgtg cctctggtga tgcttgggat ctcgccgata cgatcgatga tgatgaccac
420cgccttgtca cgtgctctcc accgactccg atcgtttccg ttgctaaatt acctaatccg
480gaacctattg ttaccgaatc gcttcctgag gaagacgagg agattgtgaa atcggttatc
540gacggagtta ttccatcgta ctcgcttgaa tctcgtctcg gtgattgcaa aagagcggcg
600tcgattcgtc gtgaggcgtt gcagagagtc accgggagat cgattgaagg gttaccgttg
660gatggatttg attatgaatc gattttgggg caatgctgtg agatgcctgt tggatacatt
720cagattcctg ttgggattgc tggtccattg ttgcttgatg gttatgagta ctctgttcct
780atggctacaa ccgaaggttg tttggttgct agcactaaca gaggctgcaa ggctatgttt
840atctctggtg gcgccaccag taccgttctt aaggacggta tgacccgagc acctgttgtt
900cggttcgctt cggcgagacg agcttcggag cttaagtttt tcttggagaa tccagagaac
960tttgatactt tggcagtagt cttcaacagg tcgagtagat ttgcaagact gcaaagtgtt
1020aaatgcacaa tcgcggggaa gaatgcttat gtaaggttct gttgtagtac tggtgatgct
1080atggggatga atatggtttc taaaggtgtg cagaatgttc ttgagtatct taccgatgat
1140ttccctgaca tggatgtgat tggaatctct ggtaacttct gttcggacaa gaaacctgct
1200gctgtgaact ggattgaggg acgtggtaaa tcagttgttt gcgaggctgt aatcagagga
1260gagatcgtga acaaggtctt gaaaacgagc gtggctgctt tagtcgagct caacatgctc
1320aagaacctag ctggctctgc tgttgcaggc tctctaggtg gattcaacgc tcatgccagt
1380aacatagtgt ctgctgtatt catagctact ggccaagatc cagctcaaaa cgtggagagt
1440tctcaatgca tcaccatgat ggaagctatt aatgacggca aagatatcca tatctcagtc
1500actatgccat ctatcgaggt ggggacagtg ggaggaggaa cacagcttgc atctcaatca
1560gcgtgtttaa acctgctcgg agttaaagga gcaagcacag agtcgccggg aatgaacgca
1620aggaggctag cgacgatcgt agccggagca gttttagctg gagagttatc tttaatgtca
1680gcaattgcag ctggacagct tgtgagaagt cacatgaaat acaatagatc cagccgagac
1740atctctggag caacgacaac gacaacaaca acaacatga
177954684DNAArtificial SequenceSchizosaccharomyces pombe IDI1 (IPP
isomerase) 54atgagttccc aacaagagaa aaaggattat gatgaagaac aattaaggtt
gatggaagaa 60gtttgtatcg ttgtagatga aaatgatgtc cctttaagat atggaacgaa
aaaggagtgt 120catttgatgg aaaatataaa taaaggtctt ttgcatagag cattctctat
gttcatcttt 180gatgagcaaa atcgcctttt acttcagcag cgtgcagaag agaaaattac
atttccatcc 240ttatggacga atacatgttg ctcccaccca ttggatgttg ctggtgaacg
tggtaatact 300ttacctgaag ctgttgaagg tgttaagaat gcagctcaac gcaagctgtt
ccatgaattg 360ggtattcaag ccaagtatat tcccaaagac aaatttcagt ttcttacacg
aatccattac 420cttgctccta gtactggtgc ttggggagag catgaaattg actacattct
tttcttcaaa 480ggtaaagttg agctggatat caatcccaat gaagttcaag cctataagta
tgttactatg 540gaagagttaa aagagatgtt ttccgatcct caatatggat tcacaccatg
gttcaaactt 600atttgtgagc attttatgtt taaatggtgg caggatgtag atcatgcgtc
aaaattccaa 660gataccttaa ttcatcgttg ctaa
68455531DNAArtificial SequenceRhodobacter capsulatus idiB
(IPP isomerase) 55atgagtgagc ttatacccgc ctgggttggt gacagactgg ctccggtgga
caagttggag 60gtgcatttga aagggctccg ccacaaggcg gtgtctgttt tcgtcatgga
tggcgaaaac 120gtgctgatcc agcgccgctc ggaggagaaa tatcactctc ccgggctttg
ggcgaacacc 180tgctgcaccc atccgggctg gaccgaacgc cccgaggaat gcgcggtgcg
gcggctgcgc 240gaggagctgg ggatcaccgg gctttatccc gcccatgccg accggctgga
atatcgcgcc 300gatgtcggcg gcggcatgat cgagcatgag gtggtcgaca tctatctggc
ctatgccaaa 360ccgcatatgc ggatcacccc cgatccgcgc gaagtggccg aggtgcgctg
gatcggcctt 420tacgatctgg cggccgaggc cggtcggcat cccgagcggt tctcgaaatg
gctcaacatc 480tatctgtcga gccatcttga ccggattttc ggatcgatcc tgcgcggctg a
531561059DNAStreptomyces sp. 56atgacggaaa cgcacgccat
agccggggtc ccgatgaggt gggtgggacc ccttcgtatt 60tccgggaacg tcgccgagac
cgagacccag gtcccgctcg ccacgtacga gtcgccgctg 120tggccgtcgg tgggccgcgg
ggcgaaggtc tcccggctga cggagaaggg catcgtcgcc 180accctcgtcg acgagcggat
gacccgctcg gtgatcgtcg aggcgacgga cgcgcagacc 240gcgtacatgg ccgcgcagac
catccacgcc cgcatcgacg agctgcgcga ggtggtgcgc 300ggctgcagcc ggttcgccca
gctgatcaac atcaagcacg agatcaacgc gaacctgctg 360ttcatccggt tcgagttcac
caccggtgac gcctccggcc acaacatggc cacgctcgcc 420tccgatgtgc tcctggggca
cctgctggag acgatccctg gcatctccta cggctcgatc 480tccggcaact actgcacgga
caagaaggcc accgcgatca acggcatcct cggccgcggc 540aagaacgtga tcaccgagct
gctggtgccg cgggacgtcg tcgagaacaa cctgcacacc 600acggctgcca agatcgtcga
gctgaacatc cgcaagaacc tgctcggcac cctgctcgcc 660ggcggcatcc gctcggccaa
cgcccacttc gcgaacatgc tgctcggctt ctacctggcc 720accggccagg acgccgccaa
catcgtcgag ggctcgcagg gcgtcgtcat ggccgaggac 780cgcgacggcg acctctactt
cgcctgcacc ctgccgaacc tgatcgtcgg cacggtcggc 840aacggcaagg gtctcggctt
cgtggagacg aacctcgccc ggctcggctg ccgagccgac 900cgcgaacccg gggagaacgc
ccgccgcctc gccgtcatcg cggcagcgac cgtgctgtgc 960ggtgaactct cgctgctcgc
ggcacagacg aacccgggcg aactcatgcg cgcgcacgtc 1020cagctggaac gcgacaacaa
gaccgcaaag gttggtgca 1059576798DNAArtificial
SequenceStreptomyces sp CL190 gene cluster containing mevalonate
pathway and IPP isomerase orfs 57tacgtacttc cctggcgtgt gcagcggttg
acgcgccgtg ccctcgctgc gagcggcgcg 60cacatctgac gtcctgcttt attgctttct
cagaactcgg gacgaagcga tcccatgatc 120acgcgatctc catgcagaaa agacaaaggg
agctgagtgc gttgacacta ccgacctcgg 180ctgagggggt atcagaaagc caccgggccc
gctcggtcgg catcggtcgc gcccacgcca 240aggccatcct gctgggagag catgcggtcg
tctacggagc gccggcactc gctctgccga 300ttccgcagct cacggtcacg gccagcgtcg
gctggtcgtc cgaggcctcc gacagtgcgg 360gtggcctgtc ctacacgatg accggtacgc
cgtcgcgggc actggtgacg caggcctccg 420acggcctgca ccggctcacc gcggaattca
tggcgcggat gggcgtgacg aacgcgccgc 480acctcgacgt gatcctggac ggcgcgatcc
cgcacggccg gggtctcggc tccagcgcgg 540ccggctcacg cgcgatcgcc ttggccctcg
ccgacctctt cggccacgaa ctggccgagc 600acacggcgta cgaactggtg cagacggccg
agaacatggc gcacggccgg gccagcggcg 660tggacgcgat gacggtcggc gcgtcccggc
cgctgctgtt ccagcagggc cgcaccgagc 720gactggccat cggctgcgac agcctgttca
tcgtcgccga cagcggcgtc ccgggcagca 780ccaaggaagc ggtcgagatg ctgcgggagg
gattcacccg cagcgccgga acacaggagc 840ggttcgtcgg ccgggcgacg gaactgaccg
aggccgcccg gcaggccctc gccgacggcc 900ggcccgagga gctgggctcg cagctgacgt
actaccacga gctgctccat gaggcccgcc 960tgagcaccga cggcatcgat gcgctggtcg
aggccgcgct gaaggcaggc agcctcggag 1020ccaagatcac cggcggtggt ctgggcggct
gcatgatcgc acaggcccgg cccgaacagg 1080cccgggaggt cacccggcag ctccacgagg
ccggtgccgt acagacctgg gtcgtaccgc 1140tgaaagggct cgacaaccat gcgcagtgaa
cacccgacca cgaccgtgct ccagtcgcgg 1200gagcagggca gcgcggccgg cgccaccgcg
gtcgcgcacc caaacatcgc gctgatcaag 1260tactggggca agcgcgacga gcggctgatc
ctgccctgca ccaccagcct gtcgatgacg 1320ctggacgtct tccccacgac caccgaggtc
cggctcgacc ccgccgccga gcacgacacg 1380gccgccctca acggcgaggt ggccacgggc
gagacgctgc gccgcatcag cgccttcctc 1440tccctggtgc gggaggtggc gggcagcgac
cagcgggccg tggtggacac ccgcaacacc 1500gtgcccaccg gggcgggcct ggcgtcctcc
gccagcgggt tcgccgccct cgccgtcgcg 1560gccgcggccg cctacgggct cgaactcgac
gaccgcgggc tgtcccggct ggcccgacgt 1620ggatccggct ccgcctcgcg gtcgatcttc
ggcggcttcg ccgtctggca cgccggcccc 1680gacggcacgg ccacggaagc ggacctcggc
tcctacgccg agccggtgcc cgcggccgac 1740ctcgacccgg cgctggtcat cgccgtggtc
aacgccggcc ccaagcccgt ctccagccgc 1800gaggccatgc gccgcaccgt cgacacctcg
ccgctgtacc ggccgtgggc cgactccagt 1860aaggacgacc tggacgagat gcgctcggcg
ctgctgcgcg gcgacctcga ggccgtgggc 1920gagatcgcgg agcgcaacgc gctcggcatg
cacgccacca tgctggccgc ccgccccgcg 1980gtgcggtacc tgtcgccggc cacggtcacc
gtgctcgaca gcgtgctcca gctccgcaag 2040gacggtgtcc tggcctacgc gaccatggac
gccggtccca acgtgaaggt gctgtgccgg 2100cgggcggacg ccgagcgggt ggccgacgtc
gtacgcgccg ccgcgtccgg cggtcaggtc 2160ctcgtcgccg ggccgggaga cggtgcccgc
ctgctgagcg agggcgcatg acgacaggtc 2220agcgcacgat cgtccggcac gcgccgggca
agctgttcgt cgcgggcgag tacgcggtcg 2280tggatccggg caacccggcg atcctggtag
cggtcgaccg gcacatcagc gtcaccgtgt 2340ccgacgccga cgcggacacc ggggccgccg
acgtcgtgat ctcctccgac ctcggtccgc 2400aggcggtcgg ctggcgctgg cacgacggcc
ggctcgtcgt ccgcgacccg gacgacgggc 2460agcaggcgcg cagcgccctg gcccacgtgg
tgtcggcgat cgagaccgtg ggccggctgc 2520tgggcgaacg cggacagaag gtccccgctc
tcaccctctc cgtcagcagc cgcctgcacg 2580aggacggccg gaagttcggc ctgggctcca
gcggcgcggt gaccgtggcg accgtagccg 2640ccgtcgccgc gttctgcgga ctcgaactgt
ccaccgacga acggttccgg ctggccatgc 2700tcgccaccgc ggaactcgac cccaagggct
ccggcgggga cctcgccgcc agcacctggg 2760gcggctggat cgcctaccag gcgcccgacc
gggcctttgt gctcgacctg gcccggcgcg 2820tgggagtcga ccggacactg aaggcgccct
ggccggggca ctcggtgcgc cgactgccgg 2880cgcccaaggg cctcaccctg gaggtcggct
ggaccggaga gcccgcctcc accgcgtccc 2940tggtgtccga tctgcaccgc cgcacctggc
ggggcagcgc ctcccaccag aggttcgtcg 3000agaccacgac cgactgtgtc cgctccgcgg
tcaccgccct ggagtccggc gacgacacga 3060gcctgctgca cgagatccgc cgggcccgcc
aggagctggc ccgcctggac gacgaggtcg 3120gcctcggcat cttcacaccc aagctgacgg
cgctgtgcga cgccgccgaa gccgtcggcg 3180gcgcggccaa gccctccggg gcaggcggcg
gcgactgcgg catcgccctg ctggacgccg 3240aggcgtcgcg ggacatcaca catgtacggc
aacggtggga gacagccggg gtgctgcccc 3300tgcccctgac tcctgccctg gaagggatct
aagaatgacc agcgcccaac gcaaggacga 3360ccacgtacgg ctcgccatcg agcagcacaa
cgcccacagc ggacgcaacc agttcgacga 3420cgtgtcgttc gtccaccacg ccctggccgg
catcgaccgg ccggacgtgt ccctggccac 3480gtccttcgcc gggatctcct ggcaggtgcc
gatctacatc aacgcgatga ccggcggcag 3540cgagaagacc ggcctcatca accgggacct
ggccaccgcc gcccgcgaga ccggcgtccc 3600catcgcgtcc gggtccatga acgcgtacat
caaggacccc tcctgcgccg acacgttccg 3660tgtgctgcgc gacgagaacc ccaacgggtt
cgtcatcgcg aacatcaacg ccaccacgac 3720ggtcgacaac gcgcagcgcg cgatcgacct
gatcgaggcg aacgccctgc agatccacat 3780caacacggcg caggagacgc cgatgccgga
gggcgaccgg tcgttcgcgt cctgggtccc 3840gcagatcgag aagatcgcgg cggccgtcga
catccccgtg atcgtcaagg aggtcggcaa 3900cggcctgagc cggcagacca tcctgctgct
cgccgacctc ggcgtgcagg cggcggacgt 3960cagcggccgc ggcggcacgg acttcgcccg
catcgagaac ggccgccggg agctcggcga 4020ctacgcgttc ctgcacggct gggggcagtc
caccgccgcc tgcctgctgg acgcccagga 4080catctccctg cccgtcctcg cctccggcgg
tgtgcgtcac ccgctcgacg tggtccgcgc 4140cctcgcgctc ggcgcccgcg ccgtcggctc
ctccgccggc ttcctgcgca ccctgatgga 4200cgacggcgtc gacgcgctga tcacgaagct
cacgacctgg ctggaccagc tggcggcgct 4260gcagaccatg ctcggcgcgc gcaccccggc
cgacctcacc cgctgcgacg tgctgctcca 4320cggcgagctg cgtgacttct gcgccgaccg
gggcatcgac acgcgccgcc tcgcccagcg 4380ctccagctcc atcgaggccc tccagacgac
gggaagcaca cgatgacgga aacgcacgcc 4440atagccgggg tcccgatgag gtgggtggga
ccccttcgta tttccgggaa cgtcgccgag 4500accgagaccc aggtcccgct cgccacgtac
gagtcgccgc tgtggccgtc ggtgggccgc 4560ggggcgaagg tctcccggct gacggagaag
ggcatcgtcg ccaccctcgt cgacgagcgg 4620atgacccgct cggtgatcgt cgaggcgacg
gacgcgcaga ccgcgtacat ggccgcgcag 4680accatccacg cccgcatcga cgagctgcgc
gaggtggtgc gcggctgcag ccggttcgcc 4740cagctgatca acatcaagca cgagatcaac
gcgaacctgc tgttcatccg gttcgagttc 4800accaccggtg acgcctccgg ccacaacatg
gccacgctcg cctccgatgt gctcctgggg 4860cacctgctgg agacgatccc tggcatctcc
tacggctcga tctccggcaa ctactgcacg 4920gacaagaagg ccaccgcgat caacggcatc
ctcggccgcg gcaagaacgt gatcaccgag 4980ctgctggtgc cgcgggacgt cgtcgagaac
aacctgcaca ccacggctgc caagatcgtc 5040gagctgaaca tccgcaagaa cctgctcggc
accctgctcg ccggcggcat ccgctcggcc 5100aacgcccact tcgcgaacat gctgctcggc
ttctacctgg ccaccggcca ggacgccgcc 5160aacatcgtcg agggctcgca gggcgtcgtc
atggccgagg accgcgacgg cgacctctac 5220ttcgcctgca ccctgccgaa cctgatcgtc
ggcacggtcg gcaacggcaa gggtctcggc 5280ttcgtggaga cgaacctcgc ccggctcggc
tgccgagccg accgcgaacc cggggagaac 5340gcccgccgcc tcgccgtcat cgcggcagcg
accgtgctgt gcggtgaact ctcgctgctc 5400gcggcacaga cgaacccggg cgaactcatg
cgcgcgcacg tccagctgga acgcgacaac 5460aagaccgcaa aggttggtgc atagggcatg
tccatctcca taggcattca cgacctgtcg 5520ttcgccacaa ccgagttcgt cctgccgcac
acggcgctcg ccgagtacaa cggcaccgag 5580atcggcaagt accacgtcgg catcggccag
cagtcgatga gcgtgccggc cgccgacgag 5640gacatcgtga ccatggccgc gaccgcggcg
cggcccatca tcgagcgcaa cggcaagagc 5700cggatccgca cggtcgtgtt cgccacggag
tcgtcgatcg accaggcgaa ggcgggcggc 5760gtgtacgtgc actccctgct ggggctggag
tcggcctgcc gggtcgtcga gctgaagcag 5820gcctgctacg gggccaccgc cgcccttcag
ttcgccatcg gcctggtgcg gcgcgacccc 5880gcccagcagg tcctggtcat cgccagtgac
gtctccaagt acgagctgga cagccccggc 5940gaggcgaccc agggcgcggc cgcggtggcc
atgctggtcg gcgccgaccc ggccctgctg 6000cgtatcgagg agccgtcggg cctgttcacc
gccgacgtca tggacttctg gcggcccaac 6060tacctcacca ccgctctggt cgacggccag
gagtccatca acgcctacct gcaggccgtc 6120gagggcgcct ggaaggacta cgcggagcag
gacggccggt cgctggagga gttcgcggcg 6180ttcgtctacc accagccgtt cacgaagatg
gcctacaagg cgcaccgcca cctgctgaac 6240ttcaacggct acgacaccga caaggacgcc
atcgagggcg ccctcggcca gacgacggcg 6300tacaacaacg tcatcggcaa cagctacacc
gcgtcggtgt acctgggcct ggccgccctg 6360ctcgaccagg cggacgacct gacgggccgt
tccatcggct tcctgagcta cggctcgggc 6420agcgtcgccg agttcttctc gggcaccgtc
gtcgccgggt accgcgagcg tctgcgcacc 6480gaggcgaacc aggaggcgat cgcccggcgc
aagagcgtcg actacgccac ctaccgcgag 6540ctgcacgagt acacgctccc gtccgacggc
ggcgaccacg ccaccccggt gcagaccacc 6600ggccccttcc ggctggccgg gatcaacgac
cacaagcgca tctacgaggc gcgctagcga 6660cacccctcgg caacggggtg cgccactgtt
cggcgcaccc cgtgccgggc tttcgcacag 6720ctattcacga ccatttgagg ggcgggcagc
cgcatgaccg acgtccgatt ccgcattatc 6780ggtacgggtg cctacgta
6798587693DNAArtificial SequenceOperon
containing A. thaliana and S. cerevisiae DNA 58ggccgcgtcg
acgccggcgg aggcacatat gtctcagaac gtttacattg tatcgactgc 60cagaacccca
attggttcat tccagggttc tctatcctcc aagacagcag tggaattggg 120tgctgttgct
ttaaaaggcg ccttggctaa ggttccagaa ttggatgcat ccaaggattt 180tgacgaaatt
atttttggta acgttctttc tgccaatttg ggccaagctc cggccagaca 240agttgctttg
gctgccggtt tgagtaatca tatcgttgca agcacagtta acaaggtctg 300tgcatccgct
atgaaggcaa tcattttggg tgctcaatcc atcaaatgtg gtaatgctga 360tgttgtcgta
gctggtggtt gtgaatctat gactaacgca ccatactaca tgccagcagc 420ccgtgcgggt
gccaaatttg gccaaactgt tcttgttgat ggtgtcgaaa gagatgggtt 480gaacgatgcg
tacgatggtc tagccatggg tgtacacgca gaaaagtgtg cccgtgattg 540ggatattact
agagaacaac aagacaattt tgccatcgaa tcctaccaaa aatctcaaaa 600atctcaaaag
gaaggtaaat tcgacaatga aattgtacct gttaccatta agggatttag 660aggtaagcct
gatactcaag tcacgaagga cgaggaacct gctagattac acgttgaaaa 720attgagatct
gcaaggactg ttttccaaaa agaaaacggt actgttactg ccgctaacgc 780ttctccaatc
aacgatggtg ctgcagccgt catcttggtt tccgaaaaag ttttgaagga 840aaagaatttg
aagcctttgg ctattatcaa aggttggggt gaggccgctc atcaaccagc 900tgattttaca
tgggctccat ctcttgcagt tccaaaggct ttgaaacatg ctggcatcga 960agacatcaat
tctgttgatt actttgaatt caatgaagcc ttttcggttg tcggtttggt 1020gaacactaag
attttgaagc tagacccatc taaggttaat gtatatggtg gtgctgttgc 1080tctaggtcac
ccattgggtt gttctggtgc tagagtggtt gttacactgc tatccatctt 1140acagcaagaa
ggaggtaaga tcggtgttgc cgccatttgt aatggtggtg gtggtgcttc 1200ctctattgtc
attgaaaaga tatgaggatc ctctagatgc gcaggaggca catatggcga 1260agaacgttgg
gattttggct atggatatct atttccctcc cacctgtgtt caacaggaag 1320ctttggaagc
acatgatgga gcaagtaaag ggaaatacac tattggactt ggccaagatt 1380gtttagcttt
ttgcactgag cttgaagatg ttatctctat gagtttcaat gcggtgacat 1440cactttttga
gaagtataag attgacccta accaaatcgg gcgtcttgaa gtaggaagtg 1500agactgttat
tgacaaaagc aagtccatca agaccttctt gatgcagctc tttgagaaat 1560gtggaaacac
tgatgtcgaa ggtgttgact cgaccaatgc ttgctatggt ggaactgcag 1620ctttgttaaa
ctgtgtcaat tgggttgaga gtaactcttg ggatggacgt tatggcctcg 1680tcatttgtac
tgacagcgcg gtttatgcag aaggacccgc aaggcccact ggaggagctg 1740cagcgattgc
tatgttgata ggtcctgatg ctcctatcgt tttcgaaagc aaattgagag 1800caagccacat
ggctcatgtc tatgactttt acaagcccaa tcttgctagc gagtacccgg 1860ttgttgatgg
taagctttca cagacttgct acctcatggc tcttgactcc tgctataaac 1920atttatgcaa
caagttcgag aagatcgagg gcaaagagtt ctccataaat gatgctgatt 1980acattgtttt
ccattctcca tacaataaac ttgtacagaa aagctttgct cgtctcttgt 2040acaacgactt
cttgagaaac gcaagctcca ttgacgaggc tgccaaagaa aagttcaccc 2100cttattcatc
tttgaccctt gacgagagtt accaaagccg tgatcttgaa aaggtgtcac 2160aacaaattgc
gaaaccgttt tatgatgcta aagtgcaacc aacgacttta ataccaaagg 2220aagtcggtaa
catgtacact gcttctctct acgctgcatt tgcttccctc atccacaaga 2280aacacaatga
tttggcggga aagcgggtgg ttatgttctc ttatggaagt ggctcaaccg 2340caacaatgtt
ctcattacgc ctcaacgaca ataagcctcc tttcagcatt tcaaacattg 2400catctgtaat
ggatgttggc ggtaaattga aagctagaca tgagtatgca cctgagaagt 2460ttgtggagac
aatgaagcta atggaacata ggtatggagc aaaggacttt gtgacaacca 2520aggagggtat
tatagatctt ttggcaccgg gaacttatta tctgaaagag gttgattcct 2580tgtaccggag
attctatggc aagaaaggtg aagatggatc tgtagccaat ggacactgag 2640gatccgtcga
gcacgtggag gcacatatgc aatgctgtga gatgcctgtt ggatacattc 2700agattcctgt
tgggattgct ggtccattgt tgcttgatgg ttatgagtac tctgttccta 2760tggctacaac
cgaaggttgt ttggttgcta gcactaacag aggctgcaag gctatgttta 2820tctctggtgg
cgccaccagt accgttctta aggacggtat gacccgagca cctgttgttc 2880ggttcgcttc
ggcgagacga gcttcggagc ttaagttttt cttggagaat ccagagaact 2940ttgatacttt
ggcagtagtc ttcaacaggt cgagtagatt tgcaagactg caaagtgtta 3000aatgcacaat
cgcggggaag aatgcttatg taaggttctg ttgtagtact ggtgatgcta 3060tggggatgaa
tatggtttct aaaggtgtgc agaatgttct tgagtatctt accgatgatt 3120tccctgacat
ggatgtgatt ggaatctctg gtaacttctg ttcggacaag aaacctgctg 3180ctgtgaactg
gattgaggga cgtggtaaat cagttgtttg cgaggctgta atcagaggag 3240agatcgtgaa
caaggtcttg aaaacgagcg tggctgcttt agtcgagctc aacatgctca 3300agaacctagc
tggctctgct gttgcaggct ctctaggtgg attcaacgct catgccagta 3360acatagtgtc
tgctgtattc atagctactg gccaagatcc agctcaaaac gtggagagtt 3420ctcaatgcat
caccatgatg gaagctatta atgacggcaa agatatccat atctcagtca 3480ctatgccatc
tatcgaggtg gggacagtgg gaggaggaac acagcttgca tctcaatcag 3540cgtgtttaaa
cctgctcgga gttaaaggag caagcacaga gtcgccggga atgaacgcaa 3600ggaggctagc
gacgatcgta gccggagcag ttttagctgg agagttatct ttaatgtcag 3660caattgcagc
tggacagctt gtgagaagtc acatgaaata caatagatcc agccgagaca 3720tctctggagc
aacgacaacg acaacaacaa caacatgacc cgggatccgg ccgcaggagg 3780agttcatatg
tcagagttga gagccttcag tgccccaggg aaagcgttac tagctggtgg 3840atatttagtt
ttagatacaa aatatgaagc atttgtagtc ggattatcgg caagaatgca 3900tgctgtagcc
catccttacg gttcattgca agggtctgat aagtttgaag tgcgtgtgaa 3960aagtaaacaa
tttaaagatg gggagtggct gtaccatata agtcctaaaa gtggcttcat 4020tcctgtttcg
ataggcggat ctaagaaccc tttcattgaa aaagttatcg ctaacgtatt 4080tagctacttt
aaacctaaca tggacgacta ctgcaataga aacttgttcg ttattgatat 4140tttctctgat
gatgcctacc attctcagga ggatagcgtt accgaacatc gtggcaacag 4200aagattgagt
tttcattcgc acagaattga agaagttccc aaaacagggc tgggctcctc 4260ggcaggttta
gtcacagttt taactacagc tttggcctcc ttttttgtat cggacctgga 4320aaataatgta
gacaaatata gagaagttat tcataattta gcacaagttg ctcattgtca 4380agctcagggt
aaaattggaa gcgggtttga tgtagcggcg gcagcatatg gatctatcag 4440atatagaaga
ttcccacccg cattaatctc taatttgcca gatattggaa gtgctactta 4500cggcagtaaa
ctggcgcatt tggttgatga agaagactgg aatattacga ttaaaagtaa 4560ccatttacct
tcgggattaa ctttatggat gggcgatatt aagaatggtt cagaaacagt 4620aaaactggtc
cagaaggtaa aaaattggta tgattcgcat atgccagaaa gcttgaaaat 4680atatacagaa
ctcgatcatg caaattctag atttatggat ggactatcta aactagatcg 4740cttacacgag
actcatgacg attacagcga tcagatattt gagtctcttg agaggaatga 4800ctgtacctgt
caaaagtatc ctgaaatcac agaagttaga gatgcagttg ccacaattag 4860acgttccttt
agaaaaataa ctaaagaatc tggtgccgat atcgaacctc ccgtacaaac 4920tagcttattg
gatgattgcc agaccttaaa aggagttctt acttgcttaa tacctggtgc 4980tggtggttat
gacgccattg cagtgattac taagcaagat gttgatctta gggctcaaac 5040cgctaatgac
aaaagatttt ctaaggttca atggctggat gtaactcagg ctgactgggg 5100tgttaggaaa
gaaaaagatc cggaaactta tcttgataaa ctgcaggagg agttttaatg 5160tcattaccgt
tcttaacttc tgcaccggga aaggttatta tttttggtga acactctgct 5220gtgtacaaca
agcctgccgt cgctgctagt gtgtctgcgt tgagaaccta cctgctaata 5280agcgagtcat
ctgcaccaga tactattgaa ttggacttcc cggacattag ctttaatcat 5340aagtggtcca
tcaatgattt caatgccatc accgaggatc aagtaaactc ccaaaaattg 5400gccaaggctc
aacaagccac cgatggcttg tctcaggaac tcgttagtct tttggatccg 5460ttgttagctc
aactatccga atccttccac taccatgcag cgttttgttt cctgtatatg 5520tttgtttgcc
tatgccccca tgccaagaat attaagtttt ctttaaagtc tactttaccc 5580atcggtgctg
ggttgggctc aagcgcctct atttctgtat cactggcctt agctatggcc 5640tacttggggg
ggttaatagg atctaatgac ttggaaaagc tgtcagaaaa cgataagcat 5700atagtgaatc
aatgggcctt cataggtgaa aagtgtattc acggtacccc ttcaggaata 5760gataacgctg
tggccactta tggtaatgcc ctgctatttg aaaaagactc acataatgga 5820acaataaaca
caaacaattt taagttctta gatgatttcc cagccattcc aatgatccta 5880acctatacta
gaattccaag gtctacaaaa gatcttgttg ctcgcgttcg tgtgttggtc 5940accgagaaat
ttcctgaagt tatgaagcca attctagatg ccatgggtga atgtgcccta 6000caaggcttag
agatcatgac taagttaagt aaatgtaaag gcaccgatga cgaggctgta 6060gaaactaata
atgaactgta tgaacaacta ttggaattga taagaataaa tcatggactg 6120cttgtctcaa
tcggtgtttc tcatcctgga ttagaactta ttaaaaatct gagcgatgat 6180ttgagaattg
gctccacaaa acttaccggt gctggtggcg gcggttgctc tttgactttg 6240ttacgaagag
acattactca agagcaaatt gacagcttca aaaagaaatt gcaagatgat 6300tttagttacg
agacatttga aacagacttg ggtgggactg gctgctgttt gttaagcgca 6360aaaaatttga
ataaagatct taaaatcaaa tccctagtat tccaattatt tgaaaataaa 6420actaccacaa
agcaacaaat tgacgatcta ttattgccag gaaacacgaa tttaccatgg 6480acttcacagg
aggagtttta atgactgtat atactgctag tgtaactgct ccggtaaata 6540ttgctactct
taagtattgg gggaaaaggg acacgaagtt gaatctgccc accaattcgt 6600ccatatcagt
gactttatcg caagatgacc tcagaacgtt gacctctgcg gctactgcac 6660ctgagtttga
acgcgacact ttgtggttaa atggagaacc acacagcatc gacaatgaaa 6720gaactcaaaa
ttgtctgcgc gacctacgcc aattaagaaa ggaaatggaa tcgaaggacg 6780cctcattgcc
cacattatct caatggaaac tccacattgt ctccgaaaat aactttccta 6840cagcagctgg
tttagcttcc tccgctgctg gctttgctgc attggtctct gcaattgcta 6900agttatacca
attaccacag tcaacttcag aaatatctag aatagcaaga aaggggtctg 6960gttcagcttg
tagatcgttg tttggcggat acgtggcctg ggaaatggga aaagctgaag 7020atggtcatga
ttccatggca gtacaaatcg cagacagctc tgactggcct cagatgaaag 7080cttgtgtcct
agttgtcagc gatattaaaa aggatgtgag ttccactcag ggtatgcaat 7140tgaccgtggc
aacctccgaa ctatttaaag aaagaattga acatgtcgta ccaaagagat 7200ttgaagtcat
gcgtaaagcc attgttgaaa aagatttcgc cacctttgca aaggaaacaa 7260tgatggattc
caactctttc catgccacat gtttggactc tttccctcca atattctaca 7320tgaatgacac
ttccaagcgt atcatcagtt ggtgccacac cattaatcag ttttacggag 7380aaacaatcgt
tgcatacacg tttgatgcag gtccaaatgc tgtgttgtac tacttagctg 7440aaaatgagtc
gaaactcttt gcatttatct ataaattgtt tggctctgtt cctggatggg 7500acaagaaatt
tactactgag cagcttgagg ctttcaacca tcaatttgaa tcatctaact 7560ttactgcacg
tgaattggat cttgagttgc aaaaggatgt tgccagagtg attttaactc 7620aagtcggttc
aggcccacaa gaaacaaacg aatctttgat tgacgcaaag actggtctac 7680caaaggaata
act
7693597695DNAArtificial SequenceOperon B containing A. thaliana and S.
