Patent application title: High Fidelity Restriction Endonucleases
Inventors:
IPC8 Class: AC12N1510FI
USPC Class:
1 1
Class name:
Publication date: 2020-02-27
Patent application number: 20200063121
Abstract:
Compositions and methods are provided for enzymes with altered properties
that involve a systematic approach to mutagenesis and a screening assay
that permits selection of the desired proteins. Embodiments of the method
are particularly suited for modifying specific properties of restriction
endonucleases such as star activity. The compositions includes
restriction endonucleases with reduced star activity as defined by an
overall fidelity index improvement factor.Claims:
1-43. (canceled)
44. A composition comprising a variant KpnI restriction endonuclease having reduced star activity, wherein the variant KpnI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent KpnI restriction endonuclease by: (a) an amino acid substitution at one or more positions corresponding to positions 2, 16, 119, 132 and 134 in SEQ ID NO:87; or (b) amino acid substitutions at positions corresponding to positions 16 and 148 in SEQ ID NO:87.
45. The composition of claim 44, wherein the variant KpnI restriction endonuclease comprises an amino acid substitution at an amino acid position corresponding to position 16 in SEQ ID NO:87.
46. The composition of claim 45, wherein the amino acid substitution is D16N.
47. The composition of claim 44, wherein the variant KpnI restriction endonuclease comprises amino acids substitutions at amino acid positions corresponding to positions 16 and 148 in SEQ ID NO:87.
48. The composition of claim 47, wherein the amino acid substitutions are D16N and D148E.
49. The composition of claim 44, wherein the variant KpnI restriction endonuclease comprises amino acids substitutions at amino acid positions corresponding to positions 16, 132 and 148 in SEQ ID NO:87.
50. The composition of claim 49, wherein the amino acid substitutions are D16N, E132A and D148E.
Description:
BACKGROUND
[0001] Restriction endonucleases are enzymes that cleave double-stranded DNAs in a sequence-specific manner (Roberts, R. J., Proc Natl Acad Sci USA, 102:5905-5908 (2005); Roberts, et al., Nucleic Acids Res, 31:1805-1812 (2003); Roberts, et al., Nucleic Acids Res, 33:D230-232 (2005); Alves, et al., Restriction Endonucleases, "Protein Engineering of Restriction Enzymes," ed. Pingoud, Springer-Verlag Berlin Heidelberg, New York, 393-407 (2004)). They are ubiquitously present among prokaryotic organisms (Raleigh, et al., Bacterial Genomes Physical Structure and Analysis, Ch. 8, eds. De Bruijin, et al., Chapman & Hall, New York, 78-92 (1998)), in which they form part of restriction-modification systems, which mainly consist of an endonuclease and a methyltransferase. The cognate methyltransferase methylates the same specific sequence that its paired endonuclease recognizes and renders the modified DNA resistant to cleavage by the endonuclease so that the host DNA can be properly protected. However, when there is an invasion of foreign DNA, in particular bacteriophage DNA, the foreign DNA will be degraded before it can be completely methylated. The major biological function of the restriction-modification system is to protect the host from bacteriophage infection (Arber, Science, 205:361-365 (1979)). Other functions have also been suggested, such as involvement in recombination and transposition (Carlson, et al., Mol Microbiol, 27:671-676 (1998); Heitman, Genet Eng (NY), 15:57-108 (1993); McKane, et al., Genetics, 139:35-43 (1995)).
[0002] The specificity of the approximately 3,000 known restriction endonucleases for their greater than 250 different target sequences could be considered their most interesting characteristic. After the discovery of the sequence-specific nature of the first restriction endonuclease (Danna, et al., Proc Natl Acad Sci USA, 68:2913-2917 (1971); Kelly, et al., J Mol Biol, 51:393-409 (1970)), it did not take long for scientists to find that certain restriction endonucleases cleave sequences which are similar but not identical to their defined recognition sequences under non-optimal conditions (Polisky, et al., Proc Natl Acad Sci USA, 72:3310-3314 (1975); Nasri, et al., Nucleic Acids Res, 14:811-821 (1986)). This relaxed specificity is referred to as star activity of the restriction endonuclease. It has been suggested that water-mediated interactions between the restriction endonuclease and DNA are the key differences between specific complexes and star complexes (Robinson, et al., J Mol Biol, 234:302-306 (1993); Robinson, et al., Proc Natl Acad Sci USA, 92:3444-3448 (1995), Sidorova, et al., Biophys J, 87:2564-2576 (2004)).
[0003] Star activity is a problem in molecular biology reactions. Star activity introduces undesirable cuts in a cloning vector or other DNA. In cases such as forensic applications, where a certain DNA substrate needs to be cleaved by a restriction endonuclease to generate a unique fingerprint, star activity will alter a cleavage pattern profile, thereby complicating analysis. Avoiding star activity is also critical in applications such as strand displacement amplification (Walker, et al., Proc Natl Acad Sci USA, 89:392-396 (1992)) and serial analysis of gene expression (Velculescu, et al., Science, 270:484-487 (1995)).
SUMMARY
[0004] In an embodiment of the invention, a composition is provided that includes a restriction endonuclease having at least one artificially introduced mutation and an overall fidelity index (FI) improvement factor of at least two, the restriction endonuclease being capable of cleaving a substrate with at least a similar cleavage activity to that of the restriction endonuclease absent the artificially introduced mutation in a predetermined buffer, the artificially introduced mutation being the product of at least one of a targeted mutation, saturation mutagenesis, or a mutation introduced through a PCR amplification procedure.
[0005] In a further embodiment of the invention, at least one of the artificially introduced mutations is a targeted mutation resulting from replacement of a naturally occurring residue with an oppositely charged residue. An Alanine or a Phenylalanine may replace the naturally occurring residue at the target site.
[0006] In a further embodiment of the invention, a composition of the type described above includes a restriction enzyme absent the artificially introduced mutation selected from the group consisting of: BamHI, EcoRI, ScaI, SalI, SphI, PstI, NcoI, NheI, SspI, NotI, SacI, PvuII, MfeI, HindIII, SbfI, EagI, EcoRV, AvrII, BstXI, PciI, HpaI, AgeI, BsmBI, BspQI, SapI, KpnI and BsaI.
[0007] Further embodiments of the invention include compositions listed in Table 4.
[0008] In a further embodiment of the invention, a DNA encoding any of the enzymes listed in Table 4 is provided, a vector comprising the DNA and a host cell for expressing the protein from the vector.
[0009] In an embodiment of the invention, a method is provided having the steps of (a) identifying which amino acid residues in an amino acid sequence of a restriction endonuclease having star activity are charged amino acids; (b) mutating one or more codons encoding one or more of the charged residues in a gene sequence encoding the restriction endonuclease; (c) generating a library of gene sequences having one or more different codon mutations in different charged residues; (d) obtaining a set of proteins expressed by the mutated gene sequences; and (e) determining an FI in a predetermined buffer and a cleavage activity for each expressed protein.
[0010] An embodiment of the method includes the step of determining an overall FI improvement factor for proteins belonging to the set of proteins in a defined set of buffers where for example, the set of buffers contains NEB1, NEB2, NEB3 and NEB4 buffers.
[0011] An embodiment of the method includes the steps described above and additionally mutating codons encoding hydroxylated amino acids or amide amino acids in a same or subsequent step to that of mutating codons for the charged amino acids.
[0012] In an embodiment of the invention described above, the codons are mutated to an Alanine except for Tyrosine which is mutated to a Phenylalanine.
[0013] In a further embodiment, the overall FI improvement factor is improved using saturation mutagenesis of one or more of the mutated codon.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] For FIGS. 1-32:
[0015] The * symbol indicates the lane to its left that contains the lowest concentration of enzyme for which star activity is observed.
[0016] The # symbol refers to the lane showing incomplete cleavage, which is adjacent to and to the right side of the lane containing a concentration of enzyme sufficient for complete cleavage of the substrate.
[0017] The gray triangle denotes the serial decrease of restriction endonuclease concentration.
[0018] "U" denotes units of enzyme.
[0019] In each of the reactions described in FIGS. 1-32, the reaction mixture contains a volume of 3 .mu.l unless otherwise specified of a buffer from New England Biolabs, Inc. (NEB), Ipswich, Mass., (see Table 1 and NEB catalog), 3 .mu.l unless otherwise specified of a specified restriction endonuclease in a diluent from NEB, Ipswich, Mass. (See Table 1 and NEB catalog) as well as variable volumes of specified substrate (containing 0.6 .mu.g) substrate and a volume of water to bring the reaction mixture to a total of 30 .mu.l. Reactions were conducted at 37.degree. C. for an incubation time of 1 hour. The results are analyzed on a 0.8% agarose gel. Where the overall volume of the reaction mix, amount of substrate, temperature of the reaction or incubation time varies from above, values are provided in the description of the figures.
[0020] The theoretical digestion pattern is provided on the right side of the gel for FIGS. 1, 5, 8, 11-18 and 20-32. Those substrates with only one restriction endonuclease site should be digested into one linear band from supercoiled form.
[0021] FIG. 1 shows the determination of the FI for wild type (WT) ScaI by digesting 1.2 .mu.l lambda DNA substrate (0.6 .mu.g) with a two-fold serial dilution using diluent A of a preparation of WT ScaI (1,200 U) in NEB3 buffer and examining the digestion products on an agarose gel. The highest concentration of a restriction endonuclease with no star activity is shown with a solid arrow; and the minimum concentration giving rise to complete digestion of substrate is shown with a hollow arrow.
[0022] FIGS. 2A-D show the results of digesting 0.5 .mu.l pUC19 substrate (0.5 .mu.g) with WT BamHI or BamHI(E86P) enzyme in a three-fold serial dilution using diluent A for 1 hour at a starting concentration of 172 U or 512 U. The middle lane is the NEB 1 kb marker (New England Biolabs, Inc. (NEB), Ipswich, Mass.).
[0023] FIG. 2A shows results using NEB1 buffer.
[0024] FIG. 2B shows results using NEB2 buffer.
[0025] FIG. 2C shows results using NEB3 buffer.
[0026] FIG. 2D shows results using NEB4 buffer.
[0027] FIGS. 3A-B show a comparison of BamHI(E86P) activity over two time periods using 0.6 .mu.l pBR322 substrate (which contains only 1 BamHI cleavage site) in NEB2 buffer using an initial concentration of 600 U of enzyme in a 2-fold serial dilution using diluent A.
[0028] FIG. 3A shows results in 1 hour.
[0029] FIG. 3B shows results in 14 hours.
[0030] FIGS. 4A-B show the cleavage of 0.6 .mu.l pBR322 substrate in a 2-fold serial dilution of BamHI-HF (E163A/E167T) using diluent A after 14 hours incubation in two different buffers on an agarose gel.
[0031] FIG. 4A shows the results with NEB2 buffer with an initial concentration of 600 U of enzyme.
[0032] FIG. 4B shows the results with NEB1 buffer with an initial concentration of 2,400 U of enzyme.
[0033] FIGS. 5A-B show a comparison of cleavage reactions using BamHI-HF and WT BamHI in NEB4 buffer. The reaction was carried out in NEB4 buffer using 1.2 .mu.l lambda DNA substrate in a 2-fold serial dilution using diluent A.
[0034] FIG. 5A shows WT BamHI with a starting concentration of 1,200 U where the FI equals 4.
[0035] FIG. 5B shows BamHI-HF with a starting concentration of 2,400 U where the FI.gtoreq.4000.