cerevisiae DNA 59ggccgcagga ggagttcata tgtcagagtt gagagccttc
agtgccccag ggaaagcgtt 60actagctggt ggatatttag ttttagatac aaaatatgaa
gcatttgtag tcggattatc 120ggcaagaatg catgctgtag cccatcctta cggttcattg
caagggtctg ataagtttga 180agtgcgtgtg aaaagtaaac aatttaaaga tggggagtgg
ctgtaccata taagtcctaa 240aagtggcttc attcctgttt cgataggcgg atctaagaac
cctttcattg aaaaagttat 300cgctaacgta tttagctact ttaaacctaa catggacgac
tactgcaata gaaacttgtt 360cgttattgat attttctctg atgatgccta ccattctcag
gaggatagcg ttaccgaaca 420tcgtggcaac agaagattga gttttcattc gcacagaatt
gaagaagttc ccaaaacagg 480gctgggctcc tcggcaggtt tagtcacagt tttaactaca
gctttggcct ccttttttgt 540atcggacctg gaaaataatg tagacaaata tagagaagtt
attcataatt tagcacaagt 600tgctcattgt caagctcagg gtaaaattgg aagcgggttt
gatgtagcgg cggcagcata 660tggatctatc agatatagaa gattcccacc cgcattaatc
tctaatttgc cagatattgg 720aagtgctact tacggcagta aactggcgca tttggttgat
gaagaagact ggaatattac 780gattaaaagt aaccatttac cttcgggatt aactttatgg
atgggcgata ttaagaatgg 840ttcagaaaca gtaaaactgg tccagaaggt aaaaaattgg
tatgattcgc atatgccaga 900aagcttgaaa atatatacag aactcgatca tgcaaattct
agatttatgg atggactatc 960taaactagat cgcttacacg agactcatga cgattacagc
gatcagatat ttgagtctct 1020tgagaggaat gactgtacct gtcaaaagta tcctgaaatc
acagaagtta gagatgcagt 1080tgccacaatt agacgttcct ttagaaaaat aactaaagaa
tctggtgccg atatcgaacc 1140tcccgtacaa actagcttat tggatgattg ccagacctta
aaaggagttc ttacttgctt 1200aatacctggt gctggtggtt atgacgccat tgcagtgatt
actaagcaag atgttgatct 1260tagggctcaa accgctaatg acaaaagatt ttctaaggtt
caatggctgg atgtaactca 1320ggctgactgg ggtgttagga aagaaaaaga tccggaaact
tatcttgata aactgcagga 1380ggagttttaa tgtcattacc gttcttaact tctgcaccgg
gaaaggttat tatttttggt 1440gaacactctg ctgtgtacaa caagcctgcc gtcgctgcta
gtgtgtctgc gttgagaacc 1500tacctgctaa taagcgagtc atctgcacca gatactattg
aattggactt cccggacatt 1560agctttaatc ataagtggtc catcaatgat ttcaatgcca
tcaccgagga tcaagtaaac 1620tcccaaaaat tggccaaggc tcaacaagcc accgatggct
tgtctcagga actcgttagt 1680cttttggatc cgttgttagc tcaactatcc gaatccttcc
actaccatgc agcgttttgt 1740ttcctgtata tgtttgtttg cctatgcccc catgccaaga
atattaagtt ttctttaaag 1800tctactttac ccatcggtgc tgggttgggc tcaagcgcct
ctatttctgt atcactggcc 1860ttagctatgg cctacttggg ggggttaata ggatctaatg
acttggaaaa gctgtcagaa 1920aacgataagc atatagtgaa tcaatgggcc ttcataggtg
aaaagtgtat tcacggtacc 1980ccttcaggaa tagataacgc tgtggccact tatggtaatg
ccctgctatt tgaaaaagac 2040tcacataatg gaacaataaa cacaaacaat tttaagttct
tagatgattt cccagccatt 2100ccaatgatcc taacctatac tagaattcca aggtctacaa
aagatcttgt tgctcgcgtt 2160cgtgtgttgg tcaccgagaa atttcctgaa gttatgaagc
caattctaga tgccatgggt 2220gaatgtgccc tacaaggctt agagatcatg actaagttaa
gtaaatgtaa aggcaccgat 2280gacgaggctg tagaaactaa taatgaactg tatgaacaac
tattggaatt gataagaata 2340aatcatggac tgcttgtctc aatcggtgtt tctcatcctg
gattagaact tattaaaaat 2400ctgagcgatg atttgagaat tggctccaca aaacttaccg
gtgctggtgg cggcggttgc 2460tctttgactt tgttacgaag agacattact caagagcaaa
ttgacagctt caaaaagaaa 2520ttgcaagatg attttagtta cgagacattt gaaacagact
tgggtgggac tggctgctgt 2580ttgttaagcg caaaaaattt gaataaagat cttaaaatca
aatccctagt attccaatta 2640tttgaaaata aaactaccac aaagcaacaa attgacgatc
tattattgcc aggaaacacg 2700aatttaccat ggacttcaga cgaggagttt taatgactgt
atatactgct agtgtaactg 2760ctccggtaaa tattgctact cttaagtatt gggggaaaag
ggacacgaag ttgaatctgc 2820ccaccaattc gtccatatca gtgactttat cgcaagatga
cctcagaacg ttgacctctg 2880cggctactgc acctgagttt gaacgcgaca ctttgtggtt
aaatggagaa ccacacagca 2940tcgacaatga aagaactcaa aattgtctgc gcgacctacg
ccaattaaga aaggaaatgg 3000aatcgaagga cgcctcattg cccacattat ctcaatggaa
actccacatt gtctccgaaa 3060ataactttcc tacagcagct ggtttagctt cctccgctgc
tggctttgct gcattggtct 3120ctgcaattgc taagttatac caattaccac agtcaacttc
agaaatatct agaatagcaa 3180gaaaggggtc tggttcagct tgtagatcgt tgtttggcgg
atacgtggcc tgggaaatgg 3240gaaaagctga agatggtcat gattccatgg cagtacaaat
cgcagacagc tctgactggc 3300ctcagatgaa agcttgtgtc ctagttgtca gcgatattaa
aaaggatgtg agttccactc 3360agggtatgca attgaccgtg gcaacctccg aactatttaa
agaaagaatt gaacatgtcg 3420taccaaagag atttgaagtc atgcgtaaag ccattgttga
aaaagatttc gccacctttg 3480caaaggaaac aatgatggat tccaactctt tccatgccac
atgtttggac tctttccctc 3540caatattcta catgaatgac acttccaagc gtatcatcag
ttggtgccac accattaatc 3600agttttacgg agaaacaatc gttgcataca cgtttgatgc
aggtccaaat gctgtgttgt 3660actacttagc tgaaaatgag tcgaaactct ttgcatttat
ctataaattg tttggctctg 3720ttcctggatg ggacaagaaa tttactactg agcagcttga
ggctttcaac catcaatttg 3780aatcatctaa ctttactgca cgtgaattgg atcttgagtt
gcaaaaggat gttgccagag 3840tgattttaac tcaagtcggt tcaggcccac aagaaacaaa
cgaatctttg attgacgcaa 3900agactggtct accaaaggaa gaggagtttt aactcgacgc
cggcggaggc acatatgtct 3960cagaacgttt acattgtatc gactgccaga accccaattg
gttcattcca gggttctcta 4020tcctccaaga cagcagtgga attgggtgct gttgctttaa
aaggcgcctt ggctaaggtt 4080ccagaattgg atgcatccaa ggattttgac gaaattattt
ttggtaacgt tctttctgcc 4140aatttgggcc aagctccggc cagacaagtt gctttggctg
ccggtttgag taatcatatc 4200gttgcaagca cagttaacaa ggtctgtgca tccgctatga
aggcaatcat tttgggtgct 4260caatccatca aatgtggtaa tgctgatgtt gtcgtagctg
gtggttgtga atctatgact 4320aacgcaccat actacatgcc agcagcccgt gcgggtgcca
aatttggcca aactgttctt 4380gttgatggtg tcgaaagaga tgggttgaac gatgcgtacg
atggtctagc catgggtgta 4440cacgcagaaa agtgtgcccg tgattgggat attactagag
aacaacaaga caattttgcc 4500atcgaatcct accaaaaatc tcaaaaatct caaaaggaag
gtaaattcga caatgaaatt 4560gtacctgtta ccattaaggg atttagaggt aagcctgata
ctcaagtcac gaaggacgag 4620gaacctgcta gattacacgt tgaaaaattg agatctgcaa
ggactgtttt ccaaaaagaa 4680aacggtactg ttactgccgc taacgcttct ccaatcaacg
atggtgctgc agccgtcatc 4740ttggtttccg aaaaagtttt gaaggaaaag aatttgaagc
ctttggctat tatcaaaggt 4800tggggtgagg ccgctcatca accagctgat tttacatggg
ctccatctct tgcagttcca 4860aaggctttga aacatgctgg catcgaagac atcaattctg
ttgattactt tgaattcaat 4920gaagcctttt cggttgtcgg tttggtgaac actaagattt
tgaagctaga cccatctaag 4980gttaatgtat atggtggtgc tgttgctcta ggtcacccat
tgggttgttc tggtgctaga 5040gtggttgtta cactgctatc catcttacag caagaaggag
gtaagatcgg tgttgccgcc 5100atttgtaatg gtggtggtgg tgcttcctct attgtcattg
aaaagatatg aggatcctct 5160agatgcgcag gaggcacata tggcgaagaa cgttgggatt
ttggctatgg atatctattt 5220ccctcccacc tgtgttcaac aggaagcttt ggaagcacat
gatggagcaa gtaaagggaa 5280atacactatt ggacttggcc aagattgttt agctttttgc
actgagcttg aagatgttat 5340ctctatgagt ttcaatgcgg tgacatcact ttttgagaag
tataagattg accctaacca 5400aatcgggcgt cttgaagtag gaagtgagac tgttattgac
aaaagcaagt ccatcaagac 5460cttcttgatg cagctctttg agaaatgtgg aaacactgat
gtcgaaggtg ttgactcgac 5520caatgcttgc tatggtggaa ctgcagcttt gttaaactgt
gtcaattggg ttgagagtaa 5580ctcttgggat ggacgttatg gcctcgtcat ttgtactgac
agcgcggttt atgcagaagg 5640acccgcaagg cccactggag gagctgcagc gattgctatg
ttgataggac ctgatgctcc 5700tatcgttttc gaaagcaaat tgagagcaag ccacatggct
catgtctatg acttttacaa 5760gcccaatctt gctagcgagt acccggttgt tgatggtaag
ctttcacaga cttgctacct 5820catggctctt gactcctgct ataaacattt atgcaacaag
ttcgagaaga tcgagggcaa 5880agagttctcc ataaatgatg ctgattacat tgttttccat
tctccataca ataaacttgt 5940acagaaaagc tttgctcgtc tcttgtacaa cgacttcttg
agaaacgcaa gctccattga 6000cgaggctgcc aaagaaaagt tcacccctta ttcatctttg
acccttgacg agagttacca 6060aagccgtgat cttgaaaagg tgtcacaaca aatttcgaaa
ccgttttatg atgctaaagt 6120gcaaccaacg actttaatac caaaggaagt cggtaacatg
tacactgctt ctctctacgc 6180tgcatttgct tccctcatcc acaataaaca caatgatttg
gcgggaaagc gggtggttat 6240gttctcttat ggaagtggct ccaccgcaac aatgttctca
ttacgcctca acgacaataa 6300gcctcctttc agcatttcaa acattgcatc tgtaatggat
gttggcggta aattgaaagc 6360tagacatgag tatgcacctg agaagtttgt ggagacaatg
aagctaatgg aacataggta 6420tggagcaaag gactttgtga caaccaagga gggtattata
gatcttttgg caccgggaac 6480ttattatctg aaagaggttg attccttgta ccggagattc
tatggcaaga aaggtgaaga 6540tggatctgta gccaatggac actgaggatc cgtcgagcac
gtggaggcac atatgcaatg 6600ctgtgagatg cctgttggat acattcagat tcctgttggg
attgctggtc cattgttgct 6660tgatggttat gagtactctg ttcctatggc tacaaccgaa
ggttgtttgg ttgctagcac 6720taacagaggc tgcaaggcta tgtttatctc tggtggcgcc
accagtaccg ttcttaagga 6780cggtatgacc cgagcacctg ttgttcggtt cgcttcggcg
agacgagctt cggagcttaa 6840gtttttcttg gagaatccag agaactttga tactttggca
gtagtcttca acaggtcgag 6900tagatttgca agactgcaaa gtgttaaatg cacaatcgcg
gggaagaatg cttatgtaag 6960gttctgttgt agtactggtg atgctatggg gatgaatatg
gtttctaaag gtgtgcagaa 7020tgttcttgag tatcttaccg atgatttccc tgacatggat
gtgattggaa tctctggtaa 7080cttctgttcg gacaagaaac ctgctgctgt gaactggatt
gagggacgtg gtaaatcagt 7140tgtttgcgag gctgtaatca gaggagagat cgtgaacaag
gtcttgaaaa cgagcgtggc 7200tgctttagtc gagctcaaca tgctcaagaa cctagctggc
tctgctgttg caggctctct 7260aggtggattc aacgctcatg ccagtaacat agtgtctgct
gtattcatag ctactggcca 7320agatccagct caaaacgtgg agagttctca atgcatcacc
atgatggaag ctattaatga 7380cggcaaagat atccatatct cagtcactat gccatctatc
gaggtgggga cagtgggagg 7440aggaacacag cttgcatctc aatcagcgtg tttaaacctg
ctcggagtta aaggagcaag 7500cacagagtcg ccgggaatga acgcaaggag gctagcgacg
atcgtagccg gagcagtttt 7560agctggagag ttatctttaa tgtcagcaat tgcagctgga
cagcttgtga gaagtcacat 7620gaaatacaat agatccagcc gagacatctc tggagcaacg
acaacgacaa caacaacaac 7680atgacccggg atccg
7695608235DNAArtificial SequenceOperon C containing
A. thaliana, S. cerevisiae, and R. caosulatus DNA 60ggccgcagga
ggagttcata tgtcagagtt gagagccttc agtgccccag ggaaagcgtt 60actagctggt
ggatatttag ttttagatac aaaatatgaa gcatttgtag tcggattatc 120ggcaagaatg
catgctgtag cccatcctta cggttcattg caagggtctg ataagtttga 180agtgcgtgtg
aaaagtaaac aatttaaaga tggggagtgg ctgtaccata taagtcctaa 240aagtggcttc
attcctgttt cgataggcgg atctaagaac cctttcattg aaaaagttat 300cgctaacgta
tttagctact ttaaacctaa catggacgac tactgcaata gaaacttgtt 360cgttattgat
attttctctg atgatgccta ccattctcag gaggatagcg ttaccgaaca 420tcgtggcaac
agaagattga gttttcattc gcacagaatt gaagaagttc ccaaaacagg 480gctgggctcc
tcggcaggtt tagtcacagt tttaactaca gctttggcct ccttttttgt 540atcggacctg
gaaaataatg tagacaaata tagagaagtt attcataatt tagcacaagt 600tgctcattgt
caagctcagg gtaaaattgg aagcgggttt gatgtagcgg cggcagcata 660tggatctatc
agatatagaa gattcccacc cgcattaatc tctaatttgc cagatattgg 720aagtgctact
tacggcagta aactggcgca tttggttgat gaagaagact ggaatattac 780gattaaaagt
aaccatttac cttcgggatt aactttatgg atgggcgata ttaagaatgg 840ttcagaaaca
gtaaaactgg tccagaaggt aaaaaattgg tatgattcgc atatgccaga 900aagcttgaaa
atatatacag aactcgatca tgcaaattct agatttatgg atggactatc 960taaactagat
cgcttacacg agactcatga cgattacagc gatcagatat ttgagtctct 1020tgagaggaat
gactgtacct gtcaaaagta tcctgaaatc acagaagtta gagatgcagt 1080tgccacaatt
agacgttcct ttagaaaaat aactaaagaa tctggtgccg atatcgaacc 1140tcccgtacaa
actagcttat tggatgattg ccagacctta aaaggagttc ttacttgctt 1200aatacctggt
gctggtggtt atgacgccat tgcagtgatt actaagcaag atgttgatct 1260tagggctcaa
accgctaatg acaaaagatt ttctaaggtt caatggctgg atgtaactca 1320ggctgactgg
ggtgttagga aagaaaaaga tccggaaact tatcttgata aactgcagga 1380ggagttttaa
tgtcattacc gttcttaact tctgcaccgg gaaaggttat tatttttggt 1440gaacactctg
ctgtgtacaa caagcctgcc gtcgctgcta gtgtgtctgc gttgagaacc 1500tacctgctaa
taagcgagtc atctgcacca gatactattg aattggactt cccggacatt 1560agctttaatc
ataagtggtc catcaatgat ttcaatgcca tcaccgagga tcaagtaaac 1620tcccaaaaat
tggccaaggc tcaacaagcc accgatggct tgtctcagga actcgttagt 1680cttttggatc
cgttgttagc tcaactatcc gaatccttcc actaccatgc agcgttttgt 1740ttcctgtata
tgtttgtttg cctatgcccc catgccaaga atattaagtt ttctttaaag 1800tctactttac
ccatcggtgc tgggttgggc tcaagcgcct ctatttctgt atcactggcc 1860ttagctatgg
cctacttggg ggggttaata ggatctaatg acttggaaaa gctgtcagaa 1920aacgataagc
atatagtgaa tcaatgggcc ttcataggtg aaaagtgtat tcacggtacc 1980ccttcaggaa
tagataacgc tgtggccact tatggtaatg ccctgctatt tgaaaaagac 2040tcacataatg
gaacaataaa cacaaacaat tttaagttct tagatgattt cccagccatt 2100ccaatgatcc
taacctatac tagaattcca aggtctacaa aagatcttgt tgctcgcgtt 2160cgtgtgttgg
tcaccgagaa atttcctgaa gttatgaagc caattctaga tgccatgggt 2220gaatgtgccc
tacaaggctt agagatcatg actaagttaa gtaaatgtaa aggcaccgat 2280gacgaggctg
tagaaactaa taatgaactg tatgaacaac tattggaatt gataagaata 2340aatcatggac
tgcttgtctc aatcggtgtt tctcatcctg gattagaact tattaaaaat 2400ctgagcgatg
atttgagaat tggctccaca aaacttaccg gtgctggtgg cggcggttgc 2460tctttgactt
tgttacgaag agacattact caagagcaaa ttgacagctt caaaaagaaa 2520ttgcaagatg
attttagtta cgagacattt gaaacagact tgggtgggac tggctgctgt 2580ttgttaagcg
caaaaaattt gaataaagat cttaaaatca aatccctagt attccaatta 2640tttgaaaata
aaactaccac aaagcaacaa attgacgatc tattattgcc aggaaacacg 2700aatttaccat
ggacttcaga cgaggagttt taatgactgt atatactgct agtgtaactg 2760ctccggtaaa
tattgctact cttaagtatt gggggaaaag ggacacgaag ttgaatctgc 2820ccaccaattc
gtccatatca gtgactttat cgcaagatga cctcagaacg ttgacctctg 2880cggctactgc
acctgagttt gaacgcgaca ctttgtggtt aaatggagaa ccacacagca 2940tcgacaatga
aagaactcaa aattgtctgc gcgacctacg ccaattaaga aaggaaatgg 3000aatcgaagga
cgcctcattg cccacattat ctcaatggaa actccacatt gtctccgaaa 3060ataactttcc
tacagcagct ggtttagctt cctccgctgc tggctttgct gcattggtct 3120ctgcaattgc
taagttatac caattaccac agtcaacttc agaaatatct agaatagcaa 3180gaaaggggtc
tggttcagct tgtagatcgt tgtttggcgg atacgtggcc tgggaaatgg 3240gaaaagctga
agatggtcat gattccatgg cagtacaaat cgcagacagc tctgactggc 3300ctcagatgaa
agcttgtgtc ctagttgtca gcgatattaa aaaggatgtg agttccactc 3360agggtatgca
attgaccgtg gcaacctccg aactatttaa agaaagaatt gaacatgtcg 3420taccaaagag
atttgaagtc atgcgtaaag ccattgttga aaaagatttc gccacctttg 3480caaaggaaac
aatgatggat tccaactctt tccatgccac atgtttggac tctttccctc 3540caatattcta
catgaatgac acttccaagc gtatcatcag ttggtgccac accattaatc 3600agttttacgg
agaaacaatc gttgcataca cgtttgatgc aggtccaaat gctgtgttgt 3660actacttagc
tgaaaatgag tcgaaactct ttgcatttat ctataaattg tttggctctg 3720ttcctggatg
ggacaagaaa tttactactg agcagcttga ggctttcaac catcaatttg 3780aatcatctaa
ctttactgca cgtgaattgg atcttgagtt gcaaaaggat gttgccagag 3840tgattttaac
tcaagtcggt tcaggcccac aagaaacaaa cgaatctttg attgacgcaa 3900agactggtct
accaaaggaa gaggagtttt aactcgacgc cggcggaggc acatatgtct 3960cagaacgttt
acattgtatc gactgccaga accccaattg gttcattcca gggttctcta 4020tcctccaaga
cagcagtgga attgggtgct gttgctttaa aaggcgcctt ggctaaggtt 4080ccagaattgg
atgcatccaa ggattttgac gaaattattt ttggtaacgt tctttctgcc 4140aatttgggcc
aagctccggc cagacaagtt gctttggctg ccggtttgag taatcatatc 4200gttgcaagca
cagttaacaa ggtctgtgca tccgctatga aggcaatcat tttgggtgct 4260caatccatca
aatgtggtaa tgctgatgtt gtcgtagctg gtggttgtga atctatgact 4320aacgcaccat
actacatgcc agcagcccgt gcgggtgcca aatttggcca aactgttctt 4380gttgatggtg
tcgaaagaga tgggttgaac gatgcgtacg atggtctagc catgggtgta 4440cacgcagaaa
agtgtgcccg tgattgggat attactagag aacaacaaga caattttgcc 4500atcgaatcct
accaaaaatc tcaaaaatct caaaaggaag gtaaattcga caatgaaatt 4560gtacctgtta
ccattaaggg atttagaggt aagcctgata ctcaagtcac gaaggacgag 4620gaacctgcta
gattacacgt tgaaaaattg agatctgcaa ggactgtttt ccaaaaagaa 4680aacggtactg
ttactgccgc taacgcttct ccaatcaacg atggtgctgc agccgtcatc 4740ttggtttccg
aaaaagtttt gaaggaaaag aatttgaagc ctttggctat tatcaaaggt 4800tggggtgagg
ccgctcatca accagctgat tttacatggg ctccatctct tgcagttcca 4860aaggctttga
aacatgctgg catcgaagac atcaattctg ttgattactt tgaattcaat 4920gaagcctttt
cggttgtcgg tttggtgaac actaagattt tgaagctaga cccatctaag 4980gttaatgtat
atggtggtgc tgttgctcta ggtcacccat tgggttgttc tggtgctaga 5040gtggttgtta
cactgctatc catcttacag caagaaggag gtaagatcgg tgttgccgcc 5100atttgtaatg
gtggtggtgg tgcttcctct attgtcattg aaaagatatg aggatcctct 5160agatgcgcag
gaggcacata tggcgaagaa cgttgggatt ttggctatgg atatctattt 5220ccctcccacc
tgtgttcaac aggaagcttt ggaagcacat gatggagcaa gtaaagggaa 5280atacactatt
ggacttggcc aagattgttt agctttttgc actgagcttg aagatgttat 5340ctctatgagt
ttcaatgcgg tgacatcact ttttgagaag tataagattg accctaacca 5400aatcgggcgt
cttgaagtag gaagtgagac tgttattgac aaaagcaagt ccatcaagac 5460cttcttgatg
cagctctttg agaaatgtgg aaacactgat gtcgaaggtg ttgactcgac 5520caatgcttgc
tatggtggaa ctgcagcttt gttaaactgt gtcaattggg ttgagagtaa 5580ctcttgggat
ggacgttatg gcctcgtcat ttgtactgac agcgcggttt atgcagaagg 5640acccgcaagg
cccactggag gagctgcagc gattgctatg ttgataggac ctgatgctcc 5700tatcgttttc
gaaagcaaat tgagagcaag ccacatggct catgtctatg acttttacaa 5760gcccaatctt
gctagcgagt acccggttgt tgatggtaag ctttcacaga cttgctacct 5820catggctctt
gactcctgct ataaacattt atgcaacaag ttcgagaaga tcgagggcaa 5880agagttctcc
ataaatgatg ctgattacat tgttttccat tctccataca ataaacttgt 5940acagaaaagc
tttgctcgtc tcttgtacaa cgacttcttg agaaacgcaa gctccattga 6000cgaggctgcc
aaagaaaagt tcacccctta ttcatctttg acccttgacg agagttacca 6060aagccgtgat
cttgaaaagg tgtcacaaca aatttcgaaa ccgttttatg atgctaaagt 6120gcaaccaacg
actttaatac caaaggaagt cggtaacatg tacactgctt ctctctacgc 6180tgcatttgct
tccctcatcc acaataaaca caatgatttg gcgggaaagc gggtggttat 6240gttctcttat
ggaagtggct ccaccgcaac aatgttctca ttacgcctca acgacaataa 6300gcctcctttc
agcatttcaa acattgcatc tgtaatggat gttggcggta aattgaaagc 6360tagacatgag
tatgcacctg agaagtttgt ggagacaatg aagctaatgg aacataggta 6420tggagcaaag
gactttgtga caaccaagga gggtattata gatcttttgg caccgggaac 6480ttattatctg
aaagaggttg attccttgta ccggagattc tatggcaaga aaggtgaaga 6540tggatctgta
gccaatggac actgaggatc cgtcgagcac gtggaggcac atatgcaatg 6600ctgtgagatg
cctgttggat acattcagat tcctgttggg attgctggtc cattgttgct 6660tgatggttat
gagtactctg ttcctatggc tacaaccgaa ggttgtttgg ttgctagcac 6720taacagaggc
tgcaaggcta tgtttatctc tggtggcgcc accagtaccg ttcttaagga 6780cggtatgacc
cgagcacctg ttgttcggtt cgcttcggcg agacgagctt cggagcttaa 6840gtttttcttg
gagaatccag agaactttga tactttggca gtagtcttca acaggtcgag 6900tagatttgca
agactgcaaa gtgttaaatg cacaatcgcg gggaagaatg cttatgtaag 6960gttctgttgt
agtactggtg atgctatggg gatgaatatg gtttctaaag gtgtgcagaa 7020tgttcttgag
tatcttaccg atgatttccc tgacatggat gtgattggaa tctctggtaa 7080cttctgttcg
gacaagaaac ctgctgctgt gaactggatt gagggacgtg gtaaatcagt 7140tgtttgcgag
gctgtaatca gaggagagat cgtgaacaag gtcttgaaaa cgagcgtggc 7200tgctttagtc
gagctcaaca tgctcaagaa cctagctggc tctgctgttg caggctctct 7260aggtggattc
aacgctcatg ccagtaacat agtgtctgct gtattcatag ctactggcca 7320agatccagct
caaaacgtgg agagttctca atgcatcacc atgatggaag ctattaatga 7380cggcaaagat
atccatatct cagtcactat gccatctatc gaggtgggga cagtgggagg 7440aggaacacag
cttgcatctc aatcagcgtg tttaaacctg ctcggagtta aaggagcaag 7500cacagagtcg
ccgggaatga acgcaaggag gctagcgacg atcgtagccg gagcagtttt 7560agctggagag
ttatctttaa tgtcagcaat tgcagctgga cagcttgtga gaagtcacat 7620gaaatacaat
agatccagcc gagacatctc tggagcaacg acaacgacaa caacaacaac 7680atgacccgta
aggaggcaca tatgagtgag cttatacccg cctgggttgg tgacagactg 7740gctccggtgg
acaagttgga ggtgcatttg aaagggctcc gccacaaggc ggtgtctgtt 7800ttcgtcatgg
atggcgaaaa cgtgctgatc cagcgccgct cggaggagaa atatcactct 7860cccgggcttt
gggcgaacac ctgctgcacc catccgggct ggaccgaacg ccccgaggaa 7920tgcgcggtgc
ggcggctgcg cgaggagctg gggatcaccg ggctttatcc cgcccatgcc 7980gaccggctgg
aatatcgcgc cgatgtcggc ggcggcatga tcgagcatga ggtggtcgac 8040atctatctgg
cctatgccaa accgcatatg cggatcaccc ccgatccgcg cgaagtggcc 8100gaggtgcgct
ggatcggcct ttacgatctg gcggccgagg ccggtcggca tcccgagcgg 8160ttctcgaaat
ggctcaacat ctatctgtcg agccatcttg accggatttt cggatcgatc 8220ctgcgcggct
gagcg
8235617681DNAArtificial SequenceOperon C containing A. thaliana, S.
cerevisiae, and Streptomyces sp CL190DNA, and R. capsulatus DNA
61ggccgcgtcg actacggccg caggaggagt tcatatgtca gagttgagag ccttcagtgc
60cccagggaaa gcgttactag ctggtggata tttagtttta gatacaaaat atgaagcatt
120tgtagtcgga ttatcggcaa gaatgcatgc tgtagcccat ccttacggtt cattgcaagg
180gtctgataag tttgaagtgc gtgtgaaaag taaacaattt aaagatgggg agtggctgta
240ccatataagt cctaaaagtg gcttcattcc tgtttcgata ggcggatcta agaacccttt
300cattgaaaaa gttatcgcta acgtatttag ctactttaaa cctaacatgg acgactactg
360caatagaaac ttgttcgtta ttgatatttt ctctgatgat gcctaccatt ctcaggagga
420tagcgttacc gaacatcgtg gcaacagaag attgagtttt cattcgcaca gaattgaaga
480agttcccaaa acagggctgg gctcctcggc aggtttagtc acagttttaa ctacagcttt
540ggcctccttt tttgtatcgg acctggaaaa taatgtagac aaatatagag aagttattca
600taatttagca caagttgctc attgtcaagc tcagggtaaa attggaagcg ggtttgatgt
660agcggcggca gcatatggat ctatcagata tagaagattc ccacccgcat taatctctaa
720tttgccagat attggaagtg ctacttacgg cagtaaactg gcgcatttgg ttgatgaaga
780agactggaat attacgatta aaagtaacca tttaccttcg ggattaactt tatggatggg
840cgatattaag aatggttcag aaacagtaaa actggtccag aaggtaaaaa attggtatga
900ttcgcatatg ccagaaagct tgaaaatata tacagaactc gatcatgcaa attctagatt
960tatggatgga ctatctaaac tagatcgctt acacgagact catgacgatt acagcgatca
1020gatatttgag tctcttgaga ggaatgactg tacctgtcaa aagtatcctg aaatcacaga
1080agttagagat gcagttgcca caattagacg ttcctttaga aaaataacta aagaatctgg
1140tgccgatatc gaacctcccg tacaaactag cttattggat gattgccaga ccttaaaagg
1200agttcttact tgcttaatac ctggtgctgg tggttatgac gccattgcag tgattactaa
1260gcaagatgtt gatcttaggg ctcaaaccgc taatgacaaa agattttcta aggttcaatg
1320gctggatgta actcaggctg actggggtgt taggaaagaa aaagatccgg aaacttatct
1380tgataaactg caggaggagt tttaatgtca ttaccgttct taacttctgc accgggaaag
1440gttattattt ttggtgaaca ctctgctgtg tacaacaagc ctgccgtcgc tgctagtgtg
1500tctgcgttga gaacctacct gctaataagc gagtcatctg caccagatac tattgaattg
1560gacttcccgg acattagctt taatcataag tggtccatca atgatttcaa tgccatcacc
1620gaggatcaag taaactccca aaaattggcc aaggctcaac aagccaccga tggcttgtct
1680caggaactcg ttagtctttt ggatccgttg ttagctcaac tatccgaatc cttccactac
1740catgcagcgt tttgtttcct gtatatgttt gtttgcctat gcccccatgc caagaatatt
1800aagttttctt taaagtctac tttacccatc ggtgctgggt tgggctcaag cgcctctatt
1860tctgtatcac tggccttagc tatggcctac ttgggggggt taataggatc taatgacttg
1920gaaaagctgt cagaaaacga taagcatata gtgaatcaat gggccttcat aggtgaaaag
1980tgtattcacg gtaccccttc aggaatagat aacgctgtgg ccacttatgg taatgccctg
2040ctatttgaaa aagactcaca taatggaaca ataaacacaa acaattttaa gttcttagat
2100gatttcccag ccattccaat gatcctaacc tatactagaa ttccaaggtc tacaaaagat
2160cttgttgctc gcgttcgtgt gttggtcacc gagaaatttc ctgaagttat gaagccaatt
2220ctagatgcca tgggtgaatg tgccctacaa ggcttagaga tcatgactaa gttaagtaaa
2280tgtaaaggca ccgatgacga ggctgtagaa actaataatg aactgtatga acaactattg
2340gaattgataa gaataaatca tggactgctt gtctcaatcg gtgtttctca tcctggatta
2400gaacttatta aaaatctgag cgatgatttg agaattggct ccacaaaact taccggtgct
2460ggtggcggcg gttgctcttt gactttgtta cgaagagaca ttactcaaga gcaaattgac
2520agcttcaaaa agaaattgca agatgatttt agttacgaga catttgaaac agacttgggt
2580gggactggct gctgtttgtt aagcgcaaaa aatttgaata aagatcttaa aatcaaatcc
2640ctagtattcc aattatttga aaataaaact accacaaagc aacaaattga cgatctatta
2700ttgccaggaa acacgaattt accatggact tcagacgagg agttttaatg actgtatata
2760ctgctagtgt aactgctccg gtaaatattg ctactcttaa gtattggggg aaaagggaca
2820cgaagttgaa tctgcccacc aattcgtcca tatcagtgac tttatcgcaa gatgacctca
2880gaacgttgac ctctgcggct actgcacctg agtttgaacg cgacactttg tggttaaatg
2940gagaaccaca cagcatcgac aatgaaagaa ctcaaaattg tctgcgcgac ctacgccaat
3000taagaaagga aatggaatcg aaggacgcct cattgcccac attatctcaa tggaaactcc
3060acattgtctc cgaaaataac tttcctacag cagctggttt agcttcctcc gctgctggct
3120ttgctgcatt ggtctctgca attgctaagt tataccaatt accacagtca acttcagaaa
3180tatctagaat agcaagaaag gggtctggtt cagcttgtag atcgttgttt ggcggatacg
3240tggcctggga aatgggaaaa gctgaagatg gtcatgattc catggcagta caaatcgcag
3300acagctctga ctggcctcag atgaaagctt gtgtcctagt tgtcagcgat attaaaaagg
3360atgtgagttc cactcagggt atgcaattga ccgtggcaac ctccgaacta tttaaagaaa
3420gaattgaaca tgtcgtacca aagagatttg aagtcatgcg taaagccatt gttgaaaaag
3480atttcgccac ctttgcaaag gaaacaatga tggattccaa ctctttccat gccacatgtt
3540tggactcttt ccctccaata ttctacatga atgacacttc caagcgtatc atcagttggt
3600gccacaccat taatcagttt tacggagaaa caatcgttgc atacacgttt gatgcaggtc
3660caaatgctgt gttgtactac ttagctgaaa atgagtcgaa actctttgca tttatctata
3720aattgtttgg ctctgttcct ggatgggaca agaaatttac tactgagcag cttgaggctt
3780tcaaccatca atttgaatca tctaacttta ctgcacgtga attggatctt gagttgcaaa
3840aggatgttgc cagagtgatt ttaactcaag tcggttcagg cccacaagaa acaaacgaat
3900ctttgattga cgcaaagact ggtctaccaa aggaagagga gttttaactc gagtaggagg
3960cacatatgtc tcagaacgtt tacattgtat cgactgccag aaccccaatt ggttcattcc
4020agggttctct atcctccaag acagcagtgg aattgggtgc tgttgcttta aaaggcgcct
4080tggctaaggt tccagaattg gatgcatcca aggattttga cgaaattatt tttggtaacg
4140ttctttctgc caatttgggc caagctccgg ccagacaagt tgctttggct gccggtttga
4200gtaatcatat cgttgcaagc acagttaaca aggtctgtgc atccgctatg aaggcaatca
4260ttttgggtgc tcaatccatc aaatgtggta atgctgatgt tgtcgtagct ggtggttgtg
4320aatctatgac taacgcacca tactacatgc cagcagcccg tgcgggtgcc aaatttggcc
4380aaactgttct tgttgatggt gtcgaaagag atgggttgaa cgatgcgtac gatggtctag
4440ccatgggtgt acacgcagaa aagtgtgccc gtgattggga tattactaga gaacaacaag
4500acaattttgc catcgaatcc taccaaaaat ctcaaaaatc tcaaaaggaa ggtaaattcg
4560acaatgaaat tgtacctgtt accattaagg gatttagagg taagcctgat actcaagtca
4620cgaaggacga ggaacctgct agattacacg ttgaaaaatt gagatctgca aggactgttt
4680tccaaaaaga aaacggtact gttactgccg ctaacgcttc tccaatcaac gatggtgctg
4740cagccgtcat cttggtttcc gaaaaagttt tgaaggaaaa gaatttgaag cctttggcta
4800ttatcaaagg ttggggtgag gccgctcatc aaccagctga ttttacatgg gctccatctc
4860ttgcagttcc aaaggctttg aaacatgctg gcatcgaaga catcaattct gttgattact
4920ttgaattcaa tgaagccttt tcggttgtcg gtttggtgaa cactaagatt ttgaagctag
4980acccatctaa ggttaatgta tatggtggtg ctgttgctct aggtcaccca ttgggttgtt
5040ctggtgctag agtggttgtt acactgctat ccatcttaca gcaagaagga ggtaagatcg
5100gtgttgccgc catttgtaat ggtggtggtg gtgcttcctc tattgtcatt gaaaagatat
5160gaggatcctc tagatgcgca ggaggcacat atggcgaaga acgttgggat tttggctatg
5220gatatctatt tccctcccac ctgtgttcaa caggaagctt tggaagcaca tgatggagca
5280agtaaaggga aatacactat tggacttggc caagattgtt tagctttttg cactgagctt
5340gaagatgtta tctctatgag tttcaatgcg gtgacatcac tttttgagaa gtataagatt
5400gaccctaacc aaatcgggcg tcttgaagta ggaagtgaga ctgttattga caaaagcaag
5460tccatcaaga ccttcttgat gcagctcttt gagaaatgtg gaaacactga tgtcgaaggt
5520gttgactcga ccaatgcttg ctatggtgga actgcagctt tgttaaactg tgtcaattgg
5580gttgagagta actcttggga tggacgttat ggcctcgtca tttgtactga cagcgcggtt
5640tatgcagaag gacccgcaag gcccactgga ggagctgcag cgattgctat gttgatagga
5700cctgatgctc ctatcgtttt cgaaagcaaa ttgagagcaa gccacatggc tcatgtctat
5760gacttttaca agcccaatct tgctagcgag tacccggttg ttgatggtaa gctttcacag
5820acttgctacc tcatggctct tgactcctgc tataaacatt tatgcaacaa gttcgagaag
5880atcgagggca aagagttctc cataaatgat gctgattaca ttgttttcca ttctccatac
5940aataaacttg tacagaaaag ctttgctcgt ctcttgtaca acgacttctt gagaaacgca
6000agctccattg acgaggctgc caaagaaaag ttcacccctt attcatcttt gacccttgac
6060gagagttacc aaagccgtga tcttgaaaag gtgtcacaac aaatttcgaa accgttttat
6120gatgctaaag tgcaaccaac gactttaata ccaaaggaag tcggtaacat gtacactgct
6180tctctctacg ctgcatttgc ttccctcatc cacaataaac acaatgattt ggcgggaaag
6240cgggtggtta tgttctctta tggaagtggc tccaccgcaa caatgttctc attacgcctc
6300aacgacaata agcctccttt cagcatttca aacattgcat ctgtaatgga tgttggcggt
6360aaattgaaag ctagacatga gtatgcacct gagaagtttg tggagacaat gaagctaatg
6420gaacataggt atggagcaaa ggactttgtg acaaccaagg agggtattat agatcttttg
6480gcaccgggaa cttattatct gaaagaggtt gattccttgt accggagatt ctatggcaag
6540aaaggtgaag atggatctgt agccaatgga cactgaggat ccgtcgactc gagcacgtga
6600ggaggcacat atgacggaaa cgcacgccat agccggggtc ccgatgaggt gggtgggacc
6660ccttcgtatt tccgggaacg tcgccgagac cgagacccag gtcccgctcg ccacgtacga
6720gtcgccgctg tggccgtcgg tgggccgcgg ggcgaaggtc tcccggctga cggagaaggg
6780catcgtcgcc accctcgtcg acgagcggat gacccgctcg gtgatcgtcg aggcgacgga
6840cgcgcagacc gcgtacatgg ccgcgcagac catccacgcc cgcatcgacg agctgcgcga
6900ggtggtgcgc ggctgcagcc ggttcgccca gctgatcaac atcaagcacg agatcaacgc
6960gaacctgctg ttcatccggt tcgagttcac caccggtgac gcctccggcc acaacatggc
7020cacgctcgcc tccgatgtgc tcctggggca cctgctggag acgatccctg gcatctccta
7080cggctcgatc tccggcaact actgcacgga caagaaggcc accgcgatca acggcatcct
7140cggccgcggc aagaacgtga tcaccgagct gctggtgccg cgggacgtcg tcgagaacaa
7200cctgcacacc acggctgcca agatcgtcga gctgaacatc cgcaagaacc tgctcggcac
7260cctgctcgcc ggcggcatcc gctcggccaa cgcccacttc gcgaacatgc tgctcggctt
7320ctacctggcc accggccagg acgccgccaa catcgtcgag ggctcgcagg gcgtcgtcat
7380ggccgaggac cgcgacggcg acctctactt cgcctgcacc ctgccgaacc tgatcgtcgg
7440cacggtcggc aacggcaagg gtctcggctt cgtggagacg aacctcgccc ggctcggctg
7500ccgagccgac cgcgaacccg gggagaacgc ccgccgcctc gccgtcatcg cggcagcgac
7560cgtgctgtgc ggtgaactct cgctgctcgc ggcacagacg aacccgggcg aactcatgcg
7620cgcgcacgtc cagctggaac gcgacaacaa gaccgcaaag gttggtgcat agacgcgtgc
7680g
7681628224DNAArtificial SequenceOperon E containing A. thaliana, S.
cerevesiae, Steptomyces sp CL190 DNA, and R. capsulatus 62ggccgcgtcg
actacggccg caggaggagt tcatatgtca gagttgagag ccttcagtgc 60cccagggaaa
gcgttactag ctggtggata tttagtttta gatacaaaat atgaagcatt 120tgtagtcgga
ttatcggcaa gaatgcatgc tgtagcccat ccttacggtt cattgcaagg 180gtctgataag
tttgaagtgc gtgtgaaaag taaacaattt aaagatgggg agtggctgta 240ccatataagt
cctaaaagtg gcttcattcc tgtttcgata ggcggatcta agaacccttt 300cattgaaaaa
gttatcgcta acgtatttag ctactttaaa cctaacatgg acgactactg 360caatagaaac
ttgttcgtta ttgatatttt ctctgatgat gcctaccatt ctcaggagga 420tagcgttacc
gaacatcgtg gcaacagaag attgagtttt cattcgcaca gaattgaaga 480agttcccaaa
acagggctgg gctcctcggc aggtttagtc acagttttaa ctacagcttt 540ggcctccttt
tttgtatcgg acctggaaaa taatgtagac aaatatagag aagttattca 600taatttagca
caagttgctc attgtcaagc tcagggtaaa attggaagcg ggtttgatgt 660agcggcggca
gcatatggat ctatcagata tagaagattc ccacccgcat taatctctaa 720tttgccagat
attggaagtg ctacttacgg cagtaaactg gcgcatttgg ttgatgaaga 780agactggaat
attacgatta aaagtaacca tttaccttcg ggattaactt tatggatggg 840cgatattaag
aatggttcag aaacagtaaa actggtccag aaggtaaaaa attggtatga 900ttcgcatatg
ccagaaagct tgaaaatata tacagaactc gatcatgcaa attctagatt 960tatggatgga
ctatctaaac tagatcgctt acacgagact catgacgatt acagcgatca 1020gatatttgag
tctcttgaga ggaatgactg tacctgtcaa aagtatcctg aaatcacaga 1080agttagagat
gcagttgcca caattagacg ttcctttaga aaaataacta aagaatctgg 1140tgccgatatc
gaacctcccg tacaaactag cttattggat gattgccaga ccttaaaagg 1200agttcttact
tgcttaatac ctggtgctgg tggttatgac gccattgcag tgattactaa 1260gcaagatgtt
gatcttaggg ctcaaaccgc taatgacaaa agattttcta aggttcaatg 1320gctggatgta
actcaggctg actggggtgt taggaaagaa aaagatccgg aaacttatct 1380tgataaactg
caggaggagt tttaatgtca ttaccgttct taacttctgc accgggaaag 1440gttattattt
ttggtgaaca ctctgctgtg tacaacaagc ctgccgtcgc tgctagtgtg 1500tctgcgttga
gaacctacct gctaataagc gagtcatctg caccagatac tattgaattg 1560gacttcccgg
acattagctt taatcataag tggtccatca atgatttcaa tgccatcacc 1620gaggatcaag
taaactccca aaaattggcc aaggctcaac aagccaccga tggcttgtct 1680caggaactcg
ttagtctttt ggatccgttg ttagctcaac tatccgaatc cttccactac 1740catgcagcgt
tttgtttcct gtatatgttt gtttgcctat gcccccatgc caagaatatt 1800aagttttctt
taaagtctac tttacccatc ggtgctgggt tgggctcaag cgcctctatt 1860tctgtatcac
tggccttagc tatggcctac ttgggggggt taataggatc taatgacttg 1920gaaaagctgt
cagaaaacga taagcatata gtgaatcaat gggccttcat aggtgaaaag 1980tgtattcacg
gtaccccttc aggaatagat aacgctgtgg ccacttatgg taatgccctg 2040ctatttgaaa
aagactcaca taatggaaca ataaacacaa acaattttaa gttcttagat 2100gatttcccag
ccattccaat gatcctaacc tatactagaa ttccaaggtc tacaaaagat 2160cttgttgctc
gcgttcgtgt gttggtcacc gagaaatttc ctgaagttat gaagccaatt 2220ctagatgcca
tgggtgaatg tgccctacaa ggcttagaga tcatgactaa gttaagtaaa 2280tgtaaaggca
ccgatgacga ggctgtagaa actaataatg aactgtatga acaactattg 2340gaattgataa
gaataaatca tggactgctt gtctcaatcg gtgtttctca tcctggatta 2400gaacttatta
aaaatctgag cgatgatttg agaattggct ccacaaaact taccggtgct 2460ggtggcggcg
gttgctcttt gactttgtta cgaagagaca ttactcaaga gcaaattgac 2520agcttcaaaa
agaaattgca agatgatttt agttacgaga catttgaaac agacttgggt 2580gggactggct
gctgtttgtt aagcgcaaaa aatttgaata aagatcttaa aatcaaatcc 2640ctagtattcc
aattatttga aaataaaact accacaaagc aacaaattga cgatctatta 2700ttgccaggaa
acacgaattt accatggact tcagacgagg agttttaatg actgtatata 2760ctgctagtgt
aactgctccg gtaaatattg ctactcttaa gtattggggg aaaagggaca 2820cgaagttgaa
tctgcccacc aattcgtcca tatcagtgac tttatcgcaa gatgacctca 2880gaacgttgac
ctctgcggct actgcacctg agtttgaacg cgacactttg tggttaaatg 2940gagaaccaca
cagcatcgac aatgaaagaa ctcaaaattg tctgcgcgac ctacgccaat 3000taagaaagga
aatggaatcg aaggacgcct cattgcccac attatctcaa tggaaactcc 3060acattgtctc
cgaaaataac tttcctacag cagctggttt agcttcctcc gctgctggct 3120ttgctgcatt
ggtctctgca attgctaagt tataccaatt accacagtca acttcagaaa 3180tatctagaat
agcaagaaag gggtctggtt cagcttgtag atcgttgttt ggcggatacg 3240tggcctggga
aatgggaaaa gctgaagatg gtcatgattc catggcagta caaatcgcag 3300acagctctga
ctggcctcag atgaaagctt gtgtcctagt tgtcagcgat attaaaaagg 3360atgtgagttc
cactcagggt atgcaattga ccgtggcaac ctccgaacta tttaaagaaa 3420gaattgaaca
tgtcgtacca aagagatttg aagtcatgcg taaagccatt gttgaaaaag 3480atttcgccac
ctttgcaaag gaaacaatga tggattccaa ctctttccat gccacatgtt 3540tggactcttt
ccctccaata ttctacatga atgacacttc caagcgtatc atcagttggt 3600gccacaccat
taatcagttt tacggagaaa caatcgttgc atacacgttt gatgcaggtc 3660caaatgctgt
gttgtactac ttagctgaaa atgagtcgaa actctttgca tttatctata 3720aattgtttgg
ctctgttcct ggatgggaca agaaatttac tactgagcag cttgaggctt 3780tcaaccatca
atttgaatca tctaacttta ctgcacgtga attggatctt gagttgcaaa 3840aggatgttgc
cagagtgatt ttaactcaag tcggttcagg cccacaagaa acaaacgaat 3900ctttgattga
cgcaaagact ggtctaccaa aggaagagga gttttaactc gagtaggagg 3960cacatatgtc
tcagaacgtt tacattgtat cgactgccag aaccccaatt ggttcattcc 4020agggttctct
atcctccaag acagcagtgg aattgggtgc tgttgcttta aaaggcgcct 4080tggctaaggt
tccagaattg gatgcatcca aggattttga cgaaattatt tttggtaacg 4140ttctttctgc
caatttgggc caagctccgg ccagacaagt tgctttggct gccggtttga 4200gtaatcatat
cgttgcaagc acagttaaca aggtctgtgc atccgctatg aaggcaatca 4260ttttgggtgc
tcaatccatc aaatgtggta atgctgatgt tgtcgtagct ggtggttgtg 4320aatctatgac
taacgcacca tactacatgc cagcagcccg tgcgggtgcc aaatttggcc 4380aaactgttct
tgttgatggt gtcgaaagag atgggttgaa cgatgcgtac gatggtctag 4440ccatgggtgt
acacgcagaa aagtgtgccc gtgattggga tattactaga gaacaacaag 4500acaattttgc
catcgaatcc taccaaaaat ctcaaaaatc tcaaaaggaa ggtaaattcg 4560acaatgaaat
tgtacctgtt accattaagg gatttagagg taagcctgat actcaagtca 4620cgaaggacga
ggaacctgct agattacacg ttgaaaaatt gagatctgca aggactgttt 4680tccaaaaaga
aaacggtact gttactgccg ctaacgcttc tccaatcaac gatggtgctg 4740cagccgtcat
cttggtttcc gaaaaagttt tgaaggaaaa gaatttgaag cctttggcta 4800ttatcaaagg
ttggggtgag gccgctcatc aaccagctga ttttacatgg gctccatctc 4860ttgcagttcc
aaaggctttg aaacatgctg gcatcgaaga catcaattct gttgattact 4920ttgaattcaa
tgaagccttt tcggttgtcg gtttggtgaa cactaagatt ttgaagctag 4980acccatctaa
ggttaatgta tatggtggtg ctgttgctct aggtcaccca ttgggttgtt 5040ctggtgctag
agtggttgtt acactgctat ccatcttaca gcaagaagga ggtaagatcg 5100gtgttgccgc
catttgtaat ggtggtggtg gtgcttcctc tattgtcatt gaaaagatat 5160gaggatcctc
tagatgcgca ggaggcacat atggcgaaga acgttgggat tttggctatg 5220gatatctatt
tccctcccac ctgtgttcaa caggaagctt tggaagcaca tgatggagca 5280agtaaaggga
aatacactat tggacttggc caagattgtt tagctttttg cactgagctt 5340gaagatgtta
tctctatgag tttcaatgcg gtgacatcac tttttgagaa gtataagatt 5400gaccctaacc
aaatcgggcg tcttgaagta ggaagtgaga ctgttattga caaaagcaag 5460tccatcaaga
ccttcttgat gcagctcttt gagaaatgtg gaaacactga tgtcgaaggt 5520gttgactcga
ccaatgcttg ctatggtgga actgcagctt tgttaaactg tgtcaattgg 5580gttgagagta
actcttggga tggacgttat ggcctcgtca tttgtactga cagcgcggtt 5640tatgcagaag
gacccgcaag gcccactgga ggagctgcag cgattgctat gttgatagga 5700cctgatgctc
ctatcgtttt cgaaagcaaa ttgagagcaa gccacatggc tcatgtctat 5760gacttttaca
agcccaatct tgctagcgag tacccggttg ttgatggtaa gctttcacag 5820acttgctacc
tcatggctct tgactcctgc tataaacatt tatgcaacaa gttcgagaag 5880atcgagggca
aagagttctc cataaatgat gctgattaca ttgttttcca ttctccatac 5940aataaacttg
tacagaaaag ctttgctcgt ctcttgtaca acgacttctt gagaaacgca 6000agctccattg
acgaggctgc caaagaaaag ttcacccctt attcatcttt gacccttgac 6060gagagttacc
aaagccgtga tcttgaaaag gtgtcacaac aaatttcgaa accgttttat 6120gatgctaaag
tgcaaccaac gactttaata ccaaaggaag tcggtaacat gtacactgct 6180tctctctacg
ctgcatttgc ttccctcatc cacaataaac acaatgattt ggcgggaaag 6240cgggtggtta
tgttctctta tggaagtggc tccaccgcaa caatgttctc attacgcctc 6300aacgacaata
agcctccttt cagcatttca aacattgcat ctgtaatgga tgttggcggt 6360aaattgaaag
ctagacatga gtatgcacct gagaagtttg tggagacaat gaagctaatg 6420gaacataggt
atggagcaaa ggactttgtg acaaccaagg agggtattat agatcttttg 6480gcaccgggaa
cttattatct gaaagaggtt gattccttgt accggagatt ctatggcaag 6540aaaggtgaag
atggatctgt agccaatgga cactgaggat ccgtcgactc gagcacgtga 6600ggaggcacat
atgacggaaa cgcacgccat agccggggtc ccgatgaggt gggtgggacc 6660ccttcgtatt
tccgggaacg tcgccgagac cgagacccag gtcccgctcg ccacgtacga 6720gtcgccgctg
tggccgtcgg tgggccgcgg ggcgaaggtc tcccggctga cggagaaggg 6780catcgtcgcc
accctcgtcg acgagcggat gacccgctcg gtgatcgtcg aggcgacgga 6840cgcgcagacc
gcgtacatgg ccgcgcagac catccacgcc cgcatcgacg agctgcgcga 6900ggtggtgcgc
ggctgcagcc ggttcgccca gctgatcaac atcaagcacg agatcaacgc 6960gaacctgctg
ttcatccggt tcgagttcac caccggtgac gcctccggcc acaacatggc 7020cacgctcgcc
tccgatgtgc tcctggggca cctgctggag acgatccctg gcatctccta 7080cggctcgatc
tccggcaact actgcacgga caagaaggcc accgcgatca acggcatcct 7140cggccgcggc
aagaacgtga tcaccgagct gctggtgccg cgggacgtcg tcgagaacaa 7200cctgcacacc
acggctgcca agatcgtcga gctgaacatc cgcaagaacc tgctcggcac 7260cctgctcgcc
ggcggcatcc gctcggccaa cgcccacttc gcgaacatgc tgctcggctt 7320ctacctggcc
accggccagg acgccgccaa catcgtcgag ggctcgcagg gcgtcgtcat 7380ggccgaggac
cgcgacggcg acctctactt cgcctgcacc ctgccgaacc tgatcgtcgg 7440cacggtcggc
aacggcaagg gtctcggctt cgtggagacg aacctcgccc ggctcggctg 7500ccgagccgac
cgcgaacccg gggagaacgc ccgccgcctc gccgtcatcg cggcagcgac 7560cgtgctgtgc
ggtgaactct cgctgctcgc ggcacagacg aacccgggcg aactcatgcg 7620cgcgcacgtc
cagctggaac gcgacaacaa gaccgcaaag gttggtgcat agacgcggta 7680aggaggcaca
tatgagtgag cttatacccg cctgggttgg tgacagactg gctccggtgg 7740acaagttgga
ggtgcatttg aaagggctcc gccacaaggc ggtgtctgtt ttcgtcatgg 7800atggcgaaaa
cgtgctgatc cagcgccgct cggaggagaa atatcactct cccgggcttt 7860gggcgaacac
ctgctgcacc catccgggct ggaccgaacg ccccgaggaa tgcgcggtgc 7920ggcggctgcg
cgaggagctg gggatcaccg ggctttatcc cgcccatgcc gaccggctgg 7980aatatcgcgc
cgatgtcggc ggcggcatga tcgagcatga ggtggtcgac atctatctgg 8040cctatgccaa
accgcatatg cggatcaccc ccgatccgcg cgaagtggcc gaggtgcgct 8100ggatcggcct
ttacgatctg gcggccgagg ccggtcggca tcccgagcgg ttctcgaaat 8160ggctcaacat
ctatctgtcg agccatcttg accggatttt cggatcgatc ctgcgcggct 8220gagc
8224638077DNAArtificial SequenceOperon F containing A. thaliana, S.