[0036] FIGS. 6A-D show a comparison of WT EcoRI and EcoRI(K62A) in NEB1-4 buffers in a 3-fold serial dilution using NEB diluent C. The reaction mixture contained 2 .mu.l lambda DNA substrate (1 .mu.g) in NEB1-4 buffers.
[0037] FIG. 6A shows the cleavage results following 2-fold serial dilution, 120 U WT EcoRI and 240 U of EcoRI (K62A) in NEB2 buffer.
[0038] FIG. 6B shows the cleavage results following 2-fold serial dilution, 120 U WT EcoRI and 240 U of EcoRI (K62A) in NEB4 buffer.
[0039] FIG. 6C shows the cleavage results following 2-fold serial dilution, 60 U WT EcoRI and 120 U of EcoRI (K62A) in NEB1 buffer.
[0040] FIG. 6D shows the cleavage results following 2-fold serial dilution, 120 U WT EcoRI and 60 U of EcoRI (K62A) in NEB3 buffer.
[0041] FIGS. 7A-C show the cleavage results with 2 different EcoRI mutants and WT EcoRI. The digestion of 100,000 U of enzyme and a 10-fold serial dilution thereof in diluent C over 10 hours using 0.6 .mu.l of Litmus28 substrate in various buffers is shown. There is only one EcoRI cleavage site in Litmus28 substrate.
[0042] FIG. 7A: EcoRI mutant K62E in NEB4 buffer.
[0043] FIG. 7B: EcoRI mutant K62A in NEB4 buffer.
[0044] FIG. 7C: WT EcoRI in EcoRI buffer (see NEB catalog 2007-8).
[0045] FIGS. 8A-B shows a comparison of EcoRI-HF and WT EcoRI in NEB4 buffer. The reaction utilized 1.2 .mu.l lambda DNA substrate in a 2-fold serial dilution using diluent C.
[0046] FIG. 8A: WT EcoRI with a starting concentration of 19,200 U reveals a FI=4 in NEB4 buffer.
[0047] FIG. 8B: EcoRI-HF with a starting concentration of 38,400 U reveals a FI=16,000 in NEB4 buffer.
[0048] FIGS. 9A-B shows a comparison of serial dilutions of WT ScaI (4.8 U), ScaI(H193A) (9.6 U), ScaI(S201F) (19.2 U), and ScaI(H193A/S201F) (19.2 U). Each sample was initially diluted by 1/10, followed by a 2-fold serial dilution in NEB2 buffer with the specified percentage of glycerol. Each reaction mixture contains 2 .mu.l of lambda DNA substrate (1 .mu.g).
[0049] FIG. 9A: 5% glycerol.
[0050] FIG. 9B: 37% glycerol.
[0051] FIGS. 10A-D shows a comparison of WT ScaI and ScaI-HF (H193A/S201F). The enzymes (unit concentration as specified) were each diluted in a 2.5-fold serial dilution with diluent A. The reaction mixture contains 2 .mu.l lambda DNA substrate and NEB1-4 buffers.
[0052] FIG. 10A: cleavage in NEB2 buffer.
[0053] FIG. 10B: cleavage in NEB4 buffer.
[0054] FIG. 10C: cleavage in NEB1 buffer.
[0055] FIG. 10D: cleavage in NEB3 buffer.
[0056] FIGS. 11A-H show the FI determination for SalI-HF and WT SalI. Both enzymes were diluted in 2-fold serial dilutions using diluent A. The reaction mixture contains 2 .mu.l HindIII-digested lambda DNA substrate.
[0057] FIGS. 11A, B, C and D show a serial dilution of 1,200 U, 1,200 U, 300 U and 1,200 U of SalI-HF demonstrating a FI.gtoreq.1,000, FI.gtoreq.2,000, FI.gtoreq.500 and FI.gtoreq.2,000 in NEB1, 2, 3 and 4 buffers, respectively.
[0058] FIGS. 11E, F, G, and H show a serial dilution of 19.2 U, 150 U, 9,600 U and 38.4 U of WT SalI demonstrating a FI=8, FI=1, FI=32 and FI=1 in NEB1, 2, 3 and 4 buffers, respectively.
[0059] FIGS. 12A-B show the results of a 2-fold serial dilution of SphI-HF (19,200 U) in diluent A and WT SphI (143,600 U) in diluent B reacted in NEB 4 buffer with 1.2 .mu.l lambda DNA substrate.
[0060] FIG. 12A shows cleavage by WT SphI.
[0061] FIG. 12B shows cleavage by SphI-HF.
[0062] FIGS. 13A-B show cleavage of 1.2 .mu.l lambda DNA substrate using 2-fold serial dilutions of PstI-HF (300 U and 150 U) and 2-fold serial dilutions of WT PstI (2,400 U and 4,800 U) in NEB3 and NEB4 buffers, respectively. Serial dilutions were performed in diluent C.
[0063] FIG. 13A shows cleavage by PstI-HF in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0064] FIG. 13B shows cleavage by WT PstI in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0065] FIGS. 14A-B show cleavage of 1.2 .mu.l lambda DNA substrate using 2-fold serial dilutions of NcoI-HF (4,800 U and 600 U) and 2-fold serial dilutions of WT NcoI (4,800 U and 1,200 U) in NEB3 and NEB4 buffers, respectively. Serial dilutions were performed in diluent A.
[0066] FIG. 14A shows cleavage by NcoI-HF in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0067] FIG. 14B shows cleavage by WT NcoI in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0068] FIGS. 15A-B show cleavage of 1.2 .mu.l pXba DNA substrate using 2-fold serial dilutions of NheI-HF (287,200 U and 76.8 U) and 2-fold serial dilutions of WT NheI (9,600 U and 300 U) in NEB3 and NEB4 buffers, respectively. Serial dilutions were performed in diluent A.
[0069] FIG. 15A shows cleavage by NheI-HF in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0070] FIG. 15B shows cleavage by WT NheI in NEB4 buffer (upper panel) and NEB3 buffer (lower panel.)
[0071] FIGS. 16A-B show cleavage of 1.2 .mu.l lambda DNA substrate using 2-fold serial dilutions of SspI-HF (9,600 U and 38.4 U) and 2-fold serial dilutions of WT SspI (19,200 U and 19,200 U) in NEB3 and NEB4 buffers, respectively. Serial dilutions were performed in diluent C.
[0072] FIG. 16A shows cleavage by SspI-HF in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0073] FIG. 16B shows cleavage by WT SspI in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0074] FIGS. 17A-B show cleavage of 1.2 .mu.l pXba DNA substrate using 2-fold serial dilutions of NotI-HF (287,200 U and 19,200 U) and 2-fold serial dilutions of WT NotI (19,200 U and 76,800 U) in NEB3 and NEB4 buffers, respectively. Serial dilutions were performed in diluent C.
[0075] FIG. 17A shows cleavage by NotI-HF in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0076] FIG. 17B shows cleavage by WT NotI I in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0077] FIGS. 18A-B show cleavage of 1.2 .mu.l pXba DNA substrate using 2-fold serial dilutions of SacI-HF (4,800 U and 76.8 U) and 2-fold serial dilutions of WT SacI (19,200 U and 1200 U) in NEB3 and NEB4 buffers, respectively. Serial dilutions were performed in diluent A.
[0078] FIG. 18A shows cleavage by SacI-HF in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0079] FIG. 18B shows cleavage by WT SacI in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0080] FIGS. 19A-B show cleavage of 0.6 .mu.l pBR322 DNA substrate using 2-fold serial dilutions of PvuII-HF (9,600 U and 19.2 U) and 2-fold serial dilutions of WT PvuII (19,200 U and 300 U) in NEB3 and NEB4 buffers, respectively. Serial dilutions were performed in diluent A.
[0081] FIG. 19A shows cleavage by PvuII-HF in NEB4 buffer (upper panel) and NEB3 buffer (lower panel) FIG. 19B shows cleavage by WT PvuII in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0082] FIGS. 20A-B show cleavage of 1.2 .mu.l lambda DNA substrate using 2-fold serial dilutions of MfeI-HF (300 U and 19.2 U) and 2-fold serial dilutions of WT MfeI (2,400 U and 38.4 U) in NEB3 and NEB4 buffers, respectively. Serial dilutions were performed in diluent A.
[0083] FIG. 20A shows cleavage by MfeI-HF in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0084] FIG. 20B shows cleavage by WT MfeI in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0085] FIGS. 21A-B show cleavage of 1.2 .mu.l lambda DNA substrate using a 2-fold serial dilution of HindIII-HF (4,800 U and 1,200 U) and a 2-fold serial dilution of WT HindIII (9,600 U and 4,800 U) in NEB3 and NEB4 buffers, respectively. Serial dilutions were performed in diluent A.
[0086] FIG. 21A shows cleavage by HindIII-HF in NEB4 buffer (upper panel) and NEB3 buffer (lower panel).
[0087] FIG. 21B shows cleavage by WT HindIII in NEB4 buffer (upper panel) and NEB3 buffer (lower panel)
[0088] FIGS. 22A-B show cleavage of 1.2 .mu.l lambda DNA substrate in a 2-fold serial dilution using diluent C of SbfI-HF (starting concentration: 76,800 U) and WT SbfI (starting concentration: 76,800 U) in NEB4 buffer.
[0089] FIG. 22A shows cleavage by WT SbfI.
[0090] FIG. 22B shows cleavage by SbfI-HF.
[0091] FIGS. 23A-B shows cleavage of 1.2 .mu.l pXba DNA substrate in a 2-fold serial dilution of EagI-HF (1,200 U and 600 U) and a 2-fold serial dilution of WT EagI (150 U and 38.4 U) in NEB2 and NEB1 buffers, respectively, using diluent C.
[0092] FIG. 23A shows cleavage by EagI-HF in NEB2 buffer (upper panel) and NEB1 buffer (lower panel).
[0093] FIG. 23B shows cleavage by WT EagI in NEB2 buffer (upper panel) and NEB1 buffer (lower panel).
[0094] FIGS. 24A-B show cleavage of 1.2 .mu.l pXba DNA substrate in a two-fold serial dilution using diluent A of EcoRV-HF (starting concentration: 38,400 U) and WT EcoRV (starting concentration: 2400 U) in NEB4 buffer.
[0095] FIG. 24A shows cleavage by WT EcoRV.
[0096] FIG. 24B shows cleavage by EcoRV-HF.
[0097] FIGS. 25A-B show cleavage of 1.2 .mu.l T7 DNA substrate in a two-fold serial dilution using diluent A of AvrII-HF (starting concentration: 1,200 U) and WT AvrII (starting concentration: 1,200 U) in NEB4 buffer.
[0098] FIG. 25A shows cleavage by WT AvrII.
[0099] FIG. 25B shows cleavage by AvrII-HF.
[0100] FIGS. 26A-B show cleavage of 1.2 .mu.l lambda DNA substrate by a two-fold serial dilution in diluent A of BstXI-HF (starting concentration: 300 U) and WT BstXI (starting concentration: 38.4 U) in NEB4 buffer. The reaction was performed at 55.degree. C.
[0101] FIG. 26A shows cleavage by WT BstXI.
[0102] FIG. 26B shows cleavage by BstXI-HF.
[0103] FIGS. 27A-B show cleavage of 1.2 .mu.l pXba DNA substrate in a two-fold serial dilution using diluent A of PciI-HF (starting concentration: 600 U) and WT PciI (starting concentration 300 U) in NEB4 buffer.
[0104] FIG. 27A shows cleavage by WT PciI.
[0105] FIG. 27B shows cleavage by PciI-HF.