cerevisiae, and Streptomyces sp CL190DNA 63ccaccgcggc ggccgcgtcg
acgccggcgg aggcacatat gtctcagaac gtttacattg 60tatcgactgc cagaacccca
attggttcat tccagggttc tctatcctcc aagacagcag 120tggaattggg tgctgttgct
ttaaaaggcg ccttggctaa ggttccagaa ttggatgcat 180ccaaggattt tgacgaaatt
atttttggta acgttctttc tgccaatttg ggccaagctc 240cggccagaca agttgctttg
gctgccggtt tgagtaatca tatcgttgca agcacagtta 300acaaggtctg tgcatccgct
atgaaggcaa tcattttggg tgctcaatcc atcaaatgtg 360gtaatgctga tgttgtcgta
gctggtggtt gtgaatctat gactaacgca ccatactaca 420tgccagcagc ccgtgcgggt
gccaaatttg gccaaactgt tcttgttgat ggtgtcgaaa 480gagatgggtt gaacgatgcg
tacgatggtc tagccatggg tgtacacgca gaaaagtgtg 540cccgtgattg ggatattact
agagaacaac aagacaattt tgccatcgaa tcctaccaaa 600aatctcaaaa atctcaaaag
gaaggtaaat tcgacaatga aattgtacct gttaccatta 660agggatttag aggtaagcct
gatactcaag tcacgaagga cgaggaacct gctagattac 720acgttgaaaa attgagatct
gcaaggactg ttttccaaaa agaaaacggt actgttactg 780ccgctaacgc ttctccaatc
aacgatggtg ctgcagccgt catcttggtt tccgaaaaag 840ttttgaagga aaagaatttg
aagcctttgg ctattatcaa aggttggggt gaggccgctc 900atcaaccagc tgattttaca
tgggctccat ctcttgcagt tccaaaggct ttgaaacatg 960ctggcatcga agacatcaat
tctgttgatt actttgaatt caatgaagcc ttttcggttg 1020tcggtttggt gaacactaag
attttgaagc tagacccatc taaggttaat gtatatggtg 1080gtgctgttgc tctaggtcac
ccattgggtt gttctggtgc tagagtggtt gttacactgc 1140tatccatctt acagcaagaa
ggaggtaaga tcggtgttgc cgccatttgt aatggtggtg 1200gtggtgcttc ctctattgtc
attgaaaaga tatgaggatc ctctaggtac ttccctggcg 1260tgtgcagcgg ttgacgcgcc
gtgccctcgc tgcgagcggc gcgcacatct gacgtcctgc 1320tttattgctt tctcagaact
cgggacgaag cgatcccatg atcacgcgat ctccatgcag 1380aaaagacaaa gggagctgag
tgcgttgaca ctaccgacct cggctgaggg ggtatcagaa 1440agccaccggg cccgctcggt
cggcatcggt cgcgcccacg ccaaggccat cctgctggga 1500gagcatgcgg tcgtctacgg
agcgccggca ctcgctctgc cgattccgca gctcacggtc 1560acggccagcg tcggctggtc
gtccgaggcc tccgacagtg cgggtggcct gtcctacacg 1620atgaccggta cgccgtcgcg
ggcactggtg acgcaggcct ccgacggcct gcaccggctc 1680accgcggaat tcatggcgcg
gatgggcgtg acgaacgcgc cgcacctcga cgtgatcctg 1740gacggcgcga tcccgcacgg
ccggggtctc ggctccagcg cggccggctc acgcgcgatc 1800gccttggccc tcgccgacct
cttcggccac gaactggccg agcacacggc gtacgaactg 1860gtgcagacgg ccgagaacat
ggcgcacggc cgggccagcg gcgtggacgc gatgacggtc 1920ggcgcgtccc ggccgctgct
gttccagcag ggccgcaccg agcgactggc catcggctgc 1980gacagcctgt tcatcgtcgc
cgacagcggc gtcccgggca gcaccaagga agcggtcgag 2040atgctgcggg agggattcac
ccgcagcgcc ggaacacagg agcggttcgt cggccgggcg 2100acggaactga ccgaggccgc
ccggcaggcc ctcgccgacg gccggcccga ggagctgggc 2160tcgcagctga cgtactacca
cgagctgctc catgaggccc gcctgagcac cgacggcatc 2220gatgcgctgg tcgaggccgc
gctgaaggca ggcagcctcg gagccaagat caccggcggt 2280ggtctgggcg gctgcatgat
cgcacaggcc cggcccgaac aggcccggga ggtcacccgg 2340cagctccacg aggccggtgc
cgtacagacc tgggtcgtac cgctgaaagg gctcgacaac 2400catgcgcagt gaacacccga
ccacgaccgt gctccagtcg cgggagcagg gcagcgcggc 2460cggcgccacc gcggtcgcgc
acccaaacat cgcgctgatc aagtactggg gcaagcgcga 2520cgagcggctg atcctgccct
gcaccaccag cctgtcgatg acgctggacg tcttccccac 2580gaccaccgag gtccggctcg
accccgccgc cgagcacgac acggccgccc tcaacggcga 2640ggtggccacg ggcgagacgc
tgcgccgcat cagcgccttc ctctccctgg tgcgggaggt 2700ggcgggcagc gaccagcggg
ccgtggtgga cacccgcaac accgtgccca ccggggcggg 2760cctggcgtcc tccgccagcg
ggttcgccgc cctcgccgtc gcggccgcgg ccgcctacgg 2820gctcgaactc gacgaccgcg
ggctgtcccg gctggcccga cgtggatccg gctccgcctc 2880gcggtcgatc ttcggcggct
tcgccgtctg gcacgccggc cccgacggca cggccacgga 2940agcggacctc ggctcctacg
ccgagccggt gcccgcggcc gacctcgacc cggcgctggt 3000catcgccgtg gtcaacgccg
gccccaagcc cgtctccagc cgcgaggcca tgcgccgcac 3060cgtcgacacc tcgccgctgt
accggccgtg ggccgactcc agtaaggacg acctggacga 3120gatgcgctcg gcgctgctgc
gcggcgacct cgaggccgtg ggcgagatcg cggagcgcaa 3180cgcgctcggc atgcacgcca
ccatgctggc cgcccgcccc gcggtgcggt acctgtcgcc 3240ggccacggtc accgtgctcg
acagcgtgct ccagctccgc aaggacggtg tcctggccta 3300cgcgaccatg gacgccggtc
ccaacgtgaa ggtgctgtgc cggcgggcgg acgccgagcg 3360ggtggccgac gtcgtacgcg
ccgccgcgtc cggcggtcag gtcctcgtcg ccgggccggg 3420agacggtgcc cgcctgctga
gcgagggcgc atgacgacag gtcagcgcac gatcgtccgg 3480cacgcgccgg gcaagctgtt
cgtcgcgggc gagtacgcgg tcgtggatcc gggcaacccg 3540gcgatcctgg tagcggtcga
ccggcacatc agcgtcaccg tgtccgacgc cgacgcggac 3600accggggccg ccgacgtcgt
gatctcctcc gacctcggtc cgcaggcggt cggctggcgc 3660tggcacgacg gccggctcgt
cgtccgcgac ccggacgacg ggcagcaggc gcgcagcgcc 3720ctggcccacg tggtgtcggc
gatcgagacc gtgggccggc tgctgggcga acgcggacag 3780aaggtccccg ctctcaccct
ctccgtcagc agccgcctgc acgaggacgg ccggaagttc 3840ggcctgggct ccagcggcgc
ggtgaccgtg gcgaccgtag ccgccgtcgc cgcgttctgc 3900ggactcgaac tgtccaccga
cgaacggttc cggctggcca tgctcgccac cgcggaactc 3960gaccccaagg gctccggcgg
ggacctcgcc gccagcacct ggggcggctg gatcgcctac 4020caggcgcccg accgggcctt
tgtgctcgac ctggcccggc gcgtgggagt cgaccggaca 4080ctgaaggcgc cctggccggg
gcactcggtg cgccgactgc cggcgcccaa gggcctcacc 4140ctggaggtcg gctggaccgg
agagcccgcc tccaccgcgt ccctggtgtc cgatctgcac 4200cgccgcacct ggcggggcag
cgcctcccac cagaggttcg tcgagaccac gaccgactgt 4260gtccgctccg cggtcaccgc
cctggagtcc ggcgacgaca cgagcctgct gcacgagatc 4320cgccgggccc gccaggagct
ggcccgcctg gacgacgagg tcggcctcgg catcttcaca 4380cccaagctga cggcgctgtg
cgacgccgcc gaagccgtcg gcggcgcggc caagccctcc 4440ggggcaggcg gcggcgactg
cggcatcgcc ctgctggacg ccgaggcgtc gcgggacatc 4500acacatgtac ggcaacggtg
ggagacagcc ggggtgctgc ccctgcccct gactcctgcc 4560ctggaaggga tctaagaatg
accagcgccc aacgcaagga cgaccacgta cggctcgcca 4620tcgagcagca caacgcccac
agcggacgca accagttcga cgacgtgtcg ttcgtccacc 4680acgccctggc cggcatcgac
cggccggacg tgtccctggc cacgtccttc gccgggatct 4740cctggcaggt gccgatctac
atcaacgcga tgaccggcgg cagcgagaag accggcctca 4800tcaaccggga cctggccacc
gccgcccgcg agaccggcgt ccccatcgcg tccgggtcca 4860tgaacgcgta catcaaggac
ccctcctgcg ccgacacgtt ccgtgtgctg cgcgacgaga 4920accccaacgg gttcgtcatc
gcgaacatca acgccaccac gacggtcgac aacgcgcagc 4980gcgcgatcga cctgatcgag
gcgaacgccc tgcagatcca catcaacacg gcgcaggaga 5040cgccgatgcc ggagggcgac
cggtcgttcg cgtcctgggt cccgcagatc gagaagatcg 5100cggcggccgt cgacatcccc
gtgatcgtca aggaggtcgg caacggcctg agccggcaga 5160ccatcctgct gctcgccgac
ctcggcgtgc aggcggcgga cgtcagcggc cgcggcggca 5220cggacttcgc ccgcatcgag
aacggccgcc gggagctcgg cgactacgcg ttcctgcacg 5280gctgggggca gtccaccgcc
gcctgcctgc tggacgccca ggacatctcc ctgcccgtcc 5340tcgcctccgg cggtgtgcgt
cacccgctcg acgtggtccg cgccctcgcg ctcggcgccc 5400gcgccgtcgg ctcctccgcc
ggcttcctgc gcaccctgat ggacgacggc gtcgacgcgc 5460tgatcacgaa gctcacgacc
tggctggacc agctggcggc gctgcagacc atgctcggcg 5520cgcgcacccc ggccgacctc
acccgctgcg acgtgctgct ccacggcgag ctgcgtgact 5580tctgcgccga ccggggcatc
gacacgcgcc gcctcgccca gcgctccagc tccatcgagg 5640ccctccagac gacgggaagc
acacgatgac ggaaacgcac gccatagccg gggtcccgat 5700gaggtgggtg ggaccccttc
gtatttccgg gaacgtcgcc gagaccgaga cccaggtccc 5760gctcgccacg tacgagtcgc
cgctgtggcc gtcggtgggc cgcggggcga aggtctcccg 5820gctgacggag aagggcatcg
tcgccaccct cgtcgacgag cggatgaccc gctcggtgat 5880cgtcgaggcg acggacgcgc
agaccgcgta catggccgcg cagaccatcc acgcccgcat 5940cgacgagctg cgcgaggtgg
tgcgcggctg cagccggttc gcccagctga tcaacatcaa 6000gcacgagatc aacgcgaacc
tgctgttcat ccggttcgag ttcaccaccg gtgacgcctc 6060cggccacaac atggccacgc
tcgcctccga tgtgctcctg gggcacctgc tggagacgat 6120ccctggcatc tcctacggct
cgatctccgg caactactgc acggacaaga aggccaccgc 6180gatcaacggc atcctcggcc
gcggcaagaa cgtgatcacc gagctgctgg tgccgcggga 6240cgtcgtcgag aacaacctgc
acaccacggc tgccaagatc gtcgagctga acatccgcaa 6300gaacctgctc ggcaccctgc
tcgccggcgg catccgctcg gccaacgccc acttcgcgaa 6360catgctgctc ggcttctacc
tggccaccgg ccaggacgcc gccaacatcg tcgagggctc 6420gcagggcgtc gtcatggccg
aggaccgcga cggcgacctc tacttcgcct gcaccctgcc 6480gaacctgatc gtcggcacgg
tcggcaacgg caagggtctc ggcttcgtgg agacgaacct 6540cgcccggctc ggctgccgag
ccgaccgcga acccggggag aacgcccgcc gcctcgccgt 6600catcgcggca gcgaccgtgc
tgtgcggtga actctcgctg ctcgcggcac agacgaaccc 6660gggcgaactc atgcgcgcgc
acgtccagct ggaacgcgac aacaagaccg caaaggttgg 6720tgcatagggc atgtccatct
ccataggcat tcacgacctg tcgttcgcca caaccgagtt 6780cgtcctgccg cacacggcgc
tcgccgagta caacggcacc gagatcggca agtaccacgt 6840cggcatcggc cagcagtcga
tgagcgtgcc ggccgccgac gaggacatcg tgaccatggc 6900cgcgaccgcg gcgcggccca
tcatcgagcg caacggcaag agccggatcc gcacggtcgt 6960gttcgccacg gagtcgtcga
tcgaccaggc gaaggcgggc ggcgtgtacg tgcactccct 7020gctggggctg gagtcggcct
gccgggtcgt cgagctgaag caggcctgct acggggccac 7080cgccgccctt cagttcgcca
tcggcctggt gcggcgcgac cccgcccagc aggtcctggt 7140catcgccagt gacgtctcca
agtacgagct ggacagcccc ggcgaggcga cccagggcgc 7200ggccgcggtg gccatgctgg
tcggcgccga cccggccctg ctgcgtatcg aggagccgtc 7260gggcctgttc accgccgacg
tcatggactt ctggcggccc aactacctca ccaccgctct 7320ggtcgacggc caggagtcca
tcaacgccta cctgcaggcc gtcgagggcg cctggaagga 7380ctacgcggag caggacggcc
ggtcgctgga ggagttcgcg gcgttcgtct accaccagcc 7440gttcacgaag atggcctaca
aggcgcaccg ccacctgctg aacttcaacg gctacgacac 7500cgacaaggac gccatcgagg
gcgccctcgg ccagacgacg gcgtacaaca acgtcatcgg 7560caacagctac accgcgtcgg
tgtacctggg cctggccgcc ctgctcgacc aggcggacga 7620cctgacgggc cgttccatcg
gcttcctgag ctacggctcg ggcagcgtcg ccgagttctt 7680ctcgggcacc gtcgtcgccg
ggtaccgcga gcgtctgcgc accgaggcga accaggaggc 7740gatcgcccgg cgcaagagcg
tcgactacgc cacctaccgc gagctgcacg agtacacgct 7800cccgtccgac ggcggcgacc
acgccacccc ggtgcagacc accggcccct tccggctggc 7860cgggatcaac gaccacaagc
gcatctacga ggcgcgctag cgacacccct cggcaacggg 7920gtgcgccact gttcggcgca
ccccgtgccg ggctttcgca cagctattca cgaccatttg 7980aggggcgggc agccgcatga
ccgacgtccg attccgcatt atcggtacgg gtgcctacct 8040agaactagtg gatcccccgg
gctgcaggaa ttcgata 8077648400DNAArtificial
SequenceOperon G containing A. thaliana, S. cerevisiae, and S. pombe
DNA 64ggccgcagga ggagttcata tgtcagagtt gagagccttc agtgccccag ggaaagcgtt
60actagctggt ggatatttag ttttagatac aaaatatgaa gcatttgtag tcggattatc
120ggcaagaatg catgctgtag cccatcctta cggttcattg caagggtctg ataagtttga
180agtgcgtgtg aaaagtaaac aatttaaaga tggggagtgg ctgtaccata taagtcctaa
240aagtggcttc attcctgttt cgataggcgg atctaagaac cctttcattg aaaaagttat
300cgctaacgta tttagctact ttaaacctaa catggacgac tactgcaata gaaacttgtt
360cgttattgat attttctctg atgatgccta ccattctcag gaggatagcg ttaccgaaca
420tcgtggcaac agaagattga gttttcattc gcacagaatt gaagaagttc ccaaaacagg
480gctgggctcc tcggcaggtt tagtcacagt tttaactaca gctttggcct ccttttttgt
540atcggacctg gaaaataatg tagacaaata tagagaagtt attcataatt tagcacaagt
600tgctcattgt caagctcagg gtaaaattgg aagcgggttt gatgtagcgg cggcagcata
660tggatctatc agatatagaa gattcccacc cgcattaatc tctaatttgc cagatattgg
720aagtgctact tacggcagta aactggcgca tttggttgat gaagaagact ggaatattac
780gattaaaagt aaccatttac cttcgggatt aactttatgg atgggcgata ttaagaatgg
840ttcagaaaca gtaaaactgg tccagaaggt aaaaaattgg tatgattcgc atatgccaga
900aagcttgaaa atatatacag aactcgatca tgcaaattct agatttatgg atggactatc
960taaactagat cgcttacacg agactcatga cgattacagc gatcagatat ttgagtctct
1020tgagaggaat gactgtacct gtcaaaagta tcctgaaatc acagaagtta gagatgcagt
1080tgccacaatt agacgttcct ttagaaaaat aactaaagaa tctggtgccg atatcgaacc
1140tcccgtacaa actagcttat tggatgattg ccagacctta aaaggagttc ttacttgctt
1200aatacctggt gctggtggtt atgacgccat tgcagtgatt actaagcaag atgttgatct
1260tagggctcaa accgctaatg acaaaagatt ttctaaggtt caatggctgg atgtaactca
1320ggctgactgg ggtgttagga aagaaaaaga tccggaaact tatcttgata aactgcagga
1380ggagttttaa tgtcattacc gttcttaact tctgcaccgg gaaaggttat tatttttggt
1440gaacactctg ctgtgtacaa caagcctgcc gtcgctgcta gtgtgtctgc gttgagaacc
1500tacctgctaa taagcgagtc atctgcacca gatactattg aattggactt cccggacatt
1560agctttaatc ataagtggtc catcaatgat ttcaatgcca tcaccgagga tcaagtaaac
1620tcccaaaaat tggccaaggc tcaacaagcc accgatggct tgtctcagga actcgttagt
1680cttttggatc cgttgttagc tcaactatcc gaatccttcc actaccatgc agcgttttgt
1740ttcctgtata tgtttgtttg cctatgcccc catgccaaga atattaagtt ttctttaaag
1800tctactttac ccatcggtgc tgggttgggc tcaagcgcct ctatttctgt atcactggcc
1860ttagctatgg cctacttggg ggggttaata ggatctaatg acttggaaaa gctgtcagaa
1920aacgataagc atatagtgaa tcaatgggcc ttcataggtg aaaagtgtat tcacggtacc
1980ccttcaggaa tagataacgc tgtggccact tatggtaatg ccctgctatt tgaaaaagac
2040tcacataatg gaacaataaa cacaaacaat tttaagttct tagatgattt cccagccatt
2100ccaatgatcc taacctatac tagaattcca aggtctacaa aagatcttgt tgctcgcgtt
2160cgtgtgttgg tcaccgagaa atttcctgaa gttatgaagc caattctaga tgccatgggt
2220gaatgtgccc tacaaggctt agagatcatg actaagttaa gtaaatgtaa aggcaccgat
2280gacgaggctg tagaaactaa taatgaactg tatgaacaac tattggaatt gataagaata
2340aatcatggac tgcttgtctc aatcggtgtt tctcatcctg gattagaact tattaaaaat
2400ctgagcgatg atttgagaat tggctccaca aaacttaccg gtgctggtgg cggcggttgc
2460tctttgactt tgttacgaag agacattact caagagcaaa ttgacagctt caaaaagaaa
2520ttgcaagatg attttagtta cgagacattt gaaacagact tgggtgggac tggctgctgt
2580ttgttaagcg caaaaaattt gaataaagat cttaaaatca aatccctagt attccaatta
2640tttgaaaata aaactaccac aaagcaacaa attgacgatc tattattgcc aggaaacacg
2700aatttaccat ggacttcaga cgaggagttt taatgactgt atatactgct agtgtaactg
2760ctccggtaaa tattgctact cttaagtatt gggggaaaag ggacacgaag ttgaatctgc
2820ccaccaattc gtccatatca gtgactttat cgcaagatga cctcagaacg ttgacctctg
2880cggctactgc acctgagttt gaacgcgaca ctttgtggtt aaatggagaa ccacacagca
2940tcgacaatga aagaactcaa aattgtctgc gcgacctacg ccaattaaga aaggaaatgg
3000aatcgaagga cgcctcattg cccacattat ctcaatggaa actccacatt gtctccgaaa
3060ataactttcc tacagcagct ggtttagctt cctccgctgc tggctttgct gcattggtct
3120ctgcaattgc taagttatac caattaccac agtcaacttc agaaatatct agaatagcaa
3180gaaaggggtc tggttcagct tgtagatcgt tgtttggcgg atacgtggcc tgggaaatgg
3240gaaaagctga agatggtcat gattccatgg cagtacaaat cgcagacagc tctgactggc
3300ctcagatgaa agcttgtgtc ctagttgtca gcgatattaa aaaggatgtg agttccactc
3360agggtatgca attgaccgtg gcaacctccg aactatttaa agaaagaatt gaacatgtcg
3420taccaaagag atttgaagtc atgcgtaaag ccattgttga aaaagatttc gccacctttg
3480caaaggaaac aatgatggat tccaactctt tccatgccac atgtttggac tctttccctc
3540caatattcta catgaatgac acttccaagc gtatcatcag ttggtgccac accattaatc
3600agttttacgg agaaacaatc gttgcataca cgtttgatgc aggtccaaat gctgtgttgt
3660actacttagc tgaaaatgag tcgaaactct ttgcatttat ctataaattg tttggctctg
3720ttcctggatg ggacaagaaa tttactactg agcagcttga ggctttcaac catcaatttg
3780aatcatctaa ctttactgca cgtgaattgg atcttgagtt gcaaaaggat gttgccagag
3840tgattttaac tcaagtcggt tcaggcccac aagaaacaaa cgaatctttg attgacgcaa
3900agactggtct accaaaggaa gaggagtttt aactcgacgc cggcggaggc acatatgtct
3960cagaacgttt acattgtatc gactgccaga accccaattg gttcattcca gggttctcta
4020tcctccaaga cagcagtgga attgggtgct gttgctttaa aaggcgcctt ggctaaggtt
4080ccagaattgg atgcatccaa ggattttgac gaaattattt ttggtaacgt tctttctgcc
4140aatttgggcc aagctccggc cagacaagtt gctttggctg ccggtttgag taatcatatc
4200gttgcaagca cagttaacaa ggtctgtgca tccgctatga aggcaatcat tttgggtgct
4260caatccatca aatgtggtaa tgctgatgtt gtcgtagctg gtggttgtga atctatgact
4320aacgcaccat actacatgcc agcagcccgt gcgggtgcca aatttggcca aactgttctt
4380gttgatggtg tcgaaagaga tgggttgaac gatgcgtacg atggtctagc catgggtgta
4440cacgcagaaa agtgtgcccg tgattgggat attactagag aacaacaaga caattttgcc
4500atcgaatcct accaaaaatc tcaaaaatct caaaaggaag gtaaattcga caatgaaatt
4560gtacctgtta ccattaaggg atttagaggt aagcctgata ctcaagtcac gaaggacgag
4620gaacctgcta gattacacgt tgaaaaattg agatctgcaa ggactgtttt ccaaaaagaa
4680aacggtactg ttactgccgc taacgcttct ccaatcaacg atggtgctgc agccgtcatc
4740ttggtttccg aaaaagtttt gaaggaaaag aatttgaagc ctttggctat tatcaaaggt
4800tggggtgagg ccgctcatca accagctgat tttacatggg ctccatctct tgcagttcca
4860aaggctttga aacatgctgg catcgaagac atcaattctg ttgattactt tgaattcaat
4920gaagcctttt cggttgtcgg tttggtgaac actaagattt tgaagctaga cccatctaag
4980gttaatgtat atggtggtgc tgttgctcta ggtcacccat tgggttgttc tggtgctaga
5040gtggttgtta cactgctatc catcttacag caagaaggag gtaagatcgg tgttgccgcc
5100atttgtaatg gtggtggtgg tgcttcctct attgtcattg aaaagatatg aggatcctct
5160agatgcgcag gaggcacata tggcgaagaa cgttgggatt ttggctatgg atatctattt
5220ccctcccacc tgtgttcaac aggaagcttt ggaagcacat gatggagcaa gtaaagggaa
5280atacactatt ggacttggcc aagattgttt agctttttgc actgagcttg aagatgttat
5340ctctatgagt ttcaatgcgg tgacatcact ttttgagaag tataagattg accctaacca
5400aatcgggcgt cttgaagtag gaagtgagac tgttattgac aaaagcaagt ccatcaagac
5460cttcttgatg cagctctttg agaaatgtgg aaacactgat gtcgaaggtg ttgactcgac
5520caatgcttgc tatggtggaa ctgcagcttt gttaaactgt gtcaattggg ttgagagtaa
5580ctcttgggat ggacgttatg gcctcgtcat ttgtactgac agcgcggttt atgcagaagg
5640acccgcaagg cccactggag gagctgcagc gattgctatg ttgataggac ctgatgctcc
5700tatcgttttc gaaagcaaat tgagagcaag ccacatggct catgtctatg acttttacaa
5760gcccaatctt gctagcgagt acccggttgt tgatggtaag ctttcacaga cttgctacct
5820catggctctt gactcctgct ataaacattt atgcaacaag ttcgagaaga tcgagggcaa
5880agagttctcc ataaatgatg ctgattacat tgttttccat tctccataca ataaacttgt
5940acagaaaagc tttgctcgtc tcttgtacaa cgacttcttg agaaacgcaa gctccattga
6000cgaggctgcc aaagaaaagt tcacccctta ttcatctttg acccttgacg agagttacca
6060aagccgtgat cttgaaaagg tgtcacaaca aatttcgaaa ccgttttatg atgctaaagt
6120gcaaccaacg actttaatac caaaggaagt cggtaacatg tacactgctt ctctctacgc
6180tgcatttgct tccctcatcc acaataaaca caatgatttg gcgggaaagc gggtggttat
6240gttctcttat ggaagtggct ccaccgcaac aatgttctca ttacgcctca acgacaataa
6300gcctcctttc agcatttcaa acattgcatc tgtaatggat gttggcggta aattgaaagc
6360tagacatgag tatgcacctg agaagtttgt ggagacaatg aagctaatgg aacataggta
6420tggagcaaag gactttgtga caaccaagga gggtattata gatcttttgg caccgggaac
6480ttattatctg aaagaggttg attccttgta ccggagattc tatggcaaga aaggtgaaga
6540tggatctgta gccaatggac actgaggatc cgtcgagcac gtggaggcac atatgcaatg
6600ctgtgagatg cctgttggat acattcagat tcctgttggg attgctggtc cattgttgct
6660tgatggttat gagtactctg ttcctatggc tacaaccgaa ggttgtttgg ttgctagcac
6720taacagaggc tgcaaggcta tgtttatctc tggtggcgcc accagtaccg ttcttaagga
6780cggtatgacc cgagcacctg ttgttcggtt cgcttcggcg agacgagctt cggagcttaa
6840gtttttcttg gagaatccag agaactttga tactttggca gtagtcttca acaggtcgag
6900tagatttgca agactgcaaa gtgttaaatg cacaatcgcg gggaagaatg cttatgtaag
6960gttctgttgt agtactggtg atgctatggg gatgaatatg gtttctaaag gtgtgcagaa
7020tgttcttgag tatcttaccg atgatttccc tgacatggat gtgattggaa tctctggtaa
7080cttctgttcg gacaagaaac ctgctgctgt gaactggatt gagggacgtg gtaaatcagt
7140tgtttgcgag gctgtaatca gaggagagat cgtgaacaag gtcttgaaaa cgagcgtggc
7200tgctttagtc gagctcaaca tgctcaagaa cctagctggc tctgctgttg caggctctct
7260aggtggattc aacgctcatg ccagtaacat agtgtctgct gtattcatag ctactggcca
7320agatccagct caaaacgtgg agagttctca atgcatcacc atgatggaag ctattaatga
7380cggcaaagat atccatatct cagtcactat gccatctatc gaggtgggga cagtgggagg
7440aggaacacag cttgcatctc aatcagcgtg tttaaacctg ctcggagtta aaggagcaag
7500cacagagtcg ccgggaatga acgcaaggag gctagcgacg atcgtagccg gagcagtttt
7560agctggagag ttatctttaa tgtcagcaat tgcagctgga cagcttgtga gaagtcacat
7620gaaatacaat agatccagcc gagacatctc tggagcaacg acaacgacaa caacaacaac
7680atgacccgta ggaggcacat atgagttccc aacaagagaa aaaggattat gatgaagaac
7740aattaaggtt gatggaagaa gtttgtatcg ttgtagatga aaatgatgtc cctttaagat
7800atggaacgaa aaaggagtgt catttgatgg aaaatataaa taaaggtctt ttgcatagag
7860cattctctat gttcatcttt gatgagcaaa atcgcctttt acttcagcag cgtgcagaag
7920agaaaattac atttccatcc ttatggacga atacatgttg ctcccaccca ttggatgttg
7980ctggtgaacg tggtaatact ttacctgaag ctgttgaagg tgttaagaat gcagctcaac
8040gcaagctgtt ccatgaattg ggtattcaag ccaagtatat tcccaaagac aaatttcagt
8100ttcttacacg aatccattac cttgctccta gtactggtgc ttggggagag catgaaattg
8160actacattct tttcttcaaa ggtaaagttg agctggatat caatcccaat gaagttcaag
8220cctataagta tgttactatg gaagagttaa aagagatgtt ttccgatcct caatatggat
8280tcacaccatg gttcaaactt atttgtgagc attttatgtt taaatggtgg caggatgtag
8340atcatgcgtc aaaattccaa gataccttaa ttcatcgttg ctaaggatcc cccgggatcc
84006555DNAArtificial SequencePCR primer containing R. capsulatus DNA
65gcgatatcgg atccaggagg accatatgat cgccgaagcg gatatggagg tctgc
556650DNAArtificial SequencePCR primer containing R. capsulatus DNA
66gcgatatcaa gcttggatcc tcaatccatc gccaggccgc ggtcgcgcgc
506760DNAArtificial SequenceOligonucleotide containing N. tabacum and R.