[0106] FIGS. 28A-B show cleavage of 1.2 .mu.l lambda DNA substrate in a two-fold serial dilution using diluent A of HpaI-HF (starting concentration: 4,800 U) and WT HpaI (starting concentration 9,600 U) in NEB2 buffer.
[0107] FIG. 28A shows cleavage by WT HpaI.
[0108] FIG. 28B shows cleavage by HpaI-HF.
[0109] FIGS. 29A-B show cleavage of 1.2 .mu.l pXba DNA substrate in a two-fold serial dilution of AgeI-HF using diluent C (starting concentration: 600 U) and WT AgeI (starting concentration 600 U) in NEB4 buffer.
[0110] FIG. 29A shows cleavage by WT AgeI.
[0111] FIG. 29B shows cleavage by AgeI-HF.
[0112] FIGS. 30A-B show cleavage of 1.2 .mu.l lambda DNA substrate in a two-fold serial dilution using diluent A of BsmBI-HF (starting concentration: 300 U) and WT BsmBI (starting concentration 4,800 U) in NEB4 buffer. The reaction is at 55.degree. C.
[0113] FIG. 30A shows cleavage by WT BsmBI.
[0114] FIG. 30B shows cleavage by BsmBI-HF.
[0115] FIGS. 31A-B show the DNA sequence (SEQ ID NO:1) and protein sequence (SEQ ID NO:2) of MluCIM, respectively, for expression of EcoRI and MfeI.
[0116] FIG. 32 shows the DNA sequence (SEQ ID NO:3) of Hpy166IIM for expression of SalI.
[0117] FIGS. 33A-B show the DNA sequence (SEQ ID NO:4) and protein sequence (SEQ ID NO:5) of MfeI, respectively.
[0118] FIGS. 34A-B show the DNA sequence (SEQ ID NO:6) and protein sequence (SEQ ID NO:7) of BstXI, respectively.
[0119] FIGS. 35A-B show the DNA sequence (SEQ ID NO:8) and protein sequence (SEQ ID NO:9) of M.BstXI, respectively.
[0120] FIGS. 36A-B show the DNA sequence (SEQ ID NO:10) and protein sequence (SEQ ID NO:11) of S.BstXI, respectively.
[0121] FIGS. 37A-B show the DNA sequence (SEQ ID NO:12) and protein sequence (SEQ ID NO:13) of PciI, respectively.
[0122] FIGS. 38A-B show the DNA sequence (SEQ ID NO:14) and protein sequence (SEQ ID NO:15) of M.PciI, respectively.
[0123] FIG. 39 shows the DNA sequence (SEQ ID NO:17) encoding MI.EarI that recognizes CTCTTC and methylates at N4 cytosine or N6 adenine for cloning SapI and BspQI.
[0124] FIG. 40 shows the DNA sequence (SEQ ID NO:18) encoding M2.EarI that recognizes CTCTTC and methylates at N4 cytosine or N6 adenine for cloning SapI and BspQI.
[0125] FIGS. 41A-B show agarose gels to determine the FI of 1 .mu.l WT BspQI and 1 .mu.l mutant (K279P/R388F) BspQI (starting concentrations 512 U and 1,024 U, respectively) by cleaving 1 .mu.l pUC19 DNA substrate in a two-fold serial dilution using diluent A and NEB1 buffer plus 10% glycerol. The reaction was conducted at 50.degree. C.
[0126] FIG. 42 shows agarose gels to determine the FI of 5 .mu.l WT SapI and 5 .mu.l mutant (K273A) SapI (starting concentrations 32 U and 16 U, respectively) by cleaving pUC19 DNA substrate in a two-fold serial dilution using diluent A and NEB2 buffer plus 25% DMSO.
[0127] FIGS. 43A-B show catalytic and star activity of pXba by 5 .mu.l WT KpnI and 5 .mu.l D16N/E132A/D148E KpnI (initial concentrations 32 U and 256 U, respectively). The enzyme digested 2 .mu.l pXba DNA substrate (0.5 .mu.g) in a 2-fold serial dilution in NEB2 buffer using a diluent containing 10 mM Tris-HCl, pH 7.4, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT and 50% glycerol with the total volume made up to 50 .mu.l with water.
[0128] FIG. 43A shows the cleavage results of KpnI.
[0129] FIG. 43B shows the cleavage results of D16N/E132A/D148E KpnI.
[0130] FIG. 44A-G show the amino acid sequences of the enzymes in Table 1 and the DNA sequences of the enzymes in Table 1 that have not been previously disclosed.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0131] Embodiments of the invention provide a general method for selecting for restriction endonucleases with desired characteristics. The general method relies on a suitable assay for determining whether the desired restriction endonuclease has been created. In particular an embodiment of the general method provides a systematic screening method with a set of steps. This method has been deduced by performing many hundreds of reactions using many restriction endonucleases. The majority of the examples provided herein relate to identifying restriction endonucleases with reduced star activity but with cleavage activity that is at least similar to the WT restriction endonuclease. However, it is expected that the same methodology can be applied successfully to modifying other properties of the restriction endonucleases relating, for example, to improved cleavage activity in desired buffers, thermostability, rate of reaction in defined conditions, etc.
[0132] As discussed above, an end point of interest is to transform restriction endonucleases with star activity into high fidelity restriction endonucleases with significantly reduced star activity. Star activity refers to promiscuity in cleavage specificity by individual restriction endonucleases. The terms "reduction in star activity" and "increase in fidelity" are used interchangeably here. Although restriction endonucleases are characterized by their property of cleaving DNA at specific sequences, some restriction endonucleases additionally cleave DNA inefficiently at secondary sites in the DNA. This secondary cleavage may occur consistently or may arise only under certain conditions such as any of: increased concentrations, certain buffers, temperature, substrate type, storage, and incubation time.
[0133] It is generally acknowledged that little is known about the complex environment generated by the hundreds of amino acids that constitute a protein and determine specificity. One approach in the prior art has been to utilize crystallography to identify contact points between an enzyme and its substrate. Nonetheless, crystallography has limitations with respect to freezing a structure in time in an unnatural chemical environment.
[0134] The rules that determine the contribution of amino acids at any site in the protein and the role played by the structure of the substrate molecule has proved elusive using existing analytical techniques. For example, it is shown here that mutating an amino acid in a restriction endonuclease can cause all or partial loss of activity.
[0135] In this context, no structural explanation has been put forward to explain why star activity could increase with high glycerol concentration (>5% v/v), high enzyme to DNA ratio (usually >100 units of enzyme per .mu.g of DNA), low ionic strength (<25 mM salt), high pH (>8.0), presence of organic solvent (such as DMSO, ethanol), and substitution of Mg.sup.2+ with other divalent cations (Mn.sup.2+, Co.sup.2+). It was here recognized that because of the diversity of factors affecting star activity, it would be necessary to conduct comparisons of WT and mutant star activity under the same reaction conditions and in the same predetermined buffer and to develop a standard reaction condition in which any high fidelity enzyme must be capable of showing the described characteristics even if these characteristics were also observed in other reaction conditions.
[0136] Present embodiments of the invention are directed to generating modified restriction endonucleases with specific improved properties, namely enhanced cleavage fidelity without significant reduction in overall cleavage activity or significant loss of yield from the host cells that make the protein. The methods that have been developed here for finding mutants with improved properties have resulted from exhaustive experimentation and the properties of the resultant enzymes have been defined in the context of specified conditions. The methods described herein may be used for altering the enzymatic properties of any restriction endonuclease under predetermined conditions, but are not limited to the specific defined conditions.
TABLE-US-00001 Restriction Steps Used to Generate a High Fidelity Restriction Endonuclease Endonuclease BamHI Comparison of isoschizomer (Ex. 1) Targeted 22 residues to mutate to Ala. 14 mutants obtained, 3 had improved fidelity Saturation mutagenesis on 2 residues-K30 and E86 Recovered E86P as preferred mutant with greatest reduced star activity in selected buffers. Added mutations to E86P. Second round of mutation (Arg, Lys, His, Asp, Glu, Ser, Thr) to Ala and Tyr to Phe. Selected E167 and Y165 for saturation mutagenesis and selected E167T and Y165F. E163A/E167T was selected as preferred high fidelity mutant (BamHI-HF). EcoRI Comparison of isoschizomer (Ex. 2) Targeted 42 charged residues to mutate to Ala. No high fidelity mutants Second round of mutation: Target additional 32 charged residues to mutate to Ala: Identified K62A. Saturation mutagenesis on K62A. EcoRI( K62E) was selected as a preferred high fidelity mutant (EcoRI-HF). ScaI Comparison of isoschizomers. (Ex. 3) Targeted 58 charged residues to mutate to Ala. Identify 4 mutants Preferred mutant of 4 is (H193A/S201F). This is selected as a preferred high fidelity mutant (ScaI-HF) SalI Target 86 charged residues and mutate to Ala. SalI (Ex. 4) (R107A) was preferentially selected as a preferred high fidelity mutant (SalI-HF). SphI Target 71 charged residues and mutate to Ala. SphI (Ex. 5) (K100A) was preferentially selected as a preferred high fidelity mutant (SphI-HF) PstI Target 92 charged amino acids and mutate to Ala. PstI (Ex. 6) (D91A) was preferentially selected as a preferred high fidelity mutant (PstI-HF) NcoI Target 66 charged residues and mutate to Ala. NcoI (Ex. 7) (A2T/R31A) was preferentially selected as a preferred high fidelity mutant (NcoI-HF). NheI Target 92 charged residues and mutate to Ala. NheI (Ex. 8) (E77A) was preferentially selected as a preferred high fidelity mutant (NheI-HF) SspI Target 81 charged residues and mutate to Ala. No (Ex. 9) preferential mutants obtained. Target 95 residues to additional charged residues and hydroxylated residues to Ala except Tyr. Tyr mutated to Phe. SspI (Y98F) was preferentially selected as a preferred high fidelity mutant (SspI-HF) NotI Target 97 charged residues and mutate to Ala. K150A (Ex. 10) was preferentially selected as a preferred high fidelity mutant (NotIHF) SacI Target 101 charged residues and mutate to Ala. SacI (Ex. 11) (Q117H/R200A) was preferentially selected as a preferred high fidelity mutant (SacI-HF) where Q117H was a carry over mutation from template with no affect on activity PvuII Target 47 charged residues and mutate to Ala. No (Ex. 12) preferred mutants obtained Target 19 hydroxylated residues--Ser/Thr and Tyr. Select T46A for further improvement Saturation mutagenesis results in a preferred mutant T46G, T46H, T46K, T46Y. PvuII(T46G) was preferentially selected as a preferred high fidelity mutant (PvuII-HF) MfeI Target 60 charged residues and mutate to Ala. No (Ex. 13) preferred mutants obtained Target 26 hydroxylated residues and mutate to Ala except for Tyr which was changed to Phe. Target 38 residues (Cys, Phe, Met, Asn, Gln, Trp) and mutate to Ala Identify Mfe (Q13A/F35Y) as a preferred high fidelity mutant (MfeI-HF) where F35Y is carried from the template HindIII Target 88 charged residues and mutate to Ala. No (Ex. 14) preferred mutants obtained Target 103 residues (Cys Met Asn, Gln, Ser Thr Trp) and mutate to Ala and Tyr changed to Phe. Identify HindIII (K198A) as a preferred high fidelity mutant (HindIII-HF) SbfI Target 78 charged residues mutated to Ala (Ex. 15) Target 41 residues (Ser Thr) mutated to Ala/Tyr to Phe Target 55 residues of Cys, Phe, Met Asn, Gln, Trp to Ala SbfI (K251A) was selected as a preferred high fidelity mutant (SbfI-HF) EagI Target 152 residues (Asp, Glu, His, Lys, Arg, Ser, thr, (Ex. 16) Asn, and Gln changed to Ala and Tyr changed to Phe). EagI H43A was selected as a preferred high fidelity mutant (EagIHF) EcoRV Target 162 residues (Cys,Asp, Glu, Phe, his, Lys, Met, (Ex. 17) Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) EcoRV (D19A/E27A) was selected as a preferred high fidelity mutant (EcoRV-HF) AvrII Target 210 residues (Cys,Asp, Glu, Phe, his, Lys, Met, (Ex. 18) Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) AvrII (Y104F) was selected as a preferred high fidelity mutant (AvrII-HF) BstXI Target 237 residues (Cys, Asp, Glu, Phe, his, Lys, Met, (Ex. 19) Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) BstXI (N65A) was selected as a preferred high fidelity mutant (BstXI-HF) PciI Target 151 residues (Cys, Asp, Glu, Phe, his, Lys, Met, (Ex. 20) Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) PciI (E78A/S133A) was selected as a preferred high fidelity mutant. (PciI-HF) This was spontaneous and not one of the 151 separate mutations HpaI Target 156 residues (Cys, Asp, Glu, Phe, his, Lys, Met, (Ex. 21) Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) HpaI (E56A) was selected as a preferred high fidelity mutant (HpaI-HF) AgeI Target 149 residues (Cys, Asp, Glu, Phe, his, Lys, Met, (Ex. 22) Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) AgeI (R139A) was selected as a preferred high fidelity mutant (AgeI-HF) BsmBI Target 358 residues (Cys, Asp, Glu, Phe, his, Lys, Met, (Ex. 23) Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) BsmBI(N185Y/R232A) was selected as a preferred high fidelity mutant (BsmBI (HF) BspQI Target 122 residues (Arg, Lys, His, Glu, Asp, Gln, Asn, (Ex. 24) Cys) Replace R at position 279 with Phe, Pro, Tyr, Glu, Asp or Leu. Preferred mutations were R388F and K279P. Created a double mutant BspQI(K279P/R388F) as preferred high fidelity mutant (BspQI-HF) SapI Find K273 and R380 in SapI corresponding to R388 and (Ex. 25) K279 in BspQI. SapI (K273P/R380F) was selected as a preferred high fidelity mutant (SapI-HF) KpnI Target all residues (Asp, Glu, Arg, Lys, His, Ser, Thr, (Ex. 26) Tyr, Asn, Gln, Phe, Trp, Cys, Met) to Ala. More mutation was done on site D16 and D148. A combined D16N/E132A/D148E was selected as a preferred high fidelity mutant (KpnI-HF). BsaI Find 11 amino acids corresponding to the site in BsmBI. (Ex. 27) BsaI (Y231F) was selected as a preferred high fidelity mutant (BsaI-HF).