caopsulatus DNA 67ctttcctgaa acataattta taatcagatc caggaggacc
atatgatcgc cgaagcggat 606860DNAArtificial SequenceOligonucleotide
containing N. tabacum and R. capsulatus DNA 68cgaccgcggc ctggcgatgg
attgaggatc taaacaaacc cggaacagac cgttgggaag 606960DNAArtificial
SequenceOligonucleotide containing N. tabacum and R. capsulatus DNA
69atttttcatc tcgaattgta ttcccacgaa ggccgcgtcg actacggccg caggaggagt
607060DNAArtificial SequenceOligonucleotide containing N. tabacum and R.
capsulatus DNA 70ttcggatcga tcctgcgcgg ctgagcggcc ggaatggtga
agttgaaaaa cgaatccttc 60711020DNARhodobacter capsulatus 71atgatcgccg
aagcggatat ggaggtctgc cgggagctga tccgcaccgg cagctactcc 60ttccatgcgg
cgtccagagt tctgccggcg cgggtccgtg accccgcgct ggcgctttac 120gccttttgcc
gcgtcgccga tgacgaagtc gacgaggttg gcgcgccgcg cgacaaggct 180gcggcggttt
tgaaacttgg cgaccggctg gaggacatct atgccggtcg tccgcgcaat 240gcgccctcgg
atcgggcttt cgcggcggtg gtcgaggaat tcgagatgcc gcgcgaattg 300cccgaggcgc
tgctggaggg cttcgcctgg gatgccgagg ggcggtggta tcacacgctt 360tcggacgtgc
aggcctattc ggcgcgggtg gcggccgccg tcggcgcgat gatgtgcgtg 420ctgatgcggg
tgcgcaaccc cgatgcgctg gcgcgggcct gcgatctcgg tcttgccatg 480cagatgtcga
acatcgcccg cgacgtgggc gaggatgccc gggcggggcg gcttttcctg 540ccgaccgact
ggatggtcga ggaggggatc gatccgcagg cgttcctggc cgatccgcag 600cccaccaagg
gcatccgccg ggtcaccgag cggttgctga accgcgccga ccggctttac 660tggcgggcgg
cgacgggggt gcggcttttg ccctttgact gccgaccggg gatcatggcc 720gcgggcaaga
tctatgccgc gatcggggcc gaggtggcga aggcgaaata cgacaacatc 780acccggcgtg
cccacacgac caagggccgc aagctgtggc tggtggcgaa ttccgcgatg 840tcggcgacgg
cgacctcgat gctgccgctc tcgccgcggg tgcatgccaa gcccgagccc 900gaagtggcgc
atctggtcga tgccgccgcg catcgcaacc tgcatcccga acggtccgag 960gtgctgatct
cggcgctgat ggcgctgaag gcgcgcgacc gcggcctggc gatggattga
10207213917DNAArtificial Sequencemisc_feature()..()Plastid transformation
vector pHKO4, containing Operon B, containi 72gcacttttcg gggaaatgtg
cgcggaaccc ctatttgttt atttttctaa atacattcaa 60atatgtatcc gctcatgaga
caataaccct gataaatgct tcaataatat tgaaaaagga 120agagtatgag tattcaacat
ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 180ttcctgtttt tgctcaccca
gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 240gtgcacgagt gggttacatc
gaactggatc tcaacagcgg taagatcctt gagagttttc 300gccccgaaga acgttttcca
atgatgagca cttttaaagt tctgctatgt ggcgcggtat 360tatcccgtat tgacgccggg
caagagcaac tcggtcgccg catacactat tctcagaatg 420acttggttga gtactcacca
gtcacagaaa agcatcttac ggatggcatg acagtaagag 480aattatgcag tgctgccata
accatgagtg ataacactgc ggccaactta cttctgacaa 540cgatcggagg accgaaggag
ctaaccgctt ttttgcacaa catgggggat catgtaactc 600gccttgatcg ttgggaaccg
gagctgaatg aagccatacc aaacgacgag cgtgacacca 660cgatgcctgt agcaatggca
acaacgttgc gcaaactatt aactggcgaa ctacttactc 720tagcttcccg gcaacaatta
atagactgga tggaggcgga taaagttgca ggaccacttc 780tgcgctcggc ccttccggct
ggctggttta ttgctgataa atctggagcc ggtgagcgtg 840ggtctcgcgg tatcattgca
gcactggggc cagatggtaa gccctcccgt atcgtagtta 900tctacacgac ggggagtcag
gcaactatgg atgaacgaaa tagacagatc gctgagatag 960gtgcctcact gattaagcat
tggtaactgt cagaccaagt ttactcatat atactttaga 1020ttgatttaaa acttcatttt
taatttaaaa ggatctaggt gaagatcctt tttgataatc 1080tcatgaccaa aatcccttaa
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1140agatcaaagg atcttcttga
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1200aaaaaccacc gctaccagcg
gtggtttgtt tgccggatca agagctacca actctttttc 1260cgaaggtaac tggcttcagc
agagcgcaga taccaaatac tgtccttcta gtgtagccgt 1320agttaggcca ccacttcaag
aactctgtag caccgcctac atacctcgct ctgctaatcc 1380tgttaccagt ggctgctgcc
agtggcgata agtcgtgtct taccgggttg gactcaagac 1440gatagttacc ggataaggcg
cagcggtcgg gctgaacggg gggttcgtgc acacagccca 1500gcttggagcg aacgacctac
accgaactga gatacctaca gcgtgagcta tgagaaagcg 1560ccacgcttcc cgaagggaga
aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1620gagagcgcac gagggagctt
ccagggggaa acgcctggta tctttatagt cctgtcgggt 1680ttcgccacct ctgacttgag
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 1740ggaaaaacgc cagcaacgcg
gcctttttac ggttcctggc cttttgctgg ccttttgctc 1800acatgttctt tcctgcgtta
tcccctgatt ctgtggataa ccgtattacc gcctttgagt 1860gagctgatac cgctcgccgc
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 1920cggaagagcg cccaatacgc
aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 1980gctggcacga caggtttccc
gactggaaag cgggcagtga gcgcaacgca attaatgtga 2040gttagctcac tcattaggca
ccccaggctt tacactttat gcttccggct cgtatgttgt 2100gtggaattgt gagcggataa
caatttcaca caggaaacag ctatgaccat gattacgcca 2160agctcgaaat taaccctcac
taaagggaac aaaagctgga gctccaccgc ggtggcggcc 2220gctctagaac tagtggatct
tcttggctgt tattcaaaag gtccaacaat gtatatatat 2280tggacatttt gaggcaatta
tagatcctgg aaggcaattc tgattggtca ataaaaatcg 2340atttcaatgc tatttttttt
ttgtttttta tgagtttagc caatttatca tgaaaggtaa 2400aaggggataa aggaaccgtg
tgttgattgt cctgtaaata taagttgtct tcctccatat 2460gtaaaaaggg aataaataaa
tcaattaaat ttcgggatgc ttcatgaagt gcttctttcg 2520gagttaaact tccgtttgtc
catatttcga gaaaaagtat ctcttgtttt tcattcccat 2580tcccataaga atgaatacta
tgattcgcgt ttcgaacagg catgaataca gcatctatag 2640gataacttcc atcttgaaag
ttatgtggcg tttttataag atatccacga tttctctcta 2700tttgtaatcc aatacaaaaa
tcaattggtt ccgttaaact ggctatatgt tgtgtattat 2760caacgatttc tacataaggc
ggcaagatga tatcttgggc agttacagat ccaggaccct 2820tgacacaaat agatgcgtca
gaagttccat atagattact tcttaatata atttctttca 2880aattcattaa aatttcatgt
accgattctt gaatgcccgt tatggtagaa tattcatgtg 2940ggactttctc agattttaca
cgtgtgatac atgttccttc tatttctcca agtaaagctc 3000ttcgcatcgc aatgcctatt
gtgtcggctt ggcctttcat aagtggagac agaataaagc 3060gtccataata aaggcgttta
ctgtctgttc ttgattcaac acacttccac tgtagtgtcc 3120gagtagatac tgttactttc
tctcgaacca tagtactatt atttgattag atcatcgaat 3180cttttatttc tcttgagatt
tcttcaatgt tcagttctac acacgtcttt ttttcggagg 3240tctacagcca ttatgtggca
taggagttac atcccgtacg aaagttaata gtataccact 3300tcgacgaata gctcgtaatg
ctgcatctct tccgagaccg ggacctttta tcatgacttc 3360tgctcgttgc ataccttgat
ccactactgt acggatagcg tttgctgctg cggtttgagc 3420agcaaacggt gttcctcttc
tcgtaccttt gaatccagaa gtaccggcgg aggaccaaga 3480aactactcga ccccgtacat
ctgtaacagt gacaatggta ttattgaaac ttgcttgaac 3540atgaataact ccctttggta
ttctacgtgc acccttacgt gaaccaatac gtccattcct 3600acgcgaacta attttcggta
tagcttttgc catattttat catctcgtaa atatgagtca 3660gagatatatg gatatatcca
tttcatgtca aaacagattc tttatttgta catcggctct 3720tctggcaagt ctgattatcc
ctgtctttgt ttatgtctcg ggttggaaca aattactata 3780attcgtcccc gcctacggat
tagtcgacat ttttcacaaa ttttacgaac ggaagctctt 3840attttcatat ttctcattcc
ttaccttaat tctgaatcta tttcttggaa gaaaataagt 3900ttcttgaaat ttttcatctc
gaattgtatt cccacgaaag gaatggtgaa gttgaaaaac 3960gaatccttca aatctttgtt
gtggagtcga taaattatac gccctttggt tgaatcataa 4020ggacttactt caattttgac
tctatctcct ggcagtatcc gtataaaact atgccggatc 4080tttcctgaaa cataatttat
aatcagatcg gccgcaggag gagttcatat gtcagagttg 4140agagccttca gtgccccagg
gaaagcgtta ctagctggtg gatatttagt tttagataca 4200aaatatgaag catttgtagt
cggattatcg gcaagaatgc atgctgtagc ccatccttac 4260ggttcattgc aagggtctga
taagtttgaa gtgcgtgtga aaagtaaaca atttaaagat 4320ggggagtggc tgtaccatat
aagtcctaaa agtggcttca ttcctgtttc gataggcgga 4380tctaagaacc ctttcattga
aaaagttatc gctaacgtat ttagctactt taaacctaac 4440atggacgact actgcaatag
aaacttgttc gttattgata ttttctctga tgatgcctac 4500cattctcagg aggatagcgt
taccgaacat cgtggcaaca gaagattgag ttttcattcg 4560cacagaattg aagaagttcc
caaaacaggg ctgggctcct cggcaggttt agtcacagtt 4620ttaactacag ctttggcctc
cttttttgta tcggacctgg aaaataatgt agacaaatat 4680agagaagtta ttcataattt
agcacaagtt gctcattgtc aagctcaggg taaaattgga 4740agcgggtttg atgtagcggc
ggcagcatat ggatctatca gatatagaag attcccaccc 4800gcattaatct ctaatttgcc
agatattgga agtgctactt acggcagtaa actggcgcat 4860ttggttgatg aagaagactg
gaatattacg attaaaagta accatttacc ttcgggatta 4920actttatgga tgggcgatat
taagaatggt tcagaaacag taaaactggt ccagaaggta 4980aaaaattggt atgattcgca
tatgccagaa agcttgaaaa tatatacaga actcgatcat 5040gcaaattcta gatttatgga
tggactatct aaactagatc gcttacacga gactcatgac 5100gattacagcg atcagatatt
tgagtctctt gagaggaatg actgtacctg tcaaaagtat 5160cctgaaatca cagaagttag
agatgcagtt gccacaatta gacgttcctt tagaaaaata 5220actaaagaat ctggtgccga
tatcgaacct cccgtacaaa ctagcttatt ggatgattgc 5280cagaccttaa aaggagttct
tacttgctta atacctggtg ctggtggtta tgacgccatt 5340gcagtgatta ctaagcaaga
tgttgatctt agggctcaaa ccgctaatga caaaagattt 5400tctaaggttc aatggctgga
tgtaactcag gctgactggg gtgttaggaa agaaaaagat 5460ccggaaactt atcttgataa
actgcaggag gagttttaat gtcattaccg ttcttaactt 5520ctgcaccggg aaaggttatt
atttttggtg aacactctgc tgtgtacaac aagcctgccg 5580tcgctgctag tgtgtctgcg
ttgagaacct acctgctaat aagcgagtca tctgcaccag 5640atactattga attggacttc
ccggacatta gctttaatca taagtggtcc atcaatgatt 5700tcaatgccat caccgaggat
caagtaaact cccaaaaatt ggccaaggct caacaagcca 5760ccgatggctt gtctcaggaa
ctcgttagtc ttttggatcc gttgttagct caactatccg 5820aatccttcca ctaccatgca
gcgttttgtt tcctgtatat gtttgtttgc ctatgccccc 5880atgccaagaa tattaagttt
tctttaaagt ctactttacc catcggtgct gggttgggct 5940caagcgcctc tatttctgta
tcactggcct tagctatggc ctacttgggg gggttaatag 6000gatctaatga cttggaaaag
ctgtcagaaa acgataagca tatagtgaat caatgggcct 6060tcataggtga aaagtgtatt
cacggtaccc cttcaggaat agataacgct gtggccactt 6120atggtaatgc cctgctattt
gaaaaagact cacataatgg aacaataaac acaaacaatt 6180ttaagttctt agatgatttc
ccagccattc caatgatcct aacctatact agaattccaa 6240ggtctacaaa agatcttgtt
gctcgcgttc gtgtgttggt caccgagaaa tttcctgaag 6300ttatgaagcc aattctagat
gccatgggtg aatgtgccct acaaggctta gagatcatga 6360ctaagttaag taaatgtaaa
ggcaccgatg acgaggctgt agaaactaat aatgaactgt 6420atgaacaact attggaattg
ataagaataa atcatggact gcttgtctca atcggtgttt 6480ctcatcctgg attagaactt
attaaaaatc tgagcgatga tttgagaatt ggctccacaa 6540aacttaccgg tgctggtggc
ggcggttgct ctttgacttt gttacgaaga gacattactc 6600aagagcaaat tgacagcttc
aaaaagaaat tgcaagatga ttttagttac gagacatttg 6660aaacagactt gggtgggact
ggctgctgtt tgttaagcgc aaaaaatttg aataaagatc 6720ttaaaatcaa atccctagta
ttccaattat ttgaaaataa aactaccaca aagcaacaaa 6780ttgacgatct attattgcca
ggaaacacga atttaccatg gacttcagac gaggagtttt 6840aatgactgta tatactgcta
gtgtaactgc tccggtaaat attgctactc ttaagtattg 6900ggggaaaagg gacacgaagt
tgaatctgcc caccaattcg tccatatcag tgactttatc 6960gcaagatgac ctcagaacgt
tgacctctgc ggctactgca cctgagtttg aacgcgacac 7020tttgtggtta aatggagaac
cacacagcat cgacaatgaa agaactcaaa attgtctgcg 7080cgacctacgc caattaagaa
aggaaatgga atcgaaggac gcctcattgc ccacattatc 7140tcaatggaaa ctccacattg
tctccgaaaa taactttcct acagcagctg gtttagcttc 7200ctccgctgct ggctttgctg
cattggtctc tgcaattgct aagttatacc aattaccaca 7260gtcaacttca gaaatatcta
gaatagcaag aaaggggtct ggttcagctt gtagatcgtt 7320gtttggcgga tacgtggcct
gggaaatggg aaaagctgaa gatggtcatg attccatggc 7380agtacaaatc gcagacagct
ctgactggcc tcagatgaaa gcttgtgtcc tagttgtcag 7440cgatattaaa aaggatgtga
gttccactca gggtatgcaa ttgaccgtgg caacctccga 7500actatttaaa gaaagaattg
aacatgtcgt accaaagaga tttgaagtca tgcgtaaagc 7560cattgttgaa aaagatttcg
ccacctttgc aaaggaaaca atgatggatt ccaactcttt 7620ccatgccaca tgtttggact
ctttccctcc aatattctac atgaatgaca cttccaagcg 7680tatcatcagt tggtgccaca
ccattaatca gttttacgga gaaacaatcg ttgcatacac 7740gtttgatgca ggtccaaatg
ctgtgttgta ctacttagct gaaaatgagt cgaaactctt 7800tgcatttatc tataaattgt
ttggctctgt tcctggatgg gacaagaaat ttactactga 7860gcagcttgag gctttcaacc
atcaatttga atcatctaac tttactgcac gtgaattgga 7920tcttgagttg caaaaggatg
ttgccagagt gattttaact caagtcggtt caggcccaca 7980agaaacaaac gaatctttga
ttgacgcaaa gactggtcta ccaaaggaag aggagtttta 8040actcgacgcc ggcggaggca
catatgtctc agaacgttta cattgtatcg actgccagaa 8100ccccaattgg ttcattccag
ggttctctat cctccaagac agcagtggaa ttgggtgctg 8160ttgctttaaa aggcgccttg
gctaaggttc cagaattgga tgcatccaag gattttgacg 8220aaattatttt tggtaacgtt
ctttctgcca atttgggcca agctccggcc agacaagttg 8280ctttggctgc cggtttgagt
aatcatatcg ttgcaagcac agttaacaag gtctgtgcat 8340ccgctatgaa ggcaatcatt
ttgggtgctc aatccatcaa atgtggtaat gctgatgttg 8400tcgtagctgg tggttgtgaa
tctatgacta acgcaccata ctacatgcca gcagcccgtg 8460cgggtgccaa atttggccaa
actgttcttg ttgatggtgt cgaaagagat gggttgaacg 8520atgcgtacga tggtctagcc
atgggtgtac acgcagaaaa gtgtgcccgt gattgggata 8580ttactagaga acaacaagac
aattttgcca tcgaatccta ccaaaaatct caaaaatctc 8640aaaaggaagg taaattcgac
aatgaaattg tacctgttac cattaaggga tttagaggta 8700agcctgatac tcaagtcacg
aaggacgagg aacctgctag attacacgtt gaaaaattga 8760gatctgcaag gactgttttc
caaaaagaaa acggtactgt tactgccgct aacgcttctc 8820caatcaacga tggtgctgca
gccgtcatct tggtttccga aaaagttttg aaggaaaaga 8880atttgaagcc tttggctatt
atcaaaggtt ggggtgaggc cgctcatcaa ccagctgatt 8940ttacatgggc tccatctctt
gcagttccaa aggctttgaa acatgctggc atcgaagaca 9000tcaattctgt tgattacttt
gaattcaatg aagccttttc ggttgtcggt ttggtgaaca 9060ctaagatttt gaagctagac
ccatctaagg ttaatgtata tggtggtgct gttgctctag 9120gtcacccatt gggttgttct
ggtgctagag tggttgttac actgctatcc atcttacagc 9180aagaaggagg taagatcggt
gttgccgcca tttgtaatgg tggtggtggt gcttcctcta 9240ttgtcattga aaagatatga
ggatcctcta gatgcgcagg aggcacatat ggcgaagaac 9300gttgggattt tggctatgga
tatctatttc cctcccacct gtgttcaaca ggaagctttg 9360gaagcacatg atggagcaag
taaagggaaa tacactattg gacttggcca agattgttta 9420gctttttgca ctgagcttga
agatgttatc tctatgagtt tcaatgcggt gacatcactt 9480tttgagaagt ataagattga
ccctaaccaa atcgggcgtc ttgaagtagg aagtgagact 9540gttattgaca aaagcaagtc
catcaagacc ttcttgatgc agctctttga gaaatgtgga 9600aacactgatg tcgaaggtgt
tgactcgacc aatgcttgct atggtggaac tgcagctttg 9660ttaaactgtg tcaattgggt
tgagagtaac tcttgggatg gacgttatgg cctcgtcatt 9720tgtactgaca gcgcggttta
tgcagaagga cccgcaaggc ccactggagg agctgcagcg 9780attgctatgt tgataggacc
tgatgctcct atcgttttcg aaagcaaatt gagagcaagc 9840cacatggctc atgtctatga
cttttacaag cccaatcttg ctagcgagta cccggttgtt 9900gatggtaagc tttcacagac
ttgctacctc atggctcttg actcctgcta taaacattta 9960tgcaacaagt tcgagaagat
cgagggcaaa gagttctcca taaatgatgc tgattacatt 10020gttttccatt ctccatacaa
taaacttgta cagaaaagct ttgctcgtct cttgtacaac 10080gacttcttga gaaacgcaag
ctccattgac gaggctgcca aagaaaagtt caccccttat 10140tcatctttga cccttgacga
gagttaccaa agccgtgatc ttgaaaaggt gtcacaacaa 10200atttcgaaac cgttttatga
tgctaaagtg caaccaacga ctttaatacc aaaggaagtc 10260ggtaacatgt acactgcttc
tctctacgct gcatttgctt ccctcatcca caataaacac 10320aatgatttgg cgggaaagcg
ggtggttatg ttctcttatg gaagtggctc caccgcaaca 10380atgttctcat tacgcctcaa
cgacaataag cctcctttca gcatttcaaa cattgcatct 10440gtaatggatg ttggcggtaa
attgaaagct agacatgagt atgcacctga gaagtttgtg 10500gagacaatga agctaatgga
acataggtat ggagcaaagg actttgtgac aaccaaggag 10560ggtattatag atcttttggc
accgggaact tattatctga aagaggttga ttccttgtac 10620cggagattct atggcaagaa
aggtgaagat ggatctgtag ccaatggaca ctgaggatcc 10680gtcgagcacg tggaggcaca
tatgcaatgc tgtgagatgc ctgttggata cattcagatt 10740cctgttggga ttgctggtcc
attgttgctt gatggttatg agtactctgt tcctatggct 10800acaaccgaag gttgtttggt
tgctagcact aacagaggct gcaaggctat gtttatctct 10860ggtggcgcca ccagtaccgt
tcttaaggac ggtatgaccc gagcacctgt tgttcggttc 10920gcttcggcga gacgagcttc
ggagcttaag tttttcttgg agaatccaga gaactttgat 10980actttggcag tagtcttcaa
caggtcgagt agatttgcaa gactgcaaag tgttaaatgc 11040acaatcgcgg ggaagaatgc
ttatgtaagg ttctgttgta gtactggtga tgctatgggg 11100atgaatatgg tttctaaagg
tgtgcagaat gttcttgagt atcttaccga tgatttccct 11160gacatggatg tgattggaat
ctctggtaac ttctgttcgg acaagaaacc tgctgctgtg 11220aactggattg agggacgtgg
taaatcagtt gtttgcgagg ctgtaatcag aggagagatc 11280gtgaacaagg tcttgaaaac
gagcgtggct gctttagtcg agctcaacat gctcaagaac 11340ctagctggct ctgctgttgc
aggctctcta ggtggattca acgctcatgc cagtaacata 11400gtgtctgctg tattcatagc
tactggccaa gatccagctc aaaacgtgga gagttctcaa 11460tgcatcacca tgatggaagc
tattaatgac ggcaaagata tccatatctc agtcactatg 11520ccatctatcg aggtggggac
agtgggagga ggaacacagc ttgcatctca atcagcgtgt 11580ttaaacctgc tcggagttaa
aggagcaagc acagagtcgc cgggaatgaa cgcaaggagg 11640ctagcgacga tcgtagccgg
agcagtttta gctggagagt tatctttaat gtcagcaatt 11700gcagctggac agcttgtgag
aagtcacatg aaatacaata gatccagccg agacatctct 11760ggagcaacga caacgacaac
aacaacaaca tgacccggga tccggccgat ctaaacaaac 11820ccggaacaga ccgttgggaa
gcgattcagt aattaaagct tcatgactcc tttttggttc 11880ttaaagtccc tttgaggtat
caactaataa gaaagatatt agacaacccc ccttttttct 11940ttttcacaaa taggaagttt
cgaatccaat ttggatatta aaaggattac cagatataac 12000acaaaatctc tccacctatt
ccttctagtc gagcctctcg gtctgtcatt atacctcgag 12060aagtagaaag aattacaatc
cccattccac ctaaaattcg cggaattcgt tgataattag 12120aatagattcg tagaccaggt
cgactgattc gttttaaatt taaaatattt ctatagggtc 12180ttttcctatt ccttctatgt
cgcagggtta aaaccaaaaa atatttgttt ttttctcgat 12240gttttctcac gttttcgata
aaaccttctc gtaaaagtat ttgaacaata ttttcggtaa 12300tattagtaga tgctattcga
accacccttt ttcgatccat atcagcattt cgtatagaag 12360ttattatctc agcaatagtg
tccctaccca tgatgaacta aaattattgg ggcctccaaa 12420tttgatataa tcaacgtgtt
ttttacttat tttttttttg aatatgatat gaattattaa 12480agatatatgc gtgagacaca
atctactaat taatctattt ctttcaaata ccccactaga 12540aacagatcac aatttcattt
tataatacct cgggagctaa tgaaactatt ttagtaaaat 12600ttaattctct caattcccgg
gcgattgcac caaaaattcg agttcctttt gatttccttc 12660cttcttgatc aataacaact
gcagcattgt catcatatcg tattatcatc ccgttgtcac 12720gtttgagttc tttacaggtc
cgcacaatta cagctctgac tacttctgat ctttctaggg 12780gcatatttgg tacggcttct
ttgatcacag caacaataac gtcaccaata tgagcatatc 12840gacgattgct agctcctatg
attcgaatac acatcaattc tcgagccccg ctgttatccg 12900ctacatttaa atgggtctga
ggttgaatca tttttttaat ccgttctttg aatgcaaagg 12960gcgaagaaaa aaaagaaata
tttttgtcca aaaaaaaaga aacatgcggt ttcgtttcat 13020atctaagagc cctttccgca
tttttttcta ttacattacg aaataatgaa ttgagttcgt 13080ataggcattt tagatgctgc
tagtgaaata gcccttctgg ctatattttc tgttactcca 13140cccatttcat aaagtattcg
acccggttta acaacagcta cccaatattc aggggatccc 13200ccgggctgca ggaattcgat
atcaagctta tcgataccgt cgacctcgag ggggggcccg 13260gtacccaatt cgccctatag
tgagtcgtat tacaattcac tggccgtcgt tttacaacgt 13320cgtgactggg aaaaccctgg
cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 13380gccagctggc gtaatagcga
agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 13440ctgaatggcg aatgggacgc
gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 13500acgcgcagcg tgaccgctac
acttgccagc gccctagcgc ccgctccttt cgctttcttc 13560ccttcctttc tcgccacgtt
cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 13620ttagggttcc gatttagtgc
tttacggcac ctcgacccca aaaaacttga ttagggtgat 13680ggttcacgta gtgggccatc
gccctgatag acggtttttc gccctttgac gttggagtcc 13740acgttcttta atagtggact
cttgttccaa actggaacaa cactcaaccc tatctcggtc 13800tattcttttg atttataagg
gattttgccg atttcggcct attggttaaa aaatgagctg 13860atttaacaaa aatttaacgc
gaattttaac aaaatattaa cgcttacaat ttaggtg 13917737252DNAArtificial
Sequencemisc_feature()..()Plastid transformation vector pHKO7,
containing Operon C, containi 73gcacttttcg gggaaatgtg cgcggaaccc
ctatttgttt atttttctaa atacattcaa 60atatgtatcc gctcatgaga caataaccct
gataaatgct tcaataatat tgaaaaagga 120agagtatgag tattcaacat ttccgtgtcg
cccttattcc cttttttgcg gcattttgcc 180ttcctgtttt tgctcaccca gaaacgctgg
tgaaagtaaa agatgctgaa gatcagttgg 240gtgcacgagt gggttacatc gaactggatc
tcaacagcgg taagatcctt gagagttttc 300gccccgaaga acgttttcca atgatgagca
cttttaaagt tctgctatgt ggcgcggtat 360tatcccgtat tgacgccggg caagagcaac
tcggtcgccg catacactat tctcagaatg 420acttggttga gtactcacca gtcacagaaa
agcatcttac ggatggcatg acagtaagag 480aattatgcag tgctgccata accatgagtg
ataacactgc ggccaactta cttctgacaa 540cgatcggagg accgaaggag ctaaccgctt
ttttgcacaa catgggggat catgtaactc 600gccttgatcg ttgggaaccg gagctgaatg
aagccatacc aaacgacgag cgtgacacca 660cgatgcctgt agcaatggca acaacgttgc
gcaaactatt aactggcgaa ctacttactc 720tagcttcccg gcaacaatta atagactgga
tggaggcgga taaagttgca ggaccacttc 780tgcgctcggc ccttccggct ggctggttta
ttgctgataa atctggagcc ggtgagcgtg 840ggtctcgcgg tatcattgca gcactggggc
cagatggtaa gccctcccgt atcgtagtta 900tctacacgac ggggagtcag gcaactatgg
atgaacgaaa tagacagatc gctgagatag 960gtgcctcact gattaagcat tggtaactgt
cagaccaagt ttactcatat atactttaga 1020ttgatttaaa acttcatttt taatttaaaa
ggatctaggt gaagatcctt tttgataatc 1080tcatgaccaa aatcccttaa cgtgagtttt
cgttccactg agcgtcagac cccgtagaaa 1140agatcaaagg atcttcttga gatccttttt
ttctgcgcgt aatctgctgc ttgcaaacaa 1200aaaaaccacc gctaccagcg gtggtttgtt
tgccggatca agagctacca actctttttc 1260cgaaggtaac tggcttcagc agagcgcaga
taccaaatac tgtccttcta gtgtagccgt 1320agttaggcca ccacttcaag aactctgtag
caccgcctac atacctcgct ctgctaatcc 1380tgttaccagt ggctgctgcc agtggcgata
agtcgtgtct taccgggttg gactcaagac 1440gatagttacc ggataaggcg cagcggtcgg
gctgaacggg gggttcgtgc acacagccca 1500gcttggagcg aacgacctac accgaactga
gatacctaca gcgtgagcta tgagaaagcg 1560ccacgcttcc cgaagggaga aaggcggaca
ggtatccggt aagcggcagg gtcggaacag 1620gagagcgcac gagggagctt ccagggggaa
acgcctggta tctttatagt cctgtcgggt 1680ttcgccacct ctgacttgag cgtcgatttt
tgtgatgctc gtcagggggg cggagcctat 1740ggaaaaacgc cagcaacgcg gcctttttac
ggttcctggc cttttgctgg ccttttgctc 1800acatgttctt tcctgcgtta tcccctgatt
ctgtggataa ccgtattacc gcctttgagt 1860gagctgatac cgctcgccgc agccgaacga
ccgagcgcag cgagtcagtg agcgaggaag 1920cggaagagcg cccaatacgc aaaccgcctc
tccccgcgcg ttggccgatt cattaatgca 1980gctggcacga caggtttccc gactggaaag
cgggcagtga gcgcaacgca attaatgtga 2040gttagctcac tcattaggca ccccaggctt
tacactttat gcttccggct cgtatgttgt 2100gtggaattgt gagcggataa caatttcaca
caggaaacag ctatgaccat gattacgcca 2160agctcgaaat taaccctcac taaagggaac
aaaagctgga gctccaccgc ggtggcggcc 2220gctctagaac tagtggatct tcttggctgt
tattcaaaag gtccaacaat gtatatatat 2280tggacatttt gaggcaatta tagatcctgg
aaggcaattc tgattggtca ataaaaatcg 2340atttcaatgc tatttttttt ttgtttttta
tgagtttagc caatttatca tgaaaggtaa 2400aaggggataa aggaaccgtg tgttgattgt
cctgtaaata taagttgtct tcctccatat 2460gtaaaaaggg aataaataaa tcaattaaat
ttcgggatgc ttcatgaagt gcttctttcg 2520gagttaaact tccgtttgtc catatttcga
gaaaaagtat ctcttgtttt tcattcccat 2580tcccataaga atgaatacta tgattcgcgt
ttcgaacagg catgaataca gcatctatag 2640gataacttcc atcttgaaag ttatgtggcg
tttttataag atatccacga tttctctcta 2700tttgtaatcc aatacaaaaa tcaattggtt
ccgttaaact ggctatatgt tgtgtattat 2760caacgatttc tacataaggc ggcaagatga
tatcttgggc agttacagat ccaggaccct 2820tgacacaaat agatgcgtca gaagttccat
atagattact tcttaatata atttctttca 2880aattcattaa aatttcatgt accgattctt
gaatgcccgt tatggtagaa tattcatgtg 2940ggactttctc agattttaca cgtgtgatac
atgttccttc tatttctcca agtaaagctc 3000ttcgcatcgc aatgcctatt gtgtcggctt
ggcctttcat aagtggagac agaataaagc 3060gtccataata aaggcgttta ctgtctgttc
ttgattcaac acacttccac tgtagtgtcc 3120gagtagatac tgttactttc tctcgaacca
tagtactatt atttgattag atcatcgaat 3180cttttatttc tcttgagatt tcttcaatgt
tcagttctac acacgtcttt ttttcggagg 3240tctacagcca ttatgtggca taggagttac
atcccgtacg aaagttaata gtataccact 3300tcgacgaata gctcgtaatg ctgcatctct
tccgagaccg ggacctttta tcatgacttc 3360tgctcgttgc ataccttgat ccactactgt
acggatagcg tttgctgctg cggtttgagc 3420agcaaacggt gttcctcttc tcgtaccttt
gaatccagaa gtaccggcgg aggaccaaga 3480aactactcga ccccgtacat ctgtaacagt
gacaatggta ttattgaaac ttgcttgaac 3540atgaataact ccctttggta ttctacgtgc
acccttacgt gaaccaatac gtccattcct 3600acgcgaacta attttcggta tagcttttgc
catattttat catctcgtaa atatgagtca 3660gagatatatg gatatatcca tttcatgtca
aaacagattc tttatttgta catcggctct 3720tctggcaagt ctgattatcc ctgtctttgt
ttatgtctcg ggttggaaca aattactata 3780attcgtcccc gcctacggat tagtcgacat
ttttcacaaa ttttacgaac ggaagctctt 3840attttcatat ttctcattcc ttaccttaat
tctgaatcta tttcttggaa gaaaataagt 3900ttcttgaaat ttttcatctc gaattgtatt
cccacgaaag gaatggtgaa gttgaaaaac 3960gaatccttca aatctttgtt gtggagtcga
taaattatac gccctttggt tgaatcataa 4020ggacttactt caattttgac tctatctcct
ggcagtatcc gtataaaact atgccggatc 4080tttcctgaaa cataatttat aatcagatcc
aggaggacca tatgatcgcc gaagcggata 4140tggaggtctg ccgggagctg atccgcaccg
gcagctactc cttccatgcg gcgtccagag 4200ttctgccggc gcgggtccgt gaccccgcgc
tggcgcttta cgccttttgc cgcgtcgccg 4260atgacgaagt cgacgaggtt ggcgcgccgc
gcgacaaggc tgcggcggtt ttgaaacttg 4320gcgaccggct ggaggacatc tatgccggtc
gtccgcgcaa tgcgccctcg gatcgggctt 4380tcgcggcggt ggtcgaggaa ttcgagatgc
cgcgcgaatt gcccgaggcg ctgctggagg 4440gcttcgcctg ggatgccgag gggcggtggt
atcacacgct ttcggacgtg caggcctatt 4500cggcgcgggt ggcggccgcc gtcggcgcga
tgatgtgcgt gctgatgcgg gtgcgcaacc 4560ccgatgcgct ggcgcgggcc tgcgatctcg
gtcttgccat gcagatgtcg aacatcgccc 4620gcgacgtggg cgaggatgcc cgggcggggc
ggcttttcct gccgaccgac tggatggtcg 4680aggaggggat cgatccgcag gcgttcctgg
ccgatccgca gcccaccaag ggcatccgcc 4740gggtcaccga gcggttgctg aaccgcgccg
accggcttta ctggcgggcg gcgacggggg 4800tgcggctttt gccctttgac tgccgaccgg
ggatcatggc cgcgggcaag atctatgccg 4860cgatcggggc cgaggtggcg aaggcgaaat
acgacaacat cacccggcgt gcccacacga 4920ccaagggccg caagctgtgg ctggtggcga
attccgcgat gtcggcgacg gcgacctcga 4980tgctgccgct ctcgccgcgg gtgcatgcca
agcccgagcc cgaagtggcg catctggtcg 5040atgccgccgc gcatcgcaac ctgcatcccg
aacggtccga ggtgctgatc tcggcgctga 5100tggcgctgaa ggcgcgcgac cgcggcctgg
cgatggattg aggatctaaa caaacccgga 5160acagaccgtt gggaagcgat tcagtaatta
aagcttcatg actccttttt ggttcttaaa 5220gtccctttga ggtatcaact aataagaaag
atattagaca accccccttt tttctttttc 5280acaaatagga agtttcgaat ccaatttgga
tattaaaagg attaccagat ataacacaaa 5340atctctccac ctattccttc tagtcgagcc
tctcggtctg tcattatacc tcgagaagta 5400gaaagaatta caatccccat tccacctaaa
attcgcggaa ttcgttgata attagaatag 5460attcgtagac caggtcgact gattcgtttt
aaatttaaaa tatttctata gggtcttttc 5520ctattccttc tatgtcgcag ggttaaaacc
aaaaaatatt tgtttttttc tcgatgtttt 5580ctcacgtttt cgataaaacc ttctcgtaaa
agtatttgaa caatattttc ggtaatatta 5640gtagatgcta ttcgaaccac cctttttcga
tccatatcag catttcgtat agaagttatt 5700atctcagcaa tagtgtccct acccatgatg
aactaaaatt attggggcct ccaaatttga 5760tataatcaac gtgtttttta cttatttttt
ttttgaatat gatatgaatt attaaagata 5820tatgcgtgag acacaatcta ctaattaatc
tatttctttc aaatacccca ctagaaacag 5880atcacaattt cattttataa tacctcggga
gctaatgaaa ctattttagt aaaatttaat 5940tctctcaatt cccgggcgat tgcaccaaaa
attcgagttc cttttgattt ccttccttct 6000tgatcaataa caactgcagc attgtcatca
tatcgtatta tcatcccgtt gtcacgtttg 6060agttctttac aggtccgcac aattacagct
ctgactactt ctgatctttc taggggcata 6120tttggtacgg cttctttgat cacagcaaca
ataacgtcac caatatgagc atatcgacga 6180ttgctagctc ctatgattcg aatacacatc
aattctcgag ccccgctgtt atccgctaca 6240tttaaatggg tctgaggttg aatcattttt
ttaatccgtt ctttgaatgc aaagggcgaa 6300gaaaaaaaag aaatattttt gtccaaaaaa
aaagaaacat gcggtttcgt ttcatatcta 6360agagcccttt ccgcattttt ttctattaca
ttacgaaata atgaattgag ttcgtatagg 6420cattttagat gctgctagtg aaatagccct
tctggctata ttttctgtta ctccacccat 6480ttcataaagt attcgacccg gtttaacaac
agctacccaa tattcagggg atcccccggg 6540ctgcaggaat tcgatatcaa gcttatcgat
accgtcgacc tcgagggggg gcccggtacc 6600caattcgccc tatagtgagt cgtattacaa
ttcactggcc gtcgttttac aacgtcgtga 6660ctgggaaaac cctggcgtta cccaacttaa
tcgccttgca gcacatcccc ctttcgccag 6720ctggcgtaat agcgaagagg cccgcaccga
tcgcccttcc caacagttgc gcagcctgaa 6780tggcgaatgg gacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg 6840cagcgtgacc gctacacttg ccagcgccct
agcgcccgct cctttcgctt tcttcccttc 6900ctttctcgcc acgttcgccg gctttccccg
tcaagctcta aatcgggggc tccctttagg 6960gttccgattt agtgctttac ggcacctcga
ccccaaaaaa cttgattagg gtgatggttc 7020acgtagtggg ccatcgccct gatagacggt
ttttcgccct ttgacgttgg agtccacgtt 7080ctttaatagt ggactcttgt tccaaactgg
aacaacactc aaccctatct cggtctattc 7140ttttgattta taagggattt tgccgatttc
ggcctattgg ttaaaaaatg agctgattta 7200acaaaaattt aacgcgaatt ttaacaaaat
attaacgctt acaatttagg tg 72527414623DNAArtificial
Sequencemisc_feature()..()Plastic transformation vector pHKO8,
containing Operon G, containi 74cacctaaatt gtaagcgtta atattttgtt
aaaattcgcg ttaaattttt gttaaatcag 60ctcatttttt aaccaatagg ccgaaatcgg
caaaatccct tataaatcaa aagaatagac 120cgagataggg ttgagtgttg ttccagtttg
gaacaagagt ccactattaa agaacgtgga 180ctccaacgtc aaagggcgaa aaaccgtcta
tcagggcgat ggcccactac gtgaaccatc 240accctaatca agttttttgg ggtcgaggtg
ccgtaaagca ctaaatcgga accctaaagg 300gagcccccga tttagagctt gacggggaaa
gccggcgaac gtggcgagaa aggaagggaa 360gaaagcgaaa ggagcgggcg ctagggcgct
ggcaagtgta gcggtcacgc tgcgcgtaac 420caccacaccc gccgcgctta atgcgccgct
acagggcgcg tcccattcgc cattcaggct 480gcgcaactgt tgggaagggc gatcggtgcg
ggcctcttcg ctattacgcc agctggcgaa 540agggggatgt gctgcaaggc gattaagttg
ggtaacgcca gggttttccc agtcacgacg 600ttgtaaaacg acggccagtg aattgtaata
cgactcacta tagggcgaat tgggtaccgg 660gccccccctc gaggtcgacg gtatcgataa
gcttgatatc gaattcctgc agcccggggg 720atcttcttgg ctgttattca aaaggtccaa
caatgtatat atattggaca ttttgaggca 780attatagatc ctggaaggca attctgattg
gtcaataaaa atcgatttca atgctatttt 840ttttttgttt tttatgagtt tagccaattt
atcatgaaag gtaaaagggg ataaaggaac 900cgtgtgttga ttgtcctgta aatataagtt
gtcttcctcc atatgtaaaa agggaataaa 960taaatcaatt aaatttcggg atgcttcatg
aagtgcttct ttcggagtta aacttccgtt 1020tgtccatatt tcgagaaaaa gtatctcttg
tttttcattc ccattcccat aagaatgaat 1080actatgattc gcgtttcgaa caggcatgaa
tacagcatct ataggataac ttccatcttg 1140aaagttatgt ggcgttttta taagatatcc
acgatttctc tctatttgta atccaataca 1200aaaatcaatt ggttccgtta aactggctat
atgttgtgta ttatcaacga tttctacata 1260aggcggcaag atgatatctt gggcagttac
agatccagga cccttgacac aaatagatgc 1320gtcagaagtt ccatatagat tacttcttaa
tataatttct ttcaaattca ttaaaatttc 1380atgtaccgat tcttgaatgc ccgttatggt
agaatattca tgtgggactt tctcagattt 1440tacacgtgtg atacatgttc cttctatttc
tccaagtaaa gctcttcgca tcgcaatgcc 1500tattgtgtcg gcttggcctt tcataagtgg
agacagaata aagcgtccat aataaaggcg 1560tttactgtct gttcttgatt caacacactt
ccactgtagt gtccgagtag atactgttac 1620tttctctcga accatagtac tattatttga
ttagatcatc gaatctttta tttctcttga 1680gatttcttca atgttcagtt ctacacacgt
ctttttttcg gaggtctaca gccattatgt 1740ggcataggag ttacatcccg tacgaaagtt
aatagtatac cacttcgacg aatagctcgt 1800aatgctgcat ctcttccgag accgggacct
tttatcatga cttctgctcg ttgcatacct 1860tgatccacta ctgtacggat agcgtttgct
gctgcggttt gagcagcaaa cggtgttcct 1920cttctcgtac ctttgaatcc agaagtaccg
gcggaggacc aagaaactac tcgaccccgt 1980acatctgtaa cagtgacaat ggtattattg
aaacttgctt gaacatgaat aactcccttt 2040ggtattctac gtgcaccctt acgtgaacca
atacgtccat tcctacgcga actaattttc 2100ggtatagctt ttgccatatt ttatcatctc
gtaaatatga gtcagagata tatggatata 2160tccatttcat gtcaaaacag attctttatt
tgtacatcgg ctcttctggc aagtctgatt 2220atccctgtct ttgtttatgt ctcgggttgg
aacaaattac tataattcgt ccccgcctac 2280ggattagtcg acatttttca caaattttac
gaacggaagc tcttattttc atatttctca 2340ttccttacct taattctgaa tctatttctt
ggaagaaaat aagtttcttg aaatttttca 2400tctcgaattg tattcccacg aaaggaatgg
tgaagttgaa aaacgaatcc ttcaaatctt 2460tgttgtggag tcgataaatt atacgccctt
tggttgaatc ataaggactt acttcaattt 2520tgactctatc tcctggcagt atccgtataa
aactatgccg gatctttcct gaaacataat 2580ttataatcag atcggccgca ggaggagttc
atatgtcaga gttgagagcc ttcagtgccc 2640cagggaaagc gttactagct ggtggatatt
tagttttaga tacaaaatat gaagcatttg 2700tagtcggatt atcggcaaga atgcatgctg
tagcccatcc ttacggttca ttgcaagggt 2760ctgataagtt tgaagtgcgt gtgaaaagta
aacaatttaa agatggggag tggctgtacc 2820atataagtcc taaaagtggc ttcattcctg
tttcgatagg cggatctaag aaccctttca 2880ttgaaaaagt tatcgctaac gtatttagct
actttaaacc taacatggac gactactgca 2940atagaaactt gttcgttatt gatattttct
ctgatgatgc ctaccattct caggaggata 3000gcgttaccga acatcgtggc aacagaagat
tgagttttca ttcgcacaga attgaagaag 3060ttcccaaaac agggctgggc tcctcggcag
gtttagtcac agttttaact acagctttgg 3120cctccttttt tgtatcggac ctggaaaata
atgtagacaa atatagagaa gttattcata 3180atttagcaca agttgctcat tgtcaagctc
agggtaaaat tggaagcggg tttgatgtag 3240cggcggcagc atatggatct atcagatata
gaagattccc acccgcatta atctctaatt 3300tgccagatat tggaagtgct acttacggca
gtaaactggc gcatttggtt gatgaagaag 3360actggaatat tacgattaaa agtaaccatt
taccttcggg attaacttta tggatgggcg 3420atattaagaa tggttcagaa acagtaaaac
tggtccagaa ggtaaaaaat tggtatgatt 3480cgcatatgcc agaaagcttg aaaatatata
cagaactcga tcatgcaaat tctagattta 3540tggatggact atctaaacta gatcgcttac
acgagactca tgacgattac agcgatcaga 3600tatttgagtc tcttgagagg aatgactgta
cctgtcaaaa gtatcctgaa atcacagaag 3660ttagagatgc agttgccaca attagacgtt
cctttagaaa aataactaaa gaatctggtg 3720ccgatatcga acctcccgta caaactagct
tattggatga ttgccagacc ttaaaaggag 3780ttcttacttg cttaatacct ggtgctggtg
gttatgacgc cattgcagtg attactaagc 3840aagatgttga tcttagggct caaaccgcta
atgacaaaag attttctaag gttcaatggc 3900tggatgtaac tcaggctgac tggggtgtta
ggaaagaaaa agatccggaa acttatcttg 3960ataaactgca ggaggagttt taatgtcatt
accgttctta acttctgcac cgggaaaggt 4020tattattttt ggtgaacact ctgctgtgta
caacaagcct gccgtcgctg ctagtgtgtc 4080tgcgttgaga acctacctgc taataagcga
gtcatctgca ccagatacta ttgaattgga 4140cttcccggac attagcttta atcataagtg
gtccatcaat gatttcaatg ccatcaccga 4200ggatcaagta aactcccaaa aattggccaa
ggctcaacaa gccaccgatg gcttgtctca 4260ggaactcgtt agtcttttgg atccgttgtt
agctcaacta tccgaatcct tccactacca 4320tgcagcgttt tgtttcctgt atatgtttgt
ttgcctatgc ccccatgcca agaatattaa 4380gttttcttta aagtctactt tacccatcgg
tgctgggttg ggctcaagcg cctctatttc 4440tgtatcactg gccttagcta tggcctactt
gggggggtta ataggatcta atgacttgga 4500aaagctgtca gaaaacgata agcatatagt
gaatcaatgg gccttcatag gtgaaaagtg 4560tattcacggt accccttcag gaatagataa
cgctgtggcc acttatggta atgccctgct 4620atttgaaaaa gactcacata atggaacaat
aaacacaaac aattttaagt tcttagatga 4680tttcccagcc attccaatga tcctaaccta
tactagaatt ccaaggtcta caaaagatct 4740tgttgctcgc gttcgtgtgt tggtcaccga
gaaatttcct gaagttatga agccaattct 4800agatgccatg ggtgaatgtg ccctacaagg
cttagagatc atgactaagt taagtaaatg 4860taaaggcacc gatgacgagg ctgtagaaac
taataatgaa ctgtatgaac aactattgga 4920attgataaga ataaatcatg gactgcttgt
ctcaatcggt gtttctcatc ctggattaga 4980acttattaaa aatctgagcg atgatttgag
aattggctcc acaaaactta ccggtgctgg 5040tggcggcggt tgctctttga ctttgttacg
aagagacatt actcaagagc aaattgacag 5100cttcaaaaag aaattgcaag atgattttag
ttacgagaca tttgaaacag acttgggtgg 5160gactggctgc tgtttgttaa gcgcaaaaaa
tttgaataaa gatcttaaaa tcaaatccct 5220agtattccaa ttatttgaaa ataaaactac
cacaaagcaa caaattgacg atctattatt 5280gccaggaaac acgaatttac catggacttc
agacgaggag ttttaatgac tgtatatact 5340gctagtgtaa ctgctccggt aaatattgct
actcttaagt attgggggaa aagggacacg 5400aagttgaatc tgcccaccaa ttcgtccata
tcagtgactt tatcgcaaga tgacctcaga 5460acgttgacct ctgcggctac tgcacctgag
tttgaacgcg acactttgtg gttaaatgga 5520gaaccacaca gcatcgacaa tgaaagaact
caaaattgtc tgcgcgacct acgccaatta 5580agaaaggaaa tggaatcgaa ggacgcctca
ttgcccacat tatctcaatg gaaactccac 5640attgtctccg aaaataactt tcctacagca
gctggtttag cttcctccgc tgctggcttt 5700gctgcattgg tctctgcaat tgctaagtta
taccaattac cacagtcaac ttcagaaata 5760tctagaatag caagaaaggg gtctggttca
gcttgtagat cgttgtttgg cggatacgtg 5820gcctgggaaa tgggaaaagc tgaagatggt
catgattcca tggcagtaca aatcgcagac 5880agctctgact ggcctcagat gaaagcttgt
gtcctagttg tcagcgatat taaaaaggat 5940gtgagttcca ctcagggtat gcaattgacc
gtggcaacct ccgaactatt taaagaaaga 6000attgaacatg tcgtaccaaa gagatttgaa
gtcatgcgta aagccattgt tgaaaaagat 6060ttcgccacct ttgcaaagga aacaatgatg
gattccaact ctttccatgc cacatgtttg 6120gactctttcc ctccaatatt ctacatgaat
gacacttcca agcgtatcat cagttggtgc 6180cacaccatta atcagtttta cggagaaaca
atcgttgcat acacgtttga tgcaggtcca 6240aatgctgtgt tgtactactt agctgaaaat
gagtcgaaac tctttgcatt tatctataaa 6300ttgtttggct ctgttcctgg atgggacaag
aaatttacta ctgagcagct tgaggctttc 6360aaccatcaat ttgaatcatc taactttact
gcacgtgaat tggatcttga gttgcaaaag 6420gatgttgcca gagtgatttt aactcaagtc
ggttcaggcc cacaagaaac aaacgaatct 6480ttgattgacg caaagactgg tctaccaaag
gaagaggagt tttaactcga cgccggcgga 6540ggcacatatg tctcagaacg tttacattgt
atcgactgcc agaaccccaa ttggttcatt 6600ccagggttct ctatcctcca agacagcagt
ggaattgggt gctgttgctt taaaaggcgc 6660cttggctaag gttccagaat tggatgcatc
caaggatttt gacgaaatta tttttggtaa 6720cgttctttct gccaatttgg gccaagctcc
ggccagacaa gttgctttgg ctgccggttt 6780gagtaatcat atcgttgcaa gcacagttaa
caaggtctgt gcatccgcta tgaaggcaat 6840cattttgggt gctcaatcca tcaaatgtgg
taatgctgat gttgtcgtag ctggtggttg 6900tgaatctatg actaacgcac catactacat