[0137] The method follows from the realization that amino acids responsible for cognate activity and star activity are different. The engineering of high fidelity restriction endonucleases described herein demonstrates that cognate activity and star activity can be separated and there are different critical amino acid residues that affect these different activities. The locations of amino acids that are here found to affect star activity are not necessarily found within the active site of the protein. The cleavage properties of any restriction endonuclease has been determined here for the first time by developing a criterion of success in the form of determining a FI (see also Wei et al. Nucleic Acid Res., 36, 9, e50 (2008)) and an overall fidelity index improvement factor.
[0138] An "overall fidelity index improvement factor" refers to the highest FI for a mutant with maximum cleavage activity divided by the highest FI of the corresponding WT endonuclease with maximum cleavage activity within a selected set of buffers. The selected set may be of any size greater than one but practically will contain less than 10 different buffers and more preferably contains 4 buffers. The set may also include less than 4 buffers. The overall FI improvement factor of at least two should preferably be applicable for any mutant restriction endonuclease in the claimed invention additionally but not exclusively to the set of buffers consisting of NEB1, NEB2, NEB3 and NEB4.
[0139] A "similar cleavage activity" can be measured by reacting the same amount of enzyme with the same amount and type of substrate under the same conditions and visually comparing the cleavage profiles on a gel after electrophoresis such that the amount of cleavage product appears to be the same within a standard margin of error and wherein the quantitative similarity is more than 10%.
[0140] "Artificial" refers to "man-made".
[0141] "Standard conditions" refers to an overall FI improvement factor calculated from results obtained in NEB1-4 buffers.
[0142] The general method described herein has been exemplified with 27 restriction endonucleases: AgeI, AvrII, BamHI, BsaI, BsmBI, BspQI, BstXI, EagI, EcoRI, EcoRV, HindIII, HpaI, KpnI, MfeI, NcoI, NheI, NotI, PciI, PstI, PvuII, SacI, SalI, SapI, SbfI, ScaI, SphI and SspI restriction endonucleases. However, as mentioned above, the method is expected to be effective for the engineering of any restriction endonuclease that has significant star activity.
[0143] Embodiments of the method utilize a general approach to create mutant restriction endonucleases with reduced star activity. For certain enzymes, it has proven useful to mutate charged residues that are determined to be conserved between two isoschizomers (see for example SapI in Example 25). In general, however, the method involves a first step of identifying all the charged and polar residues in a protein sequence for the endonuclease. For example, charged amino acids and polar residues include the acidic residues Glu and Asp, the basic residues His, Lys and Arg, the amide residues Asn and Gln, the aromatic residues Phe, Tyr and Trp and the nucleophilic residue Cys. Individual residues are targeted and mutated to an Ala and the products of these targeted mutations are screened for the desired properties of increased fidelity. If none of the mutants obtained provide a satisfactory result, the next step is to target mutations to all the hydroxylated amino acids, namely, Ser, Thr and Tyr, the preferred mutation being Ser and Thr to Ala and Tyr to Phe. It is also possible to target mutations to both classes of residues at one time as was done for Examples 16-23. The mutation to Ala may be substituted by mutations to Val, Leu or Ile.
[0144] After these analyses, if one or more of the preferred mutants generated in the above steps still have substandard performance under the selected tests, these mutants can be selected and mutated again to each of the additional possible 18 amino acids. This is called saturation mutagenesis. Saturation mutagenesis provided the preferred high fidelity mutants for EcoRI (Example 2), BamHI in part (Example 1) and PvuII (Example 12). Depending on the results of saturation mutagenesis, the next step would be to introduce additional mutations either targeted or random or both into the restriction endonuclease. In Example 11, SacI-HF includes a random mutation generated fortuitously during inverse PCR. In Example 20, PciI-HF resulted from a random mutation and not from targeted mutations. In Example 26, BspQI-HF contains two mutations that were found to act synergistically in enhancing fidelity.
[0145] The use of various methods of targeted mutagenesis such as inverse PCR may involve the introduction of non-target mutations at secondary sites in the protein. These secondary mutations may fortuitously provide the desired properties (see Example 20). It is desirable to examine those mutated enzymes with multiple mutations to establish whether all the mutations are required for the observed effect. In Example 11, Q117H in the double mutant had no effect on activity. In Example 20, the additional spontaneous mutation appears to be solely responsible for the observed improved fidelity, whereas in Example 24, the individual mutations acted synergistically.
[0146] In some cases, a mutation may provide an additional advantage other than improved fidelity (see for example BamHI in which either E163A or P173A causes the enzyme to become more thermolabile).
[0147] The high fidelity/reduced star activity properties of the mutants provided in the Examples were selected according to their function in a set of standard buffers. Other mutations may be preferable if different buffer compositions were selected. However, the same methodology for finding mutants would apply. Table 4 lists mutations which apply to each restriction endonuclease and provide an overall FI improvement factor in the standard buffer.
[0148] The engineering of the high fidelity restriction endonucleases to provide an overall FI improvement factor of at least 2 involves one or more of the following steps:
1. Assessment of the Star Activity of the WT Restriction Endonuclease
[0149] In an embodiment of the invention, the extent of star activity of a restriction endonuclease is tested by means of the following protocol: the endonuclease activity is determined for an appropriate substrate using a high initial concentration of a stock endonuclease and serial dilutions thereof (for example, two-fold or three-fold dilutions). The initial concentration of restriction endonuclease is not important as long as it is sufficient to permit an observation of star activity in at least one concentration such that on dilution, the star activity is no longer detected.
[0150] An appropriate substrate contains nucleotide sequences that are cleaved by cognate endonuclease activity and where star activity can be observed. This substrate may be the vector containing the gene for the restriction endonuclease or a second DNA substrate. Examples of substrates used in Table 2 are pBC4, pXba, T7, lambda, and pBR322.
[0151] The concentration of stock restriction endonuclease is initially selected so that the star activity can be readily recognized and assayed in WT and mutated restriction endonucleases. Appropriate dilution buffers such as NEB diluent A, B or C is selected for performing the serial dilutions according to guidelines in the 2007-08 NEB catalog. The serially diluted restriction endonuclease is reacted with a predetermined concentration of the appropriate substrate in a total reaction volume that is determined by the size of the reaction vessel. For example, it is convenient to perform multiple reactions in microtiter plates where a 30 .mu.l reaction mixture is an appropriate volume for each well. Hence, the examples generally utilize 0.6 .mu.g of substrate in 30 .mu.l, which is equivalent to 1 .mu.g of substrate in 50 .mu.l. The amount of substrate in the reaction mixture is not critical, but it is preferred that it be constant between reactions. The cleavage reaction occurs at a predetermined temperature (for example 25.degree. C., 30.degree. C., 37.degree. C., 50.degree. C., 55.degree. C. or 65.degree. C.) for a standard time such as one hour. The cleavage products can be determined by any standard technique, for example, by 0.8% agarose gel electrophoresis to determine the fidelity indices as defined above.
[0152] Not all restriction endonucleases have significant star activity as determined from their FI. However, if an endonuclease has a highest FI of no more than about 250 and a lowest FI of less than 100, the restriction endonuclease is classified as having significant star activity. Such endonucleases are selected as a target of enzyme engineering to increase fidelity for a single substrate. In some cases, the restriction endonucleases with both FI over about 500 and FI less than about 100 are also engineered for better cleavage activity.
[0153] Table 2 below lists the FI of some engineered restriction endonucleases before engineering. All samples were analyzed on 0.8% agarose gel.
TABLE-US-00002 TABLE 2 Diluent Temp Enzyme (NEB)*** Substrate* .degree. C. FI-1** FI-2** FI-3** FI-4** AgeI C pXba 37 16(1) 8(1/2) 64(1/8) 8(1/2) AvrII B T7 37 64(1) 8(1) 32(1/4) 32(1) BamHI A .lamda. 37 4(1/2) 4(1) 32(1) 4(1/2) BsaI B pBC4 50 8(1/4) 120(1) 16(1/4) 32(1) BsmBI B .lamda. 55 1(1/8) 8(1/2) 120(1) 4(1/4) BspQI B .lamda. 50 2(1/8) 16(1) 32(1) 4(1/2) BstXI B .lamda. 55 2(1/2) 2(1/2) 2(1/8) 4(1) EagI B pXba 37 4(1/4) 8(1/2) 250(1) 16(1) EcoRI C .lamda. 37 250(1/2) 4(1) 250(1) 4(1) EcoRV A pXba 37 32(1/16) 120(1/2) 1000(1) 64(1/4) HindIII B .lamda. 37 32(1/4) 250(1) 4000(1/4) 32(1/2) HpaI A .lamda. 37 32(1/16) 1(1/4) 2(1/8) 16(1) KpnI A pXba 37 16(1) 16(1/4) 8(1/16) 4(1/2) MfeI A .lamda. 37 32(1) 16(1/8) 8(1/16) 32(1) NcoI A .lamda. 37 120(1) 32(1) 120(1/4) 32(1) NheI C pXba 37 32(1) 120(1/4) 120(1/8) 32(1) NotI C pXba 37 32000(1/16) 64(1) 500(1) 32(1/4) PciI A pXba 37 2000(1/2) 16(1/4) 120(1) 8(1/8) PstI C .lamda. 37 64(1) 32(1) 120(1) 8(1/2) PvuII A pBR32 37 250(1) 16(1/4) 8(1/32) 1/4(1) 2 SacI A pXba 37 120(1) 120(1/2) 120(1/32) 32(1/2) Sall A .lamda. (H3) 37 8(1/500) 1(1/16) 32(1) 1(1/120) SapI C .lamda. 37 16(1/4) 64(1/2) 32(1/4) 16(1) SbfI A .lamda. 37 32(1) 8(1/4) 8(1/16) 8(1/2) ScaI A .lamda. 37 1/16(1/32) 1/8(1) 4(1/2) 1/64(1/16) SphI B .lamda. 37 64(1) 32(1) 64(1/4) 16(1/2) SspI C .lamda. 37 64(1) 16(1) 32(1/4) 16(1) *Substrate: +80 +0 is lambda phage DNA; +80 +0 (H3) is HindIII-digested lambda phage DNA; pXba is pUC19 with XbaI-digested fragment of Adeno Virus; pBC4: a shorter version of pXBa; T7: T7 DNA **FI-1 to FI-4: fidelity index of the enzyme in NEBuffer 1, 2, 3 and 4. The number in parenthesis is a value for relative cleavage activity of the mutant restriction endonuclease in a specified buffer in a set of buffers compared with the +37 best+38 +0 cleavage activity of the same mutant restriction endonuclease in any of the buffers in the set of buffers. The compositions of NEB buffers follow: NEB1: 10 mM Bis Tris Propane-HCl, 10 mM MgCl+hd 2+l , 1 mM dithiohreitol (pH 7.0 at at 25+20 +0 C.); NEB2: 50 mM NaCI, 10 mM Tris-HCl, 10 mM MgCl+hd 2+l , 1 mM dithiothreitol (pH 7.9 at 25+20 +0 C.); NEB3: 100 mM NaCl, 50 mM Tris-HCl, 10 MM MgCl+hd 2+l , 1 mM dithiothreitol (pH 7.9 at 25+20 +0 C.); NEB4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol (pH 7.9 at 25+20 +0 C.). ***The compositions of NEB diluents follow. (Using diluents in the dilution instead of water will keep the glycerol concentration in the reaction as a constant.) Diluent A: 50 mM KCl, 10 mg/ml Tris-HCl, 0.1 mN EDTA, 1 mM dithiothreitol, 220 mg/ml BSA. 50% glycerol (pH 7.4 at 25+20 +0 C.); Diluent B: 300 mM NaCl, 10 mM Tris-HCI, 0.1 mM EDTA, 1 mg/ml dithiothreitol, 500 mg/ml BSA, 50% glycerol (pH 7.4 at 25+20 +0 C.); Diluent C: 250 mM NaCl, 10 mM Tris-HCI, 0.1 mM EDTA, 1 mM dithiothreitol, 0.15% Triton X-100, 200 mg/ml BSA, 50% glycerol (pH 7.4 at 25+20 +0 C.).
2. Construction of High Expression Host Cell Strains
[0154] It is convenient if a host cell is capable of over-expressing the mutant restriction endonuclease for which reduced star activity is sought. If the restriction enzyme is highly expressed in E. coli, the star activity can be readily detected in the crude extract, which simplifies the screening for the high fidelity restriction endonuclease. However, the mutated restriction endonuclease can be expressed in any host cell providing that the host cell is protected in some way from toxicity arising from enzyme cleavage. This might include: the presence of a methylase; production in a compartment of the cell which provides a barrier to access to the genome (such as an inclusion body or the periplasm); in vitro synthesis; production in an emulsion (see U.S. patent application Ser. No. 12/035,872) absence of cleavage sites in the host genome; manufacture of the enzyme in component parts subject to intein mediated ligation (see U.S. Pat. No. 6,849,428), etc.
[0155] Over-expression of the mutated restriction endonucleases for purposes of production can be achieved using standard techniques of cloning, for example, use of an E. coli host, insertion of the endonuclease into a pUC19-derived expression vector, which is a high copy, and use of a relatively small plasmid that is capable of constant expression of recombinant protein. The vector may preferably contain a suitable promoter such as the lac promoter and a multicopy insertion site placed adjacent to the promoter. Alternatively, a promoter can be selected that requires IPTG induction of gene expression. If the activity in the crude extract is not sufficient, a column purification step for the restriction endonuclease in crude extract may be performed.
3. Mutagenesis of Restriction Endonuclease
[0156] DNA encoding each charged or polar group in the restriction endonuclease may be individually targeted and the mutated DNA cloned and prepared for testing. Multiple mutations may be introduced into individual restriction endonuclease genes. Targeted mutagenesis of restriction endonucleases may be achieved by any method known in the art. A convenient method used here is inverse PCR. In this approach, a pair of complementary primers that contains the targeted codon plus a plurality of nucleotides (for Example 18 nt) on both the 5' and 3' side of the codon is synthesized. The selection of suitable primers can be readily achieved by reviewing the gene sequence of the endonuclease of interest around the amino acid residue of interest. Access to gene sequences is provided through REBASE and GenBank. The sequences for the endonucleases described herein in the Examples are provided in FIGS. 31 to 38 and 44. The template for PCR is a plasmid containing the restriction endonuclease gene. The polymerase is preferably a high fidelity polymerase such as Vent.RTM. or Deep Vent.TM. DNA polymerase. By varying the annealing temperature and Mg.sup.2+ concentration, successful introduction of most mutations can be achieved. The PCR amplification product is then purified and preferably digested by DpnI. In an embodiment of the invention, the digested product was transformed into competent host cells (for example, E. coli), which have been pre-modified with a corresponding methylase. Colonies from each mutant were picked and grown under similar conditions to those in which the WT is grown (for example, using similar growth medium, drug selection, and temperature). The resulting restriction endonucleases were screened for reduced star activity.
4. Screening for Mutant Restriction Endonucleases with Reduced Star Activity
[0157] Conditions such as buffer composition, temperature and diluent should be defined for determining star activity in a mutant restriction endonuclease. Tables 2 and 3 show the FI of recombinant endonucleases before and after mutation in four different buffers using three different diluents at 37.degree. C. Accordingly, it is possible to determine which mutants have an overall desirable improved fidelity index factor of at least 2, more than 10, at least 50 or more than 500 and to select enzymes as preferred high fidelity mutants.
[0158] In an embodiment of the invention, the mutant restriction endonucleases were screened for activity in normal buffer conditions (no more than 5% glycerol) first. For those mutants with at least about 10% of activity of WT restriction endonuclease, activity was also determined in star activity promotion conditions that promoted star activity, for example, high glycerol concentration and optionally high pH. Preferably, the mutant with the least star activity but with acceptable cognate activity in normal buffers is selected. Plasmid can then be extracted and sequenced for the confirmation of the mutant. In some cases, the star activity is not easily measured, even with high glycerol and high pH conditions. Instead, the activity in different buffers is measured and compared, and the one with the highest cleavage activity ratio in NEB4 compared with NEB3 can be tested further for star activity improvement.
5. Saturation Mutagenesis on One Single Residue
[0159] As described in the previous section, the first step is to mutate a target amino acid in the restriction endonuclease to Ala. If the results are not satisfactory, saturation mutagenesis is performed. This is preferably performed by one of two methods. One method is to change the intended codon into NNN. After mutagenesis, multiple colonies are assayed under normal conditions and under conditions that promote star activity. Alternatively, a different codon can be selected for mutagenesis of each of the targeted amino acids for example: Ala: GCT; Cys: TGC; Asp: GAC; Glu: GAA; His: CAC; Ile: ATC; Lys: AAA; Leu: CTG; Met: ATG; Asn: AAC; Pro: CCG; Gln: CAG; Arg: CGT; Ser: TCC; Thr: ACC; Val: GTT; Trp: TGG and Tyr: TAC
6. Combination
[0160] More than one mutation can be introduced into the restriction endonuclease gene if a single mutation does not sufficiently reduce the star activity. Mutation combination and saturation mutagenesis can be performed in any order.
7. Mutant Purification and Assessment of the Improvement
[0161] The high fidelity mutants may be purified in a variety of ways including use of different chromatography columns. For normal quality assessment, one FPLC heparin column is enough to eliminate the DNA and non-specific nucleases from the preparation. Multiple columns including ion exchange, hydrophobic, size exclusion and affinity columns can be used for further purification.
[0162] Purified high fidelity restriction endonucleases are measured for FI in four NEB buffers and compared with the FIs of the WT restriction endonuclease. The ratio of FI for the high fidelity restriction endonuclease in its optimal buffer to that of WT is the overall improvement factor.