gccagcagcc cgtgcgggtg ccaaatttgg 6960ccaaactgtt cttgttgatg gtgtcgaaag
agatgggttg aacgatgcgt acgatggtct 7020agccatgggt gtacacgcag aaaagtgtgc
ccgtgattgg gatattacta gagaacaaca 7080agacaatttt gccatcgaat cctaccaaaa
atctcaaaaa tctcaaaagg aaggtaaatt 7140cgacaatgaa attgtacctg ttaccattaa
gggatttaga ggtaagcctg atactcaagt 7200cacgaaggac gaggaacctg ctagattaca
cgttgaaaaa ttgagatctg caaggactgt 7260tttccaaaaa gaaaacggta ctgttactgc
cgctaacgct tctccaatca acgatggtgc 7320tgcagccgtc atcttggttt ccgaaaaagt
tttgaaggaa aagaatttga agcctttggc 7380tattatcaaa ggttggggtg aggccgctca
tcaaccagct gattttacat gggctccatc 7440tcttgcagtt ccaaaggctt tgaaacatgc
tggcatcgaa gacatcaatt ctgttgatta 7500ctttgaattc aatgaagcct tttcggttgt
cggtttggtg aacactaaga ttttgaagct 7560agacccatct aaggttaatg tatatggtgg
tgctgttgct ctaggtcacc cattgggttg 7620ttctggtgct agagtggttg ttacactgct
atccatctta cagcaagaag gaggtaagat 7680cggtgttgcc gccatttgta atggtggtgg
tggtgcttcc tctattgtca ttgaaaagat 7740atgaggatcc tctagatgcg caggaggcac
atatggcgaa gaacgttggg attttggcta 7800tggatatcta tttccctccc acctgtgttc
aacaggaagc tttggaagca catgatggag 7860caagtaaagg gaaatacact attggacttg
gccaagattg tttagctttt tgcactgagc 7920ttgaagatgt tatctctatg agtttcaatg
cggtgacatc actttttgag aagtataaga 7980ttgaccctaa ccaaatcggg cgtcttgaag
taggaagtga gactgttatt gacaaaagca 8040agtccatcaa gaccttcttg atgcagctct
ttgagaaatg tggaaacact gatgtcgaag 8100gtgttgactc gaccaatgct tgctatggtg
gaactgcagc tttgttaaac tgtgtcaatt 8160gggttgagag taactcttgg gatggacgtt
atggcctcgt catttgtact gacagcgcgg 8220tttatgcaga aggacccgca aggcccactg
gaggagctgc agcgattgct atgttgatag 8280gacctgatgc tcctatcgtt ttcgaaagca
aattgagagc aagccacatg gctcatgtct 8340atgactttta caagcccaat cttgctagcg
agtacccggt tgttgatggt aagctttcac 8400agacttgcta cctcatggct cttgactcct
gctataaaca tttatgcaac aagttcgaga 8460agatcgaggg caaagagttc tccataaatg
atgctgatta cattgttttc cattctccat 8520acaataaact tgtacagaaa agctttgctc
gtctcttgta caacgacttc ttgagaaacg 8580caagctccat tgacgaggct gccaaagaaa
agttcacccc ttattcatct ttgacccttg 8640acgagagtta ccaaagccgt gatcttgaaa
aggtgtcaca acaaatttcg aaaccgtttt 8700atgatgctaa agtgcaacca acgactttaa
taccaaagga agtcggtaac atgtacactg 8760cttctctcta cgctgcattt gcttccctca
tccacaataa acacaatgat ttggcgggaa 8820agcgggtggt tatgttctct tatggaagtg
gctccaccgc aacaatgttc tcattacgcc 8880tcaacgacaa taagcctcct ttcagcattt
caaacattgc atctgtaatg gatgttggcg 8940gtaaattgaa agctagacat gagtatgcac
ctgagaagtt tgtggagaca atgaagctaa 9000tggaacatag gtatggagca aaggactttg
tgacaaccaa ggagggtatt atagatcttt 9060tggcaccggg aacttattat ctgaaagagg
ttgattcctt gtaccggaga ttctatggca 9120agaaaggtga agatggatct gtagccaatg
gacactgagg atccgtcgag cacgtggagg 9180cacatatgca atgctgtgag atgcctgttg
gatacattca gattcctgtt gggattgctg 9240gtccattgtt gcttgatggt tatgagtact
ctgttcctat ggctacaacc gaaggttgtt 9300tggttgctag cactaacaga ggctgcaagg
ctatgtttat ctctggtggc gccaccagta 9360ccgttcttaa ggacggtatg acccgagcac
ctgttgttcg gttcgcttcg gcgagacgag 9420cttcggagct taagtttttc ttggagaatc
cagagaactt tgatactttg gcagtagtct 9480tcaacaggtc gagtagattt gcaagactgc
aaagtgttaa atgcacaatc gcggggaaga 9540atgcttatgt aaggttctgt tgtagtactg
gtgatgctat ggggatgaat atggtttcta 9600aaggtgtgca gaatgttctt gagtatctta
ccgatgattt ccctgacatg gatgtgattg 9660gaatctctgg taacttctgt tcggacaaga
aacctgctgc tgtgaactgg attgagggac 9720gtggtaaatc agttgtttgc gaggctgtaa
tcagaggaga gatcgtgaac aaggtcttga 9780aaacgagcgt ggctgcttta gtcgagctca
acatgctcaa gaacctagct ggctctgctg 9840ttgcaggctc tctaggtgga ttcaacgctc
atgccagtaa catagtgtct gctgtattca 9900tagctactgg ccaagatcca gctcaaaacg
tggagagttc tcaatgcatc accatgatgg 9960aagctattaa tgacggcaaa gatatccata
tctcagtcac tatgccatct atcgaggtgg 10020ggacagtggg aggaggaaca cagcttgcat
ctcaatcagc gtgtttaaac ctgctcggag 10080ttaaaggagc aagcacagag tcgccgggaa
tgaacgcaag gaggctagcg acgatcgtag 10140ccggagcagt tttagctgga gagttatctt
taatgtcagc aattgcagct ggacagcttg 10200tgagaagtca catgaaatac aatagatcca
gccgagacat ctctggagca acgacaacga 10260caacaacaac aacatgaccc gtaggaggca
catatgagtt cccaacaaga gaaaaaggat 10320tatgatgaag aacaattaag gttgatggaa
gaagtttgta tcgttgtaga tgaaaatgat 10380gtccctttaa gatatggaac gaaaaaggag
tgtcatttga tggaaaatat aaataaaggt 10440cttttgcata gagcattctc tatgttcatc
tttgatgagc aaaatcgcct tttacttcag 10500cagcgtgcag aagagaaaat tacatttcca
tccttatgga cgaatacatg ttgctcccac 10560ccattggatg ttgctggtga acgtggtaat
actttacctg aagctgttga aggtgttaag 10620aatgcagctc aacgcaagct gttccatgaa
ttgggtattc aagccaagta tattcccaaa 10680gacaaatttc agtttcttac acgaatccat
taccttgctc ctagtactgg tgcttgggga 10740gagcatgaaa ttgactacat tcttttcttc
aaaggtaaag ttgagctgga tatcaatccc 10800aatgaagttc aagcctataa gtatgttact
atggaagagt taaaagagat gttttccgat 10860cctcaatatg gattcacacc atggttcaaa
cttatttgtg agcattttat gtttaaatgg 10920tggcaggatg tagatcatgc gtcaaaattc
caagatacct taattcatcg ttgctaagga 10980tcccccggga tccggccgat ctaaacaaac
ccggaacaga ccgttgggaa gcgattcagt 11040aattaaagct tcatgactcc tttttggttc
ttaaagtccc tttgaggtat caactaataa 11100gaaagatatt agacaacccc ccttttttct
ttttcacaaa taggaagttt cgaatccaat 11160ttggatatta aaaggattac cagatataac
acaaaatctc tccacctatt ccttctagtc 11220gagcctctcg gtctgtcatt atacctcgag
aagtagaaag aattacaatc cccattccac 11280ctaaaattcg cggaattcgt tgataattag
aatagattcg tagaccaggt cgactgattc 11340gttttaaatt taaaatattt ctatagggtc
ttttcctatt ccttctatgt cgcagggtta 11400aaaccaaaaa atatttgttt ttttctcgat
gttttctcac gttttcgata aaaccttctc 11460gtaaaagtat ttgaacaata ttttcggtaa
tattagtaga tgctattcga accacccttt 11520ttcgatccat atcagcattt cgtatagaag
ttattatctc agcaatagtg tccctaccca 11580tgatgaacta aaattattgg ggcctccaaa
tttgatataa tcaacgtgtt ttttacttat 11640tttttttttg aatatgatat gaattattaa
agatatatgc gtgagacaca atctactaat 11700taatctattt ctttcaaata ccccactaga
aacagatcac aatttcattt tataatacct 11760cgggagctaa tgaaactatt ttagtaaaat
ttaattctct caattcccgg gcgattgcac 11820caaaaattcg agttcctttt gatttccttc
cttcttgatc aataacaact gcagcattgt 11880catcatatcg tattatcatc ccgttgtcac
gtttgagttc tttacaggtc cgcacaatta 11940cagctctgac tacttctgat ctttctaggg
gcatatttgg tacggcttct ttgatcacag 12000caacaataac gtcaccaata tgagcatatc
gacgattgct agctcctatg attcgaatac 12060acatcaattc tcgagccccg ctgttatccg
ctacatttaa atgggtctga ggttgaatca 12120tttttttaat ccgttctttg aatgcaaagg
gcgaagaaaa aaaagaaata tttttgtcca 12180aaaaaaaaga aacatgcggt ttcgtttcat
atctaagagc cctttccgca tttttttcta 12240ttacattacg aaataatgaa ttgagttcgt
ataggcattt tagatgctgc tagtgaaata 12300gcccttctgg ctatattttc tgttactcca
cccatttcat aaagtattcg acccggttta 12360acaacagcta cccaatattc aggggatcca
ctagttctag agcggccgcc accgcggtgg 12420agctccagct tttgttccct ttagtgaggg
ttaatttcga gcttggcgta atcatggtca 12480tagctgtttc ctgtgtgaaa ttgttatccg
ctcacaattc cacacaacat acgagccgga 12540agcataaagt gtaaagcctg gggtgcctaa
tgagtgagct aactcacatt aattgcgttg 12600cgctcactgc ccgctttcca gtcgggaaac
ctgtcgtgcc agctgcatta atgaatcggc 12660caacgcgcgg ggagaggcgg tttgcgtatt
gggcgctctt ccgcttcctc gctcactgac 12720tcgctgcgct cggtcgttcg gctgcggcga
gcggtatcag ctcactcaaa ggcggtaata 12780cggttatcca cagaatcagg ggataacgca
ggaaagaaca tgtgagcaaa aggccagcaa 12840aaggccagga accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct ccgcccccct 12900gacgagcatc acaaaaatcg acgctcaagt
cagaggtggc gaaacccgac aggactataa 12960agataccagg cgtttccccc tggaagctcc
ctcgtgcgct ctcctgttcc gaccctgccg 13020cttaccggat acctgtccgc ctttctccct
tcgggaagcg tggcgctttc tcatagctca 13080cgctgtaggt atctcagttc ggtgtaggtc
gttcgctcca agctgggctg tgtgcacgaa 13140ccccccgttc agcccgaccg ctgcgcctta
tccggtaact atcgtcttga gtccaacccg 13200gtaagacacg acttatcgcc actggcagca
gccactggta acaggattag cagagcgagg 13260tatgtaggcg gtgctacaga gttcttgaag
tggtggccta actacggcta cactagaagg 13320acagtatttg gtatctgcgc tctgctgaag
ccagttacct tcggaaaaag agttggtagc 13380tcttgatccg gcaaacaaac caccgctggt
agcggtggtt tttttgtttg caagcagcag 13440attacgcgca gaaaaaaagg atctcaagaa
gatcctttga tcttttctac ggggtctgac 13500gctcagtgga acgaaaactc acgttaaggg
attttggtca tgagattatc aaaaaggatc 13560ttcacctaga tccttttaaa ttaaaaatga
agttttaaat caatctaaag tatatatgag 13620taaacttggt ctgacagtta ccaatgctta
atcagtgagg cacctatctc agcgatctgt 13680ctatttcgtt catccatagt tgcctgactc
cccgtcgtgt agataactac gatacgggag 13740ggcttaccat ctggccccag tgctgcaatg
ataccgcgag acccacgctc accggctcca 13800gatttatcag caataaacca gccagccgga
agggccgagc gcagaagtgg tcctgcaact 13860ttatccgcct ccatccagtc tattaattgt
tgccgggaag ctagagtaag tagttcgcca 13920gttaatagtt tgcgcaacgt tgttgccatt
gctacaggca tcgtggtgtc acgctcgtcg 13980tttggtatgg cttcattcag ctccggttcc
caacgatcaa ggcgagttac atgatccccc 14040atgttgtgca aaaaagcggt tagctccttc
ggtcctccga tcgttgtcag aagtaagttg 14100gccgcagtgt tatcactcat ggttatggca
gcactgcata attctcttac tgtcatgcca 14160tccgtaagat gcttttctgt gactggtgag
tactcaacca agtcattctg agaatagtgt 14220atgcggcgac cgagttgctc ttgcccggcg
tcaatacggg ataataccgc gccacatagc 14280agaactttaa aagtgctcat cattggaaaa
cgttcttcgg ggcgaaaact ctcaaggatc 14340ttaccgctgt tgagatccag ttcgatgtaa
cccactcgtg cacccaactg atcttcagca 14400tcttttactt tcaccagcgt ttctgggtga
gcaaaaacag gaaggcaaaa tgccgcaaaa 14460aagggaataa gggcgacacg gaaatgttga
atactcatac tcttcctttt tcaatattat 14520tgaagcattt atcagggtta ttgtctcatg
agcggataca tatttgaatg tatttagaaa 14580aataaacaaa taggggttcc gcgcacattt
ccccgaaaag tgc 14623757252DNAArtificial
Sequencemisc_feature()..()Plastid transformation vector pFHO5 containing
R. capsulatus DNA e 75gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
atttttctaa atacattcaa 60atatgtatcc gctcatgaga caataaccct gataaatgct
tcaataatat tgaaaaagga 120agagtatgag tattcaacat ttccgtgtcg cccttattcc
cttttttgcg gcattttgcc 180ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
agatgctgaa gatcagttgg 240gtgcacgagt gggttacatc gaactggatc tcaacagcgg
taagatcctt gagagttttc 300gccccgaaga acgttttcca atgatgagca cttttaaagt
tctgctatgt ggcgcggtat 360tatcccgtat tgacgccggg caagagcaac tcggtcgccg
catacactat tctcagaatg 420acttggttga gtactcacca gtcacagaaa agcatcttac
ggatggcatg acagtaagag 480aattatgcag tgctgccata accatgagtg ataacactgc
ggccaactta cttctgacaa 540cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
catgggggat catgtaactc 600gccttgatcg ttgggaaccg gagctgaatg aagccatacc
aaacgacgag cgtgacacca 660cgatgcctgt agcaatggca acaacgttgc gcaaactatt
aactggcgaa ctacttactc 720tagcttcccg gcaacaatta atagactgga tggaggcgga
taaagttgca ggaccacttc 780tgcgctcggc ccttccggct ggctggttta ttgctgataa
atctggagcc ggtgagcgtg 840ggtctcgcgg tatcattgca gcactggggc cagatggtaa
gccctcccgt atcgtagtta 900tctacacgac ggggagtcag gcaactatgg atgaacgaaa
tagacagatc gctgagatag 960gtgcctcact gattaagcat tggtaactgt cagaccaagt
ttactcatat atactttaga 1020ttgatttaaa acttcatttt taatttaaaa ggatctaggt
gaagatcctt tttgataatc 1080tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
agcgtcagac cccgtagaaa 1140agatcaaagg atcttcttga gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa 1200aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
agagctacca actctttttc 1260cgaaggtaac tggcttcagc agagcgcaga taccaaatac
tgtccttcta gtgtagccgt 1320agttaggcca ccacttcaag aactctgtag caccgcctac
atacctcgct ctgctaatcc 1380tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
taccgggttg gactcaagac 1440gatagttacc ggataaggcg cagcggtcgg gctgaacggg
gggttcgtgc acacagccca 1500gcttggagcg aacgacctac accgaactga gatacctaca
gcgtgagcta tgagaaagcg 1560ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
aagcggcagg gtcggaacag 1620gagagcgcac gagggagctt ccagggggaa acgcctggta
tctttatagt cctgtcgggt 1680ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
gtcagggggg cggagcctat 1740ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
cttttgctgg ccttttgctc 1800acatgttctt tcctgcgtta tcccctgatt ctgtggataa
ccgtattacc gcctttgagt 1860gagctgatac cgctcgccgc agccgaacga ccgagcgcag
cgagtcagtg agcgaggaag 1920cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
ttggccgatt cattaatgca 1980gctggcacga caggtttccc gactggaaag cgggcagtga
gcgcaacgca attaatgtga 2040gttagctcac tcattaggca ccccaggctt tacactttat
gcttccggct cgtatgttgt 2100gtggaattgt gagcggataa caatttcaca caggaaacag
ctatgaccat gattacgcca 2160agctcgaaat taaccctcac taaagggaac aaaagctgga
gctccaccgc ggtggcggcc 2220gctctagaac tagtggatct tcttggctgt tattcaaaag
gtccaacaat gtatatatat 2280tggacatttt gaggcaatta tagatcctgg aaggcaattc
tgattggtca ataaaaatcg 2340atttcaatgc tatttttttt ttgtttttta tgagtttagc
caatttatca tgaaaggtaa 2400aaggggataa aggaaccgtg tgttgattgt cctgtaaata
taagttgtct tcctccatat 2460gtaaaaaggg aataaataaa tcaattaaat ttcgggatgc
ttcatgaagt gcttctttcg 2520gagttaaact tccgtttgtc catatttcga gaaaaagtat
ctcttgtttt tcattcccat 2580tcccataaga atgaatacta tgattcgcgt ttcgaacagg
catgaataca gcatctatag 2640gataacttcc atcttgaaag ttatgtggcg tttttataag
atatccacga tttctctcta 2700tttgtaatcc aatacaaaaa tcaattggtt ccgttaaact
ggctatatgt tgtgtattat 2760caacgatttc tacataaggc ggcaagatga tatcttgggc
agttacagat ccaggaccct 2820tgacacaaat agatgcgtca gaagttccat atagattact
tcttaatata atttctttca 2880aattcattaa aatttcatgt accgattctt gaatgcccgt
tatggtagaa tattcatgtg 2940ggactttctc agattttaca cgtgtgatac atgttccttc
tatttctcca agtaaagctc 3000ttcgcatcgc aatgcctatt gtgtcggctt ggcctttcat
aagtggagac agaataaagc 3060gtccataata aaggcgttta ctgtctgttc ttgattcaac
acacttccac tgtagtgtcc 3120gagtagatac tgttactttc tctcgaacca tagtactatt
atttgattag atcatcgaat 3180cttttatttc tcttgagatt tcttcaatgt tcagttctac
acacgtcttt ttttcggagg 3240tctacagcca ttatgtggca taggagttac atcccgtacg
aaagttaata gtataccact 3300tcgacgaata gctcgtaatg ctgcatctct tccgagaccg
ggacctttta tcatgacttc 3360tgctcgttgc ataccttgat ccactactgt acggatagcg
tttgctgctg cggtttgagc 3420agcaaacggt gttcctcttc tcgtaccttt gaatccagaa
gtaccggcgg aggaccaaga 3480aactactcga ccccgtacat ctgtaacagt gacaatggta
ttattgaaac ttgcttgaac 3540atgaataact ccctttggta ttctacgtgc acccttacgt
gaaccaatac gtccattcct 3600acgcgaacta attttcggta tagcttttgc catattttat
catctcgtaa atatgagtca 3660gagatatatg gatatatcca tttcatgtca aaacagattc
tttatttgta catcggctct 3720tctggcaagt ctgattatcc ctgtctttgt ttatgtctcg
ggttggaaca aattactata 3780attcgtcccc gcctacggat tagtcgacat ttttcacaaa
ttttacgaac ggaagctctt 3840attttcatat ttctcattcc ttaccttaat tctgaatcta
tttcttggaa gaaaataagt 3900ttcttgaaat ttttcatctc gaattgtatt cccacgaaag
gaatggtgaa gttgaaaaac 3960gaatccttca aatctttgtt gtggagtcga taaattatac
gccctttggt tgaatcataa 4020ggacttactt caattttgac tctatctcct ggcagtatcc
gtataaaact atgccggatc 4080tttcctgaaa cataatttat aatcagatcc aggaggacca
tatgatcgcc gaagcggata 4140tggaggtctg ccgggagctg atccgcaccg gcagctactc
cttccatgcg gcgtccagag 4200ttctgccggc gcgggtccgt gaccccgcgc tggcgcttta
cgccttttgc cgcgtcgccg 4260atgacgaagt cgacgaggtt ggcgcgccgc gcgacaaggc
tgcggcggtt ttgaaacttg 4320gcgaccggct ggaggacatc tatgccggtc gtccgcgcaa
tgcgccctcg gatcgggctt 4380tcgcggcggt ggtcgaggaa ttcgagatgc cgcgcgaatt
gcccgaggcg ctgctggagg 4440gcttcgcctg ggatgccgag gggcggtggt atcacacgct
ttcggacgtg caggcctatt 4500cggcgcgggt ggcggccgcc gtcggcgcga tgatgtgcgt
gctgatgcgg gtgcgcaacc 4560ccgatgcgct ggcgcgggcc tgcgatctcg gtcttgccat
gcagatgtcg aacatcgccc 4620gcgacgtggg cgaggatgcc cgggcggggc ggcttttcct
gccgaccgac tggatggtcg 4680aggaggggat cgatccgcag gcgttcctgg ccgatccgca
gcccaccaag ggcatccgcc 4740gggtcaccga gcggttgctg aaccgcgccg accggcttta
ctggcgggcg gcgacggggg 4800tgcggctttt gccctttgac tgccgaccgg ggatcatggc
cgcgggcaag atctatgccg 4860cgatcggggc cgaggtggcg aaggcgaaat acgacaacat
cacccggcgt gcccacacga 4920ccaagggccg caagctgtgg ctggtggcga attccgcgat
gtcggcgacg gcgacctcga 4980tgctgccgct ctcgccgcgg gtgcatgcca agcccgagcc
cgaagtggcg catctggtcg 5040atgccgccgc gcatcgcaac ctgcatcccg aacggtccga
ggtgctgatc tcggcgctga 5100tggcgctgaa ggcgcgcgac cgcggcctgg cgatggattg
aggatctaaa caaacccgga 5160acagaccgtt gggaagcgat tcagtaatta aagcttcatg
actccttttt ggttcttaaa 5220gtccctttga ggtatcaact aataagaaag atattagaca
accccccttt tttctttttc 5280acaaatagga agtttcgaat ccaatttgga tattaaaagg
attaccagat ataacacaaa 5340atctctccac ctattccttc tagtcgagcc tctcggtctg
tcattatacc tcgagaagta 5400gaaagaatta caatccccat tccacctaaa attcgcggaa
ttcgttgata attagaatag 5460attcgtagac caggtcgact gattcgtttt aaatttaaaa
tatttctata gggtcttttc 5520ctattccttc tatgtcgcag ggttaaaacc aaaaaatatt
tgtttttttc tcgatgtttt 5580ctcacgtttt cgataaaacc ttctcgtaaa agtatttgaa
caatattttc ggtaatatta 5640gtagatgcta ttcgaaccac cctttttcga tccatatcag
catttcgtat agaagttatt 5700atctcagcaa tagtgtccct acccatgatg aactaaaatt
attggggcct ccaaatttga 5760tataatcaac gtgtttttta cttatttttt ttttgaatat
gatatgaatt attaaagata 5820tatgcgtgag acacaatcta ctaattaatc tatttctttc
aaatacccca ctagaaacag 5880atcacaattt cattttataa tacctcggga gctaatgaaa
ctattttagt aaaatttaat 5940tctctcaatt cccgggcgat tgcaccaaaa attcgagttc
cttttgattt ccttccttct 6000tgatcaataa caactgcagc attgtcatca tatcgtatta
tcatcccgtt gtcacgtttg 6060agttctttac aggtccgcac aattacagct ctgactactt
ctgatctttc taggggcata 6120tttggtacgg cttctttgat cacagcaaca ataacgtcac
caatatgagc atatcgacga 6180ttgctagctc ctatgattcg aatacacatc aattctcgag
ccccgctgtt atccgctaca 6240tttaaatggg tctgaggttg aatcattttt ttaatccgtt
ctttgaatgc aaagggcgaa 6300gaaaaaaaag aaatattttt gtccaaaaaa aaagaaacat
gcggtttcgt ttcatatcta 6360agagcccttt ccgcattttt ttctattaca ttacgaaata
atgaattgag ttcgtatagg 6420cattttagat gctgctagtg aaatagccct tctggctata
ttttctgtta ctccacccat 6480ttcataaagt attcgacccg gtttaacaac agctacccaa
tattcagggg atcccccggg 6540ctgcaggaat tcgatatcaa gcttatcgat accgtcgacc
tcgagggggg gcccggtacc 6600caattcgccc tatagtgagt cgtattacaa ttcactggcc
gtcgttttac aacgtcgtga 6660ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
gcacatcccc ctttcgccag 6720ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
caacagttgc gcagcctgaa 6780tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
gcgggtgtgg tggttacgcg 6840cagcgtgacc gctacacttg ccagcgccct agcgcccgct
cctttcgctt tcttcccttc 6900ctttctcgcc acgttcgccg gctttccccg tcaagctcta
aatcgggggc tccctttagg 6960gttccgattt agtgctttac ggcacctcga ccccaaaaaa
cttgattagg gtgatggttc 7020acgtagtggg ccatcgccct gatagacggt ttttcgccct