TABLE-US-00003 TABLE 3 FI* for exemplified restriction endonucleases Diluent Temp Enzyme (NEB) Substrate* .degree. C. FI-1** FI-2** FI-3** FI-4** AgeI-HF C pXba 37 .gtoreq.500(1) .gtoreq.250(1/2) .gtoreq.16(1/16) .gtoreq.250(1) AvrII-HF B T7 37 500(1) .gtoreq.500(1/2) .gtoreq.16(1/64) .gtoreq.1.000(1) BamHI- A .lamda. 37 .gtoreq.4000(1) .gtoreq.4000(1) .gtoreq.250(1/16) .gtoreq.4000(1) HF BsaI B pBC4 50 .gtoreq.4000(1/2) .gtoreq.8000(1) 120(1) .gtoreq.8000(1) BsmBI B .lamda. 55 2(1) .gtoreq.500(1) .gtoreq.64(1/8) .gtoreq.500(1) BspQI-HF A pUC19 50 .gtoreq.1.000(1/4) .gtoreq.1.000(1/4) .gtoreq.64(1/64) .gtoreq.4000(1) BstXI-HF A .lamda. 55 .gtoreq.120(1/2) .gtoreq.250(1) .gtoreq.16(1/16) .gtoreq.250(1) EagI-HF C pXba 37 250(1/2) 250(1) 250(1/2) 500(1) EcoRI-HF C .lamda. 37 2000(1/8) 4000(1/4) 250(1/250) 16000(1) EcoRV- A pXba 37 .gtoreq.16000(1/4) .gtoreq.64000(1) .gtoreq.32000(1/2) .gtoreq.64000(1) HF HindIII- B .lamda. 37 .gtoreq.16000(1/4) .gtoreq.64000(1) .gtoreq.16000(1/4) .gtoreq.32000(1/2) HF HpaI-HF A .lamda. 37 .gtoreq.32(1/32) .gtoreq.2000(1) .gtoreq.2(1/8) .gtoreq.2000(1/2) KpnI-HF A pXba 37 .gtoreq.4000(1) 1.gtoreq.000(1/4) .gtoreq.64(1/64) .gtoreq.4000(1) MfeI-HF A .lamda. 37 .gtoreq.1000(1) .gtoreq.250(1/4) .gtoreq.16(1/64) .gtoreq.500(1/2) NcoI-HF A .lamda. 37 .gtoreq.4000(1/4) .gtoreq.4000(1/4) .gtoreq.1000(1/16) .gtoreq.64000(1) NheI-HF C pXba 37 .gtoreq.128000(1) .gtoreq.4000(1/32) .gtoreq.32(1/2000) .gtoreq.32000(1/2) NotI-HF C pXba 37 .gtoreq.8000(1/16) .gtoreq.128000(1) .gtoreq.4000(1/64) .gtoreq.64000(1/2) PciI-HF A pXba 37 NC .gtoreq.2000(1) .gtoreq.2000(1) .gtoreq.1.000(1) PstI-HF C .lamda. 37 1000(1/8) 4000(1/2) 4000(1/4) 4000(1) PvuII-HF A pBR322 37 .gtoreq.250(1/120) .gtoreq.2000(1/16) .gtoreq.250(1/120) .gtoreq.500(1) SacI-HF A pXba 37 .gtoreq.32000(1) .gtoreq.16000(1/2) .gtoreq.500(1/64) .gtoreq.32000(1) SalI-HF A .lamda.(H3) 37 .gtoreq.8000(1/8) .gtoreq.64000(1) .gtoreq.4000(1/16) .gtoreq.32000(1/2) SbfI-HF C .lamda. 37 1000(1) 120(1/2) 8(1/32) 250(1) ScaI-HF A .lamda. 37 4000(1/8) 1000(1) 2000(1/32) 1000(1) SphI-HF B .lamda. 37 4000(1/8) 2000(1/16) 250(1/250) 8000(1) SspI-HF C .lamda. 37 .gtoreq.4000(1/2) 120(1/2) .gtoreq.32(1/128) 500(1) *The FI is a ratio of the highest concentration that does not show star activity to the lowest concentration that completes digestion of the substrate. **The number in parenthesis is a value for relative cleavage activity of the mutant restriction endonuclease in a specified buffer in a set of buffers compared with the greatest cleavage activity of the same mutant restriction endonuclease in any of the buffers in the set of buffers
TABLE-US-00004 TABLE 4 Mutations providing restriction endonucleases with high fidelity Restriction Endonuclease Examples of mutants with overall improved FI factor .gtoreq. 2 AgeI R139A; S201A* AvrII Y104F; M29A; E96A; K106A; S127A; F142A BamHI E163A/E167T; K30A; E86A; E86P; K87A; K87E; K87V; K87N; P144A; Y165F; E167A; E167R; E167K; E167L; E1671 K30A/E86A; E86A/K106A; K30A/E86A/K106A; K30A/K87A; E86P/K87E; E86A/Y165F; K30A/E167A; E163S/E170T/P173A; E163S/E170T/P173A; E86P/K87T/K88N/E163S/E170T/P173A; E86P/K87R/K88G/E163S/E170T/P173A; E86P/K87P/ K88R/E163S/E170T/P173A/E211K; E86P/K87T/K88R/ E163S/E170T/P173A/N158S; E86P/K87S/K88P/ E163S/E170T/P173A; E86P/K87G/K88S/ E163S/E170T/P173A; E86P/K87R/K88Q/ E163S/E170T/P173A; E86P/K87W/K88V; E86P/P173A BsaI Y231F BsmBI N185Y/R232A; H230A; D231A; R232A; BspQI K279P/R388F; K279A; K279F; K279P; K279Y; K279E; K279D R388A; R388F; R388Y; R388L; K279P/R388F; K279A/R388A; D244A BstXI N65A; Y57F; E75A; N76A; K199A; EagI H43A EcoRI K62A; K62S; K62L; R9A; K15A; R123A; K130A; R131A; R183A; S2Y; D135A; R187A; K62E EcoRV D19A; E27A; D19A/E27A HindIII S188P/E190A; K198A HpaI Y29F; E56A KpnI D148E; D16N/R119A/D148E; D2A/D16N/D148E; D16N/E134A/D148E; D16N/E132A/D148E MfeI Y173F; Q13A/F35Y NcoI D56A; H143A; E166A; R212A; D268A; A2T/R31A NheI E77A NotI K176A; R177A; R253A; K150A PciI E78A/S133A PstI E204G; K228A; K228A/A289V; D91A PvuII T46A; T46H; T46K; T46Y; T46G SacI Q117H/R154A/L284P; Q117H/R200A SalI R82A; K93A; K101A; R107A SapI K273P; R380A; K273P/R380A SbfI K251A ScaI R18A; R112A; E119A; H193A; S201F; H193A/S201F SphI D91A; D139A; D164A; K100A SspI H65A; K74A; E78A; E85A; E89A; K109A; E118A; R177A; K197A; Y98F The mutations for each enzyme are separated by a semicolon.
[0163] All references cited above and below, as well as U.S. provisional application Ser. No. 60/959,203, are incorporated by reference.
EXAMPLES
[0164] Where amino acids are referred to by a single letter code, this is intended to be standard nomenclature. The key to the code is provided for example in the NEB catalog 2007/2008 on page 280.
[0165] Plasmids used for cloning and as substrates have sequences as follows:
[0166] pLaczz2 (SEQ ID NO:102), pSyx20-lacIq (SEQ ID NO:105), pBC4 (SEQ ID NO:103), pXba (SEQ ID. NO:104) and pAGR3 (SEQ ID NO:106). pACYC is described in GenBank XO 6403, T7 in GenBank NC001604, pUC18 in GenBank L09136, and pRRS in Skoglund et al. Gene, 88:1-5 (1990. pSX33 was constructed by inserting lad gene into pLG339 at EcoRI site. pLG339 is described in Stoker, et al. Gene 19, 335-341 (1982).
[0167] All buffers identified as NEB buffers used herein are obtainable from New England Biolabs, Inc. (NEB), Ipswich, Mass.
Example 1: Engineering of High Fidelity BamHI
1. Extraction of Plasmids Containing BamHI Methylase and BamHI Endonuclease
[0168] Competent E. coli host cells were transformed with pUC18-BamHIR and pACYC184-BamHIM and BamHIR was extracted by a standard Qiagen Mini-prep method using standard miniprep techniques (Qiagen, Valencia, Calif.).
2. Selection of Mutagenesis Target
[0169] BamHI and related restriction endonuclease OkrAI were cloned and sequenced. OkrAI was found to have significant star activity if the reaction occurred at 37.degree. C. in NEB buffers (1, 2 and 4). The present analysis tested the assumption that the amino acid residue(s) responsible for the star activity were similar between BamHI and OkrAI endonuclease.
[0170] The complete protein sequence of BamHI (SEQ ID NO:19) is:
TABLE-US-00005 1 MEVEKEFITD EAKELLSKDK LIQQAYNEVK TSICSPIWPA TSKTFTINNT 51 EKNCNGVVPI KELCYTLLED TYNWYREKPL DILKLEKKKG GPIDVYKEFI 101 ENSELKRVGM EFETGNISSA HRSMNKLLLG LKHGEIDLAI ILMPIKQLAY 151 YLTDRVTNFE ELEPYFELTE GQPFIFIGFN AEAYNSNVPL IPKGSDGMSK 201 RSIKKWKDKV ENK
[0171] The complete protein sequence of OkrAI (SEQ ID NO:20) is:
TABLE-US-00006 1 MKIKRIEVLI NNGSVPGIPM ILNEIQDAIK TVSWPEGNNS FVINPVRKGN 51 GVKPIKNSCM RHLHQKGWAL EHPVRIKAEM RPGPLDAVKM IGGKAFALEW 101 ETGNISSSHR AINKMVMGML ERVIIGGVLI LPSRDMYNYL TDRVGNFREL 151 EPYFSVWRQF NLKDAYLAIV EIEHDSVDAQ VSLIPKGTDG RAIR
[0172] A "Bestfit" similarity analysis done by GCG for the protein sequence of BamHI and OkrAI endonuclease showed the following result where the upper protein sequence is BamHI and the bottom protein sequence is OkrAI:
TABLE-US-00007 bamhir.pep .times. okrair.pep . . . . . 22 IQQAYNEVKTSICSPIWPATSKTFTINNTEKNCNGVVPIKELCYTLLEDT 71 | ||:..|. || ..||| || | ||| ||| | | 18 IPMILNEIQDAIKTVSWPEGNNSFVINPVRKG.NGVKPIKNSCMRHLHQK 66 . . . . . 72 YNWYREKPLDILKLEKKKGGPIDVYKEFIENSELKRVGMEFETGNISSAH 121 | ||. | | | : ||:| | | | :|.|||||||.| 67 .GWALEHPVRI.KAEMRP.GPLDAVK.MIGG...KAFALEWETGNISSSH 109 . . . . . 122 RSMNKLLLGLKHGEIDLAIILMPIKQLAYYLTDRVTNFEELEPYFEL... 168 |..||:.:|: | ::::| : : |||||| || |||||| . 1 10 RAINKMVMGMLERVIIGGVLILPSRDMYNYLTDRVGNFRELEPYFSVWRQ 159 . . . 1 69 ..TEGQPFIFIGFNAEAYNSNVPLIPKGSDGMSKR 201 (SEQ ID NO: 21) . : :. ..| |||||.|| . | 160 FNLKDAYLAIVEIEHDSVDAQVSLIPKGTDGRAIR 194 (SEQ ID NO: 22)
[0173] The similar charged residues (D, E, H, K, R) in BamHI were found to be E28, K30, K52, K61, E77, K84, E86, K88, D94, K97, K106, E111, E113, H121, R122, K126, K146, D154, R155, E161, E163, E170, E182, K193, D196 and R201. These residues are underlined in the above comparison. Known mutants E77K, D94N, E111K and E113K were previously reported to be inactive (Xu, Shuang-yong et al. J. Bacteriol. 266: 4425-4429 (1991)) so they were excluded. The initial mutagenesis selection targeted 22 shared charged amino acid residue for mutation to Alanine: E28A, K30A, K52A, K61A, K84A, E86A, K88A, K97A, K106A, H121A, R122A, K126A, K146A, D154A, R155A, E161A, E163A, E170A, E182A, K193A, D196A and R201A.
3. Mutagenesis of BamHI
[0174] The point mutagenesis of the selected mutations was done by inverse PCR. The corresponding codons were all changed to GCA (alanine). The following primers were used for mutagenesis:
TABLE-US-00008 E28A (SEQ ID NO: 23) 5'ATTCAACAAGCATACAATGCAGTTAAAACATCTATTGT3' (SEQ ID NO: 24) 5'ACAAATAGATGTTTTAACTGCATTGTATGCTTGTTGAAT3' K30A (SEQ ID NO: 25) 5'CAAGCATACAATGAAGTTGCAACATCTATTTGTTCACCT3' (SEQ ID NO: 26) 5'AGGTGAACAAATAGATGTTGCAACTTCATTGTATGCTTG3' K52A (SEQ ID NO: 27) 5'ACGATTAACAACACCGAAGCAAATTGTAACGGTGTAGTA3' (SEQ ID NO: 28) 5'TACTACACCGTTACAATTTGCTTCGGTGTTGTTAATCGT3' K61A (SEQ ID NO: 29) 5'AACGGTGTAGTACCAATTGCAGAACTATGTTACACCTTA3' (SEQ ID NO: 30) 5'TAAGGTGTAACATAGTTCTGCAATTGGTACTACACCGTT3' K84A (SEQ ID NO: 31) 5'AACCCCCTTGATATACTTGCACTTGAAAAGAAAAAAGGT3' (SEQ ID NO: 32) 5'ACCTTTTTTCTTTTCAAGTGCAAGTATATCAAGGGGTTT3' E86A (SEQ ID NO: 33) 5'GATATACTTAAACTTGCAAAGAAAAAAGGTGGTCCG3' (SEQ ID NO: 34) 5'CGGACCACCTTTTTTCTTTGCAAGTTTAAGTATATCAAG3' K88A (SEQ ID NO: 35) 5'ATACTTAAACTTGAAAAGGCAAAAGGTGGTCCGATTGAT3' (SEQ ID NO: 36) 5'ATCAATCGGACCACCTTTTGCCTTTTTCAAGTTTAAGTAT3' K97A (SEQ ID NO: 37) 5'GGTCCGATTGATGTTTATGCAGAGTTCATAGAAAACAGT3' (SEQ ID NO: 38) 5'ACTGTTTTCTATGAACTCTGCATAAACATCAATCGGACC3' K106A (SEQ ID NO: 39) 5'ATAGAAAAACAGTGAACTTGCACGTGTAGGTATGGAA3' (SEQ ID NO: 40) 5'AAATTCCATACCTACACGTGCAAGTTCACTGTTTTCTAT3' H121A (SEQ ID NO: 41) 5'GGAAATATTAGTTCTGCCGCACGTTCAATGAACAAACTT3' (SEQ ID NO: 42) 5'AAGTTTGTTCATTGAAACGTGCGGCAGAACTAATATTCC3' R122A (SEQ ID NO: 43) 5'AATATTAGTTCTGCCCACGCATCAATGAACAAACTTCTA3' (SEQ ID NO: 44) 5'TAGAAGTTTGTTCATTGATGCGTGGGCAGAACTAATATT3' K126A (SEQ ID NO: 45) 5'GCCCACCGTTCAATGAACGCACTTCTATTAGGATTAAAACAT3' (SEQ ID NO: 46) 5'ATGTTTTAATCCTAATAGAAGTGCGGTCATTGAACGGTGGGC3' K146A (SEQ ID NO: 47) 5'ATTATCCTTATGCCTATTGCACAATTGGCCTATTATCTT3' (SEQ ID NO: 48) 5'AAGATAATAGGCCAATTGTGCAATAGGCATAAGGATAAT3' D154A (SEQ ID NO: 49) 5'TTGGCCTATTATCTTACAGCACGTGTTACCAATTTCGAG3' (SEQ ID NO: 50) 5'CTCGAAATTGGTAACACGTGCTGTAAGATAATAGGCCAA3' R155A (SEQ ID NO: 51) 5'GCCTATTATCTTACAGATGCAGTTACCAATTTCGAGGAA3' (SEQ ID NO: 52) 5'TTCCTCGAAATTGGTAACTGCATCTGTAAGATAATAGGC3' E161A (SEQ ID NO: 53) 5'CGTGTTACCAATTTCGAGGCATTAGAACCTTATTTTGAA3' (SEQ ID NO: 54) 5'TTCAAAATAAGGTTCTAATGCCTCGAAATTGGTAACACG3' E163A (SEQ ID NO: 55) 5'ACCAATTTCGAGGAATTAGCACCTTATTTTGAACTTACT3' (SEQ ID NO: 56) 5'AGTAAGTTCAAAATAAGGTGCTAATTCCTCGAAATTGGT3' E170A (SEQ ID NO: 57) 5'CCTTATTTTGAACTTACTGCAGGACAACCATTTATTTTTATT3' (SEQ ID NO: 58) 5'AATAAAAATAAATGGTTGTGCTGCAGTAAGTTCAAAATAAGG3' E182A (SEQ ID NO: 59) 5'TTTATTTTTATTGGATTTAATGCTGCAGCTTATAATTCTAATGTC3' (SEQ ID NO: 60) 5'GACATTAGAATTATAAGCTGCAGCATTAAATCCAATAAAAATAAA3' K193A (SEQ ID NO: 61) 5'AATGTCCCTTTAATTCCCGCAGGTTCTGACGGTATGTCA3' (SEQ ID NO: 62) 5'TGACATACCGTCAGAACCTGCGGGAATTAAAGGGACATT3' D196A (SEQ ID NO: 63) 5'TTAATTCCCAAAGGTTCTGCAGGTATGTCAAAACGCTCA3' (SEQ ID NO: 64) 5'TGAGCGTTTTGACATACCTGCAGAACCTTTGGGAATTAA3' R201A (SEQ ID NO: 65) 5'TCTGACGGTATGTCAAAAGCATCAATTAAGAAATGGAAA3' (SEQ ID NO: 66) 5'TTTCCATTTCTTAATTGATGCTTTTGACATACCGTCAGA3'
[0175] The PCR reaction in a reaction volume of 100 .mu.l, contained 2 .mu.l of each PCR primer, 1 .mu.l pUC18-bamhiR, 400 .mu.M dNTP, 4 units of Deep Vent.TM. DNA polymerase, and 10 .mu.l 10.times. Thermopol buffer containing 0, 2, or 6 .mu.l MgSO.sub.4 with additional water.
[0176] The PCR reaction conditions were 94.degree. C. for 5 min, followed by 25 cycles of 94.degree. C. 30 sec, 55.degree. C. 30 sec, 72.degree. C. 4 min and a final extension time at 72.degree. C. for 7 mins. The PCR product was purified on a standard Qiagen spin column (Qiagen, Valencia, Calif.). Six to sixteen .mu.l of PCR product was digested by 20 units of DpnI for 1 hour. The digested product was transformed into E. coli (pACYC-bamHIM).
[0177] After six PCR reactions, 14 out of the engineered 22 mutations were obtained: E28A, K30A, K61A, E86A, K97A, H121A, K126A, K146A, E161A, E163A, E170A, E182A, and R201A. Mutant proteins were extracted from cell lysates in an overnight culture and the activity was compared to WT BamHI. Normal enzyme activity was assayed in NEB2 buffer with or without 5% glycerol, while star activity was determined in NEB2 with 39.2% glycerol, though initially, lower percentage glycerol could be used. The substrate used for different reactions was pBR322, pUC19 or lambda DNA. The cleavage reaction was performed at 37.degree. C. for 30 min or 1 hour. It was found that mutants K97A, H121A, K126A, E161A, E182A, R201A were inactive (less than 1% of the WT BamHI activity) while E28A, K146A, E163A, E170A mutants had a similar level of activity including star activity to that of WT enzyme. Three mutants K30A, E86A and K126A were found to have significantly reduced star activity compared with WT BamHI. It was also found that K30A and E86A had similar overall cleavage activity to the WT enzyme while showing significant reduction in star activity. In contrast, K126A had only 25% of the overall cleavage activity of the WT enzyme and less significant improvement on star activity than observed for K30A an E86A.
[0178] A recheck on the pUC18-bamHIR plasmid revealed that the normal high copy plasmid had mutated to a low copy plasmid. A pair of primers was designed to transfer the bamHIR gene into the high copy plasmid:
TABLE-US-00009 (SEQ ID NO: 67) 5'GGTGGTGCATGCGGAGGTAAATAAATGGAAGTAGAAAAAGAGTTTATT ACTGAT3' (SEQ ID NO: 68) 5'GGTGGTGGTACCCTATTTGTTTTCAACTTTATCTTTCCATTTCTTAAT TGA3'
[0179] The template was pUC18-bamhIR WT, with mutations at K30A, E86A or K126A. The PCR composition contained: 5 .mu.l template, 2 .mu.l primers each, 400 .mu.M dNTP, 10 .mu.l 10.times. Thermopol buffer, 4 units 2 .mu.l Deep Vent.TM. polymerase, 72 .mu.l H.sub.2O with 0, 2, 6 .mu.l MgSO.sub.4. The PCR conditions were 94.degree. C. for 5 min, followed by 25 cycles of 94.degree. C. at 30 sec, 55.degree. C. at 30 sec and 72.degree. C. at 40 sec and a final extension period of 7 min. The PCR product was digested with SphI and KpnI and was ligated to pUC19 with the same pair of enzyme digestion. The ligated product was transformed into competent E. coli-containing pACYC-bamHIM. 26 colonies that contained the pUC19 version of BamHIR K30A and 12 of those that contained E86A were identified and grown. The activity of BamHI from these cultures was checked. All of them were active. Plasmids from five colonies of each mutation were extracted and the BamHIR plasmids from three of each mutation were sequenced. The identity of plasmids pUC19-BamHI(K30A) and pUC19-BamHI(E86A) were confirmed.
[0180] Those mutations that were unsuccessful in pUC18-BamHIR were repeated using the pUC19-BamHI(K30A) vector. The PCR mixture contained: 1 .mu.l template and an amplification mixture containing 2 .mu.l primers each, 400 .mu.M dNTP, 10 .mu.l 10.times. Thermopol buffer, 4 units 2 .mu.l Deep Vent.TM. polymerase, 76 .mu.l H.sub.2O with 0, 2, 6 .mu.l 100 .mu.M MgSO.sub.4. The PCR condition was 94.degree. C. for 5 min, followed by 25 cycles of 94.degree. C. for 30 sec, 55.degree. C. for 30 sec and 72.degree. C. for 3 min and 30 sec and a final extension period of 7 min. The PCR products were digested by DpnI and transformed to competent E. coli transformed with pACYC-BamHIM. The enzyme activities were checked on pUC19 substrate. The reaction composition was: 3 .mu.l cell extract, 3 .mu.l NEB2, 3 .mu.l 50% glycerol, 0.5 .mu.l 0.5 .mu.g pUC19, 20.5 .mu.l H2O. Reaction was at 37.degree. C. for 1 hour. K30A/R122A, K30A/R155A and K30A/K193A were inactive. K30A/K52A and K30A/K88A were about 1/10 of the K30A activity. The normal activity of K30A/K106A, K30A/D154A and K30A/D196A were similar to that of K30A BamHI. The comparison of star activity of these three mutants with K30A at the high concentration glycerol (39.2%) showed that K30A/D196A had similar star activity as K30A, K30A/D154A even has more star activity than K30A, and K30A/K106A had less star activity than K30A. Attempts to isolate the K106A mutation of BamHI in the pUC19 vector failed because of cytotoxicity.
[0181] The mutation on the K30, E86 and K106 sites was combined using the inverse PCR: K30A/E86A, E86A/K106A, K30A/K106A and K30A/E86A/K106A. K30A/E86A appeared to be the preferred mutant. After purification, the FI was found to be improved for the BamHI mutant by 25% in all NEB buffers.
[0182] Further mutagenesis was done on the site of K30 and E86 randomly:
TABLE-US-00010 For K30: (SEQ ID NO: 69) 5'CAAGCATACAATGAAGTTNNNACATCTATTTGTTCACCT3' (SEQ ID NO: 70) 5'AGGTGAACAAATAGATGTNNNAACTTCATTGTATGCTTG3' For E86: (SEQ ID NO: 71) 5'GATATACTTAAACTTNNNAAGAAAAAAAGGTGGTCCG3' (SEQ ID NO: 72) 5'CGGACCACCTTTTTTCTTNNNAAGTTTAAGTATATCAAG3'
[0183] The PCR composition was: 1 .mu.l template (pUC19-BamHIR(K30A) or pUC19-BamHIR(E86A)) and the amplification mixture as described above was used. The PCR was performed at 94.degree. C. 5 min, followed by 25 cycles of 94.degree. C. 30 sec, 55.degree. C. 30 sec and 72.degree. C. 3 min and 30 sec and a final extension period of 7 min. The PCR products were digested by DpnI and transformed into E. coli (pACYC-BamHIM).