ttgacgttgg agtccacgtt 7080ctttaatagt ggactcttgt tccaaactgg aacaacactc
aaccctatct cggtctattc 7140ttttgattta taagggattt tgccgatttc ggcctattgg
ttaaaaaatg agctgattta 7200acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
acaatttagg tg 72527614623DNAArtificial
Sequencemisc_feature()..()Plastid transformation vector pFHO6,
containing Operon E, containi 76cacctaaatt gtaagcgtta atattttgtt
aaaattcgcg ttaaattttt gttaaatcag 60ctcatttttt aaccaatagg ccgaaatcgg
caaaatccct tataaatcaa aagaatagac 120cgagataggg ttgagtgttg ttccagtttg
gaacaagagt ccactattaa agaacgtgga 180ctccaacgtc aaagggcgaa aaaccgtcta
tcagggcgat ggcccactac gtgaaccatc 240accctaatca agttttttgg ggtcgaggtg
ccgtaaagca ctaaatcgga accctaaagg 300gagcccccga tttagagctt gacggggaaa
gccggcgaac gtggcgagaa aggaagggaa 360gaaagcgaaa ggagcgggcg ctagggcgct
ggcaagtgta gcggtcacgc tgcgcgtaac 420caccacaccc gccgcgctta atgcgccgct
acagggcgcg tcccattcgc cattcaggct 480gcgcaactgt tgggaagggc gatcggtgcg
ggcctcttcg ctattacgcc agctggcgaa 540agggggatgt gctgcaaggc gattaagttg
ggtaacgcca gggttttccc agtcacgacg 600ttgtaaaacg acggccagtg aattgtaata
cgactcacta tagggcgaat tgggtaccgg 660gccccccctc gaggtcgacg gtatcgataa
gcttgatatc gaattcctgc agcccggggg 720atcttcttgg ctgttattca aaaggtccaa
caatgtatat atattggaca ttttgaggca 780attatagatc ctggaaggca attctgattg
gtcaataaaa atcgatttca atgctatttt 840ttttttgttt tttatgagtt tagccaattt
atcatgaaag gtaaaagggg ataaaggaac 900cgtgtgttga ttgtcctgta aatataagtt
gtcttcctcc atatgtaaaa agggaataaa 960taaatcaatt aaatttcggg atgcttcatg
aagtgcttct ttcggagtta aacttccgtt 1020tgtccatatt tcgagaaaaa gtatctcttg
tttttcattc ccattcccat aagaatgaat 1080actatgattc gcgtttcgaa caggcatgaa
tacagcatct ataggataac ttccatcttg 1140aaagttatgt ggcgttttta taagatatcc
acgatttctc tctatttgta atccaataca 1200aaaatcaatt ggttccgtta aactggctat
atgttgtgta ttatcaacga tttctacata 1260aggcggcaag atgatatctt gggcagttac
agatccagga cccttgacac aaatagatgc 1320gtcagaagtt ccatatagat tacttcttaa
tataatttct ttcaaattca ttaaaatttc 1380atgtaccgat tcttgaatgc ccgttatggt
agaatattca tgtgggactt tctcagattt 1440tacacgtgtg atacatgttc cttctatttc
tccaagtaaa gctcttcgca tcgcaatgcc 1500tattgtgtcg gcttggcctt tcataagtgg
agacagaata aagcgtccat aataaaggcg 1560tttactgtct gttcttgatt caacacactt
ccactgtagt gtccgagtag atactgttac 1620tttctctcga accatagtac tattatttga
ttagatcatc gaatctttta tttctcttga 1680gatttcttca atgttcagtt ctacacacgt
ctttttttcg gaggtctaca gccattatgt 1740ggcataggag ttacatcccg tacgaaagtt
aatagtatac cacttcgacg aatagctcgt 1800aatgctgcat ctcttccgag accgggacct
tttatcatga cttctgctcg ttgcatacct 1860tgatccacta ctgtacggat agcgtttgct
gctgcggttt gagcagcaaa cggtgttcct 1920cttctcgtac ctttgaatcc agaagtaccg
gcggaggacc aagaaactac tcgaccccgt 1980acatctgtaa cagtgacaat ggtattattg
aaacttgctt gaacatgaat aactcccttt 2040ggtattctac gtgcaccctt acgtgaacca
atacgtccat tcctacgcga actaattttc 2100ggtatagctt ttgccatatt ttatcatctc
gtaaatatga gtcagagata tatggatata 2160tccatttcat gtcaaaacag attctttatt
tgtacatcgg ctcttctggc aagtctgatt 2220atccctgtct ttgtttatgt ctcgggttgg
aacaaattac tataattcgt ccccgcctac 2280ggattagtcg acatttttca caaattttac
gaacggaagc tcttattttc atatttctca 2340ttccttacct taattctgaa tctatttctt
ggaagaaaat aagtttcttg aaatttttca 2400tctcgaattg tattcccacg aaaggaatgg
tgaagttgaa aaacgaatcc ttcaaatctt 2460tgttgtggag tcgataaatt atacgccctt
tggttgaatc ataaggactt acttcaattt 2520tgactctatc tcctggcagt atccgtataa
aactatgccg gatctttcct gaaacataat 2580ttataatcag atcggccgca ggaggagttc
atatgtcaga gttgagagcc ttcagtgccc 2640cagggaaagc gttactagct ggtggatatt
tagttttaga tacaaaatat gaagcatttg 2700tagtcggatt atcggcaaga atgcatgctg
tagcccatcc ttacggttca ttgcaagggt 2760ctgataagtt tgaagtgcgt gtgaaaagta
aacaatttaa agatggggag tggctgtacc 2820atataagtcc taaaagtggc ttcattcctg
tttcgatagg cggatctaag aaccctttca 2880ttgaaaaagt tatcgctaac gtatttagct
actttaaacc taacatggac gactactgca 2940atagaaactt gttcgttatt gatattttct
ctgatgatgc ctaccattct caggaggata 3000gcgttaccga acatcgtggc aacagaagat
tgagttttca ttcgcacaga attgaagaag 3060ttcccaaaac agggctgggc tcctcggcag
gtttagtcac agttttaact acagctttgg 3120cctccttttt tgtatcggac ctggaaaata
atgtagacaa atatagagaa gttattcata 3180atttagcaca agttgctcat tgtcaagctc
agggtaaaat tggaagcggg tttgatgtag 3240cggcggcagc atatggatct atcagatata
gaagattccc acccgcatta atctctaatt 3300tgccagatat tggaagtgct acttacggca
gtaaactggc gcatttggtt gatgaagaag 3360actggaatat tacgattaaa agtaaccatt
taccttcggg attaacttta tggatgggcg 3420atattaagaa tggttcagaa acagtaaaac
tggtccagaa ggtaaaaaat tggtatgatt 3480cgcatatgcc agaaagcttg aaaatatata
cagaactcga tcatgcaaat tctagattta 3540tggatggact atctaaacta gatcgcttac
acgagactca tgacgattac agcgatcaga 3600tatttgagtc tcttgagagg aatgactgta
cctgtcaaaa gtatcctgaa atcacagaag 3660ttagagatgc agttgccaca attagacgtt
cctttagaaa aataactaaa gaatctggtg 3720ccgatatcga acctcccgta caaactagct
tattggatga ttgccagacc ttaaaaggag 3780ttcttacttg cttaatacct ggtgctggtg
gttatgacgc cattgcagtg attactaagc 3840aagatgttga tcttagggct caaaccgcta
atgacaaaag attttctaag gttcaatggc 3900tggatgtaac tcaggctgac tggggtgtta
ggaaagaaaa agatccggaa acttatcttg 3960ataaactgca ggaggagttt taatgtcatt
accgttctta acttctgcac cgggaaaggt 4020tattattttt ggtgaacact ctgctgtgta
caacaagcct gccgtcgctg ctagtgtgtc 4080tgcgttgaga acctacctgc taataagcga
gtcatctgca ccagatacta ttgaattgga 4140cttcccggac attagcttta atcataagtg
gtccatcaat gatttcaatg ccatcaccga 4200ggatcaagta aactcccaaa aattggccaa
ggctcaacaa gccaccgatg gcttgtctca 4260ggaactcgtt agtcttttgg atccgttgtt
agctcaacta tccgaatcct tccactacca 4320tgcagcgttt tgtttcctgt atatgtttgt
ttgcctatgc ccccatgcca agaatattaa 4380gttttcttta aagtctactt tacccatcgg
tgctgggttg ggctcaagcg cctctatttc 4440tgtatcactg gccttagcta tggcctactt
gggggggtta ataggatcta atgacttgga 4500aaagctgtca gaaaacgata agcatatagt
gaatcaatgg gccttcatag gtgaaaagtg 4560tattcacggt accccttcag gaatagataa
cgctgtggcc acttatggta atgccctgct 4620atttgaaaaa gactcacata atggaacaat
aaacacaaac aattttaagt tcttagatga 4680tttcccagcc attccaatga tcctaaccta
tactagaatt ccaaggtcta caaaagatct 4740tgttgctcgc gttcgtgtgt tggtcaccga
gaaatttcct gaagttatga agccaattct 4800agatgccatg ggtgaatgtg ccctacaagg
cttagagatc atgactaagt taagtaaatg 4860taaaggcacc gatgacgagg ctgtagaaac
taataatgaa ctgtatgaac aactattgga 4920attgataaga ataaatcatg gactgcttgt
ctcaatcggt gtttctcatc ctggattaga 4980acttattaaa aatctgagcg atgatttgag
aattggctcc acaaaactta ccggtgctgg 5040tggcggcggt tgctctttga ctttgttacg
aagagacatt actcaagagc aaattgacag 5100cttcaaaaag aaattgcaag atgattttag
ttacgagaca tttgaaacag acttgggtgg 5160gactggctgc tgtttgttaa gcgcaaaaaa
tttgaataaa gatcttaaaa tcaaatccct 5220agtattccaa ttatttgaaa ataaaactac
cacaaagcaa caaattgacg atctattatt 5280gccaggaaac acgaatttac catggacttc
agacgaggag ttttaatgac tgtatatact 5340gctagtgtaa ctgctccggt aaatattgct
actcttaagt attgggggaa aagggacacg 5400aagttgaatc tgcccaccaa ttcgtccata
tcagtgactt tatcgcaaga tgacctcaga 5460acgttgacct ctgcggctac tgcacctgag
tttgaacgcg acactttgtg gttaaatgga 5520gaaccacaca gcatcgacaa tgaaagaact
caaaattgtc tgcgcgacct acgccaatta 5580agaaaggaaa tggaatcgaa ggacgcctca
ttgcccacat tatctcaatg gaaactccac 5640attgtctccg aaaataactt tcctacagca
gctggtttag cttcctccgc tgctggcttt 5700gctgcattgg tctctgcaat tgctaagtta
taccaattac cacagtcaac ttcagaaata 5760tctagaatag caagaaaggg gtctggttca
gcttgtagat cgttgtttgg cggatacgtg 5820gcctgggaaa tgggaaaagc tgaagatggt
catgattcca tggcagtaca aatcgcagac 5880agctctgact ggcctcagat gaaagcttgt
gtcctagttg tcagcgatat taaaaaggat 5940gtgagttcca ctcagggtat gcaattgacc
gtggcaacct ccgaactatt taaagaaaga 6000attgaacatg tcgtaccaaa gagatttgaa
gtcatgcgta aagccattgt tgaaaaagat 6060ttcgccacct ttgcaaagga aacaatgatg
gattccaact ctttccatgc cacatgtttg 6120gactctttcc ctccaatatt ctacatgaat
gacacttcca agcgtatcat cagttggtgc 6180cacaccatta atcagtttta cggagaaaca
atcgttgcat acacgtttga tgcaggtcca 6240aatgctgtgt tgtactactt agctgaaaat
gagtcgaaac tctttgcatt tatctataaa 6300ttgtttggct ctgttcctgg atgggacaag
aaatttacta ctgagcagct tgaggctttc 6360aaccatcaat ttgaatcatc taactttact
gcacgtgaat tggatcttga gttgcaaaag 6420gatgttgcca gagtgatttt aactcaagtc
ggttcaggcc cacaagaaac aaacgaatct 6480ttgattgacg caaagactgg tctaccaaag
gaagaggagt tttaactcga cgccggcgga 6540ggcacatatg tctcagaacg tttacattgt
atcgactgcc agaaccccaa ttggttcatt 6600ccagggttct ctatcctcca agacagcagt
ggaattgggt gctgttgctt taaaaggcgc 6660cttggctaag gttccagaat tggatgcatc
caaggatttt gacgaaatta tttttggtaa 6720cgttctttct gccaatttgg gccaagctcc
ggccagacaa gttgctttgg ctgccggttt 6780gagtaatcat atcgttgcaa gcacagttaa
caaggtctgt gcatccgcta tgaaggcaat 6840cattttgggt gctcaatcca tcaaatgtgg
taatgctgat gttgtcgtag ctggtggttg 6900tgaatctatg actaacgcac catactacat
gccagcagcc cgtgcgggtg ccaaatttgg 6960ccaaactgtt cttgttgatg gtgtcgaaag
agatgggttg aacgatgcgt acgatggtct 7020agccatgggt gtacacgcag aaaagtgtgc
ccgtgattgg gatattacta gagaacaaca 7080agacaatttt gccatcgaat cctaccaaaa
atctcaaaaa tctcaaaagg aaggtaaatt 7140cgacaatgaa attgtacctg ttaccattaa
gggatttaga ggtaagcctg atactcaagt 7200cacgaaggac gaggaacctg ctagattaca
cgttgaaaaa ttgagatctg caaggactgt 7260tttccaaaaa gaaaacggta ctgttactgc
cgctaacgct tctccaatca acgatggtgc 7320tgcagccgtc atcttggttt ccgaaaaagt
tttgaaggaa aagaatttga agcctttggc 7380tattatcaaa ggttggggtg aggccgctca
tcaaccagct gattttacat gggctccatc 7440tcttgcagtt ccaaaggctt tgaaacatgc
tggcatcgaa gacatcaatt ctgttgatta 7500ctttgaattc aatgaagcct tttcggttgt
cggtttggtg aacactaaga ttttgaagct 7560agacccatct aaggttaatg tatatggtgg
tgctgttgct ctaggtcacc cattgggttg 7620ttctggtgct agagtggttg ttacactgct
atccatctta cagcaagaag gaggtaagat 7680cggtgttgcc gccatttgta atggtggtgg
tggtgcttcc tctattgtca ttgaaaagat 7740atgaggatcc tctagatgcg caggaggcac
atatggcgaa gaacgttggg attttggcta 7800tggatatcta tttccctccc acctgtgttc
aacaggaagc tttggaagca catgatggag 7860caagtaaagg gaaatacact attggacttg
gccaagattg tttagctttt tgcactgagc 7920ttgaagatgt tatctctatg agtttcaatg
cggtgacatc actttttgag aagtataaga 7980ttgaccctaa ccaaatcggg cgtcttgaag
taggaagtga gactgttatt gacaaaagca 8040agtccatcaa gaccttcttg atgcagctct
ttgagaaatg tggaaacact gatgtcgaag 8100gtgttgactc gaccaatgct tgctatggtg
gaactgcagc tttgttaaac tgtgtcaatt 8160gggttgagag taactcttgg gatggacgtt
atggcctcgt catttgtact gacagcgcgg 8220tttatgcaga aggacccgca aggcccactg
gaggagctgc agcgattgct atgttgatag 8280gacctgatgc tcctatcgtt ttcgaaagca
aattgagagc aagccacatg gctcatgtct 8340atgactttta caagcccaat cttgctagcg
agtacccggt tgttgatggt aagctttcac 8400agacttgcta cctcatggct cttgactcct
gctataaaca tttatgcaac aagttcgaga 8460agatcgaggg caaagagttc tccataaatg
atgctgatta cattgttttc cattctccat 8520acaataaact tgtacagaaa agctttgctc
gtctcttgta caacgacttc ttgagaaacg 8580caagctccat tgacgaggct gccaaagaaa
agttcacccc ttattcatct ttgacccttg 8640acgagagtta ccaaagccgt gatcttgaaa
aggtgtcaca acaaatttcg aaaccgtttt 8700atgatgctaa agtgcaacca acgactttaa
taccaaagga agtcggtaac atgtacactg 8760cttctctcta cgctgcattt gcttccctca
tccacaataa acacaatgat ttggcgggaa 8820agcgggtggt tatgttctct tatggaagtg
gctccaccgc aacaatgttc tcattacgcc 8880tcaacgacaa taagcctcct ttcagcattt
caaacattgc atctgtaatg gatgttggcg 8940gtaaattgaa agctagacat gagtatgcac
ctgagaagtt tgtggagaca atgaagctaa 9000tggaacatag gtatggagca aaggactttg
tgacaaccaa ggagggtatt atagatcttt 9060tggcaccggg aacttattat ctgaaagagg
ttgattcctt gtaccggaga ttctatggca 9120agaaaggtga agatggatct gtagccaatg
gacactgagg atccgtcgag cacgtggagg 9180cacatatgca atgctgtgag atgcctgttg
gatacattca gattcctgtt gggattgctg 9240gtccattgtt gcttgatggt tatgagtact
ctgttcctat ggctacaacc gaaggttgtt 9300tggttgctag cactaacaga ggctgcaagg
ctatgtttat ctctggtggc gccaccagta 9360ccgttcttaa ggacggtatg acccgagcac
ctgttgttcg gttcgcttcg gcgagacgag 9420cttcggagct taagtttttc ttggagaatc
cagagaactt tgatactttg gcagtagtct 9480tcaacaggtc gagtagattt gcaagactgc
aaagtgttaa atgcacaatc gcggggaaga 9540atgcttatgt aaggttctgt tgtagtactg
gtgatgctat ggggatgaat atggtttcta 9600aaggtgtgca gaatgttctt gagtatctta
ccgatgattt ccctgacatg gatgtgattg 9660gaatctctgg taacttctgt tcggacaaga
aacctgctgc tgtgaactgg attgagggac 9720gtggtaaatc agttgtttgc gaggctgtaa
tcagaggaga gatcgtgaac aaggtcttga 9780aaacgagcgt ggctgcttta gtcgagctca
acatgctcaa gaacctagct ggctctgctg 9840ttgcaggctc tctaggtgga ttcaacgctc
atgccagtaa catagtgtct gctgtattca 9900tagctactgg ccaagatcca gctcaaaacg
tggagagttc tcaatgcatc accatgatgg 9960aagctattaa tgacggcaaa gatatccata
tctcagtcac tatgccatct atcgaggtgg 10020ggacagtggg aggaggaaca cagcttgcat
ctcaatcagc gtgtttaaac ctgctcggag 10080ttaaaggagc aagcacagag tcgccgggaa
tgaacgcaag gaggctagcg acgatcgtag 10140ccggagcagt tttagctgga gagttatctt
taatgtcagc aattgcagct ggacagcttg 10200tgagaagtca catgaaatac aatagatcca
gccgagacat ctctggagca acgacaacga 10260caacaacaac aacatgaccc gtaggaggca
catatgagtt cccaacaaga gaaaaaggat 10320tatgatgaag aacaattaag gttgatggaa
gaagtttgta tcgttgtaga tgaaaatgat 10380gtccctttaa gatatggaac gaaaaaggag
tgtcatttga tggaaaatat aaataaaggt 10440cttttgcata gagcattctc tatgttcatc
tttgatgagc aaaatcgcct tttacttcag 10500cagcgtgcag aagagaaaat tacatttcca
tccttatgga cgaatacatg ttgctcccac 10560ccattggatg ttgctggtga acgtggtaat
actttacctg aagctgttga aggtgttaag 10620aatgcagctc aacgcaagct gttccatgaa
ttgggtattc aagccaagta tattcccaaa 10680gacaaatttc agtttcttac acgaatccat
taccttgctc ctagtactgg tgcttgggga 10740gagcatgaaa ttgactacat tcttttcttc
aaaggtaaag ttgagctgga tatcaatccc 10800aatgaagttc aagcctataa gtatgttact
atggaagagt taaaagagat gttttccgat 10860cctcaatatg gattcacacc atggttcaaa
cttatttgtg agcattttat gtttaaatgg 10920tggcaggatg tagatcatgc gtcaaaattc
caagatacct taattcatcg ttgctaagga 10980tcccccggga tccggccgat ctaaacaaac
ccggaacaga ccgttgggaa gcgattcagt 11040aattaaagct tcatgactcc tttttggttc
ttaaagtccc tttgaggtat caactaataa 11100gaaagatatt agacaacccc ccttttttct
ttttcacaaa taggaagttt cgaatccaat 11160ttggatatta aaaggattac cagatataac
acaaaatctc tccacctatt ccttctagtc 11220gagcctctcg gtctgtcatt atacctcgag
aagtagaaag aattacaatc cccattccac 11280ctaaaattcg cggaattcgt tgataattag
aatagattcg tagaccaggt cgactgattc 11340gttttaaatt taaaatattt ctatagggtc
ttttcctatt ccttctatgt cgcagggtta 11400aaaccaaaaa atatttgttt ttttctcgat
gttttctcac gttttcgata aaaccttctc 11460gtaaaagtat ttgaacaata ttttcggtaa
tattagtaga tgctattcga accacccttt 11520ttcgatccat atcagcattt cgtatagaag
ttattatctc agcaatagtg tccctaccca 11580tgatgaacta aaattattgg ggcctccaaa
tttgatataa tcaacgtgtt ttttacttat 11640tttttttttg aatatgatat gaattattaa
agatatatgc gtgagacaca atctactaat 11700taatctattt ctttcaaata ccccactaga
aacagatcac aatttcattt tataatacct 11760cgggagctaa tgaaactatt ttagtaaaat
ttaattctct caattcccgg gcgattgcac 11820caaaaattcg agttcctttt gatttccttc
cttcttgatc aataacaact gcagcattgt 11880catcatatcg tattatcatc ccgttgtcac
gtttgagttc tttacaggtc cgcacaatta 11940cagctctgac tacttctgat ctttctaggg
gcatatttgg tacggcttct ttgatcacag 12000caacaataac gtcaccaata tgagcatatc
gacgattgct agctcctatg attcgaatac 12060acatcaattc tcgagccccg ctgttatccg
ctacatttaa atgggtctga ggttgaatca 12120tttttttaat ccgttctttg aatgcaaagg
gcgaagaaaa aaaagaaata tttttgtcca 12180aaaaaaaaga aacatgcggt ttcgtttcat
atctaagagc cctttccgca tttttttcta 12240ttacattacg aaataatgaa ttgagttcgt
ataggcattt tagatgctgc tagtgaaata 12300gcccttctgg ctatattttc tgttactcca
cccatttcat aaagtattcg acccggttta 12360acaacagcta cccaatattc aggggatcca
ctagttctag agcggccgcc accgcggtgg 12420agctccagct tttgttccct ttagtgaggg
ttaatttcga gcttggcgta atcatggtca 12480tagctgtttc ctgtgtgaaa ttgttatccg
ctcacaattc cacacaacat acgagccgga 12540agcataaagt gtaaagcctg gggtgcctaa
tgagtgagct aactcacatt aattgcgttg 12600cgctcactgc ccgctttcca gtcgggaaac
ctgtcgtgcc agctgcatta atgaatcggc 12660caacgcgcgg ggagaggcgg tttgcgtatt
gggcgctctt ccgcttcctc gctcactgac 12720tcgctgcgct cggtcgttcg gctgcggcga
gcggtatcag ctcactcaaa ggcggtaata 12780cggttatcca cagaatcagg ggataacgca
ggaaagaaca tgtgagcaaa aggccagcaa 12840aaggccagga accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct ccgcccccct 12900gacgagcatc acaaaaatcg acgctcaagt
cagaggtggc gaaacccgac aggactataa 12960agataccagg cgtttccccc tggaagctcc
ctcgtgcgct ctcctgttcc gaccctgccg 13020cttaccggat acctgtccgc ctttctccct
tcgggaagcg tggcgctttc tcatagctca 13080cgctgtaggt atctcagttc ggtgtaggtc
gttcgctcca agctgggctg tgtgcacgaa 13140ccccccgttc agcccgaccg ctgcgcctta
tccggtaact atcgtcttga gtccaacccg 13200gtaagacacg acttatcgcc actggcagca
gccactggta acaggattag cagagcgagg 13260tatgtaggcg gtgctacaga gttcttgaag
tggtggccta actacggcta cactagaagg 13320acagtatttg gtatctgcgc tctgctgaag
ccagttacct tcggaaaaag agttggtagc 13380tcttgatccg gcaaacaaac caccgctggt
agcggtggtt tttttgtttg caagcagcag 13440attacgcgca gaaaaaaagg atctcaagaa
gatcctttga tcttttctac ggggtctgac 13500gctcagtgga acgaaaactc acgttaaggg
attttggtca tgagattatc aaaaaggatc 13560ttcacctaga tccttttaaa ttaaaaatga
agttttaaat caatctaaag tatatatgag 13620taaacttggt ctgacagtta ccaatgctta
atcagtgagg cacctatctc agcgatctgt 13680ctatttcgtt catccatagt tgcctgactc
cccgtcgtgt agataactac gatacgggag 13740ggcttaccat ctggccccag tgctgcaatg
ataccgcgag acccacgctc accggctcca 13800gatttatcag caataaacca gccagccgga
agggccgagc gcagaagtgg tcctgcaact 13860ttatccgcct ccatccagtc tattaattgt
tgccgggaag ctagagtaag tagttcgcca 13920gttaatagtt tgcgcaacgt tgttgccatt
gctacaggca tcgtggtgtc acgctcgtcg 13980tttggtatgg cttcattcag ctccggttcc
caacgatcaa ggcgagttac atgatccccc 14040atgttgtgca aaaaagcggt tagctccttc
ggtcctccga tcgttgtcag aagtaagttg 14100gccgcagtgt tatcactcat ggttatggca
gcactgcata attctcttac tgtcatgcca 14160tccgtaagat gcttttctgt gactggtgag
tactcaacca agtcattctg agaatagtgt 14220atgcggcgac cgagttgctc ttgcccggcg
tcaatacggg ataataccgc gccacatagc 14280agaactttaa aagtgctcat cattggaaaa
cgttcttcgg ggcgaaaact ctcaaggatc 14340ttaccgctgt tgagatccag ttcgatgtaa
cccactcgtg cacccaactg atcttcagca 14400tcttttactt tcaccagcgt ttctgggtga
gcaaaaacag gaaggcaaaa tgccgcaaaa 14460aagggaataa gggcgacacg gaaatgttga
atactcatac tcttcctttt tcaatattat 14520tgaagcattt atcagggtta ttgtctcatg
agcggataca tatttgaatg tatttagaaa 14580aataaacaaa taggggttcc gcgcacattt
ccccgaaaag tgc 14623
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