[0184] Total of 155 colonies were picked on K30 random mutations, and 158 colonies on E86 site. The colonies were grown overnight and made into cell extract. 0.5 .mu.g pUC19 was digested with 1 .mu.l cell extract in NEB 2 buffer with 42.5% glycerol, 37.degree. C. 1 hour. The cell extract with apparent less star activity was re-assayed under 1, 4, 16 fold dilution on 0.5 .mu.g pUC19 in NEB 2 buffer with 39.2% glycerol, 37.degree. C. 30 min. For those mutants observed to have reduced star activity, the corresponding plasmids were extracted and sequenced to confirm the mutation. A total of 3 clones (#12, #66 and #82) contained the K30 mutation, and a total of 33 clones (#5, #15, #16, #19, #29, #47, #51, #55, #56, #58, #61, #69, #71, #73, #76, #82, #86, #88, #93, #94, #97, #98, #100, #104, #107, #113, #117, #118, #129, #132, #136, #139 and #151) were sequenced. After sequencing, #12 and #66 were found to contain the K30G mutation, and #82 the K30N mutation. Surprisingly, all 33 mutations are E86P mutation, just in different codons (CCA, CCT, CCC, CCG). Among these codons, the CCG occurred at the highest frequency in E. coli (clones #98, #136 and #139).
[0185] The cell extracts corresponding to K30G, K30N and K30A were serially diluted as 1, 2, 4, 8, 16 and 32 folds, while E86P and E86A were serially diluted 1, 2, 4, 8, 16, 32, 64, 128 and 256 fold. The serially diluted extracts were reacted with 0.5 .mu.g pUC19 in NEB2 with 39.2% glycerol, 37.degree. C. 30 min. Under extreme conditions, E86P appeared to be much superior to other mutants. At up to 32 times fold digestion, there was no significant star activity band. The difference between E86P and the K30 mutants (K30G, K30N and K30A) was so large that it was not additionally necessary to combine any of these mutations in the E86P mutant.
[0186] The activity of BamHI(E86P) was determined for 1 .mu.g lambda DNA, substrate (also used for WT BamHI activity determination). The assay was performed in NEB1 buffer at 37.degree. C. for 1 hour.
4. Detailed Comparison of BamHI(E86P) and WT BamHI
A. The Activity of BamHI(E86P) in Different NEB Buffers
[0187] The activity of purified BamHI(E86P) was determined in NEB1, NEB2, NEB3, NEB4 and NEB BamHI buffer, using lambda DNA substrate at 37.degree. C. for 1 hour. BamHI(E86P) was most active in NEB1 buffer and NEB2, while having 50%, 50% and 25% activity levels in NEB3, NEB4, and BamHI buffer.
B. A Comparison of Cleavage Activity of BamHI(E86P) and WT BamHI on pUC19
[0188] There is one GGATCC site (BamHI site) and 6 AGATCC sites (BamHI star activity site) in pUC19 so that pUC19 was selected as a preferred substrate for comparison of the BamHI(E86P) and WT BamHI.
[0189] 0.5 mg pUC19 was digested by WT BamHI and BamHI(E86P) in a serial dilution of 1, 3, 9, 27, 81, 243, 729, 2181, 6561, and 19683 folds with NEB dilution buffer A, in different buffers. WT BamHI showed star activity in every NEB normal buffer, while BamHI(E86P) showed no star activity bands at all (FIGS. 2-5). This demonstrated that BamHI(E86P) had greatly reduced star activity while retaining the cognate cleavage activity.
C. A Comparison of Cleavage Activity of BamHI(E86P) and WT BamHI on Lambda DNA Substrate
[0190] To calculate the Fidelity Index, the restriction enzyme was diluted with dilution buffer, and the glycerol concentration was kept constantly at 5%. In the standard reaction condition used here, lambda DNA substrate concentration was 1 .mu.g and the total reaction volume was 50 .mu.l. In order to keep the enzyme volume at 10%, the enzyme was added in a volume of 5 .mu.l. This is equivalent to 0.6 .mu.g of substrate digested by 3 .mu.l of restriction enzyme in a total volume of 30 .mu.l. 0.6 mg lambda DNA was digested by 3 .mu.l WT BamHI and BamHI(E86P) in a 1:2 serial dilution from 1 to 32768, in NEB1, NEB2, NEB3, NEB4 and NEB BamHI buffer at 37.degree. C. for 1 hour.
TABLE-US-00011 TABLE 5 Fidelity Indices for WT and mutant BamHI in various buffers BamHI(E86P) WT BamHI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 100% .gtoreq.4000 50% 4 .gtoreq.1000 NEB2 100% .gtoreq.4000 100% 16 .gtoreq.250 NEB3 50% .gtoreq.4000 100% 32 .gtoreq.125 NEB4 50% .gtoreq.4000 50% 4 .gtoreq.1000 BamHI 25% .gtoreq.2000 50% 32 .gtoreq.125 buffer
5. Further Improvement of BamHI for High Fidelity Mutants
[0191] At one hour level, the BamHI(E86P) appeared to be a good high fidelity BamHI mutant. However, when the reaction time was extended (e.g. overnight, or 14 hours), star activity bands appeared even though the star activity of E86P was not detected at one hour. (FIG. 3) The search for improved high fidelity BamHI was continued.
6. Mutations of Other Charged and Polar Residues
[0192] The other charged residues (Arg, Lys, His, Asp, Glu) were mutated to Ala at the positions of 2, 4, 5, 6, 10, 11, 13, 14, 18, 19, 20, 43, 51, 62, 69, 70, 76, 77, 78, 81, 87, 89, 94, 98, 101, 104, 107, 111, 113, 132, 133, 135, 137, 160, 167, 200, 204, 205, 207, 208, 209, 211, 213 in SEQ ID NO:19. The mutations were done on the template of pUC19-BamHI(K30A).
[0193] Other polar residues (Ser, Thr and Tyr) were mutated to Ala while Tyr was mutated to Phe at the positions of 9, 17, 26, 32, 36, 41, 42, 44, 46, 50, 65, 66, 71, 72, 75, 96, 103, 114, 118, 119, 123, 150, 151, 153, 157, 165, 169, 184, 186, 195, 199, 202 in SEQ ID NO:19.
[0194] By using similar mutation and screen methods, the following mutations were discovered to have reduced star activity, K30A/K87A, E86P/K87E, E86A/Y165F, and K30A/E167A. E86P/K87E was identified as a mutant with improved properties in the presence of additional DMSO. However, the activity of this mutant in normal reaction buffer was much lower than that of WT BamHI.
[0195] The following combination of mutations was made: E86P/Y165F, E86P/E167A, E86P/Y165F/E167A, K30A/Y165F/E167A, K30G/Y165F/E167A, K30A/Y165F/E167A, E86A/Y165F/E167A. All had low activity.
[0196] Up to this point, it was found that E167A and Y165F had a strong effect, K87A had medium effect, and K30A and E86A had weak effect on the BamHI star activity. E86P is a special mutation that reduces star activity at 1 hour level but not overnight.
7. Mutation of E167 and Y165 to all Other Residues
[0197] E167 was mutated to all other residues in pUC19-BamHI by changing the codon to GCA for Ala, TGC for Cys, GAC for Asp, TTC for Phe, GGT for Gly, CAC for His, ATC for Ile, AAA for Lys, CTG for Leu, ATG for Met, AAC for Asn, CCG for Pro, CAG for Gln, CGT for Arg, TCC for Ser, ACC for Thr, GTT for Val, TGG for Trp, and TAC for Tyr.
[0198] After comparison of all the mutants, the E167T mutation was preferred, while E167R, E167K, E167L and E167I mutations showed improvement in reduced star activity compared with E167A.
[0199] Y165 was also mutated to all other amino acid residues by changing the corresponding codon to GCT for Ala, TGC for Cys, GAC for Asp, GAA for Glu, GGT for Gly, CAC for His, ATC for Ile, AAA for Lys, CTG for Leu, ATG for Met, AAC for Asn, CCG for Pro, CAG for Gln, CGT for Arg, TCC for Ser, ACC for Thr, GTT for Val, TGG for Trp.
[0200] After comparison of all the mutants, the presence of Y165F resulted in significant cleavage activity while other mutations of listed immediately above showed low activity or no cleavage activity.
8. Further Mutations on BamHI(E167T)
[0201] All charged and polar residues were mutated to Ala, on the template of puc19-BamHI(E167T), as the same procedure as above.
[0202] E163A/E167T as the preferred mutation was identified as BamHI-HF.
9. Comparison of BamHI-HF to WT BamHI
[0203] Introduction of a mutation at E163 resulted in reduced thermostability of the BamHI mutant, as did mutation P173A when added to other mutations responsible for reducing star activity.
[0204] BamHI-HF, unlike the BamHI(E86P), had no significant star activity in an overnight reaction in NEB1-4 buffers. FIG. 4 shows the results in NEB1 and NEB2. Hence BamHI(E163A/E167T) was selected as the preferred high fidelity BamHI.
[0205] The fidelity indices of BamHI-HF were measured in all of the four NEB buffers on lambda DNA substrate, with diluent A, at 37.degree. C. and compared with the WT enzyme.
TABLE-US-00012 TABLE 6 Comparison of BamHI-HF and WT BamHI BamHI-HF WT BamHI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 100% .gtoreq.8000 50% 4 .gtoreq.1000 NEB2 50% .gtoreq.4000 100% 16 .gtoreq.250 NEB3 12.5% .gtoreq.250 100% 32 .gtoreq.8 NEB4 50% .gtoreq.4000 50% 4 .gtoreq.1000
[0206] BamHI-HF has a highest activity in NEB1, the fidelity index is .gtoreq.8000, WT BamHI has the highest activity in NEB2 and NEB3, and the highest FI is 32. The overall FI improvement factor, which is the ratio of the FI in the best buffer for each of the mutant and the WT enzyme, is .gtoreq.8000/32=250 fold.
10. Additional Mutations of BamHI
[0207] E163A/E167T/P173A was predicted to have a preferred reduction in star activity and additionally to be thermolabile.
[0208] (E86P/K87S/K88P/E163S/E170T/P173A) was tested. This mutant displayed 10-fold reduction in specific activity but had a compensating increased yield of protein from host cells.
[0209] Other BamHI mutants that shared reduced thermostability, reduced star activity and acceptable specific activity include:
[0210] E86P/K87R/K88G/E163S/E170T/P173A
[0211] E86P/K87P/K88R/E163S/E170T/P173A/E211K
[0212] E86P/K87T/K88R/E163S/E170T/P173A/N158S
[0213] E86P/K87S/K88P/E163S/E170T/P173A
[0214] E86P/K87G/K88S/E163S/E170T/P173A
[0215] E86P/K87R/K88Q/E163S/E170T/P173A
Example 2: Preparation of a High Fidelity EcoRI
1. Expression of EcoRI
[0216] PCR on EcoRI used the following primers:
TABLE-US-00013 (SEQ ID NO: 73) GGTGGTGCATGCGGAGGTAAATAAATGTCTAATAAAAAACAGTCAAATAG GCTA (SEQ ID NO: 74) GGTGGTGGTACCTCACTTAGATCTAAGCTGTTCAAACAA
[0217] The PCR product was then digested with a second pair of restriction endonucleases--SphI and Acc65I, and ligated into the pUC19 digested with the same second pair of restriction endonucleases. The ligated plasmid was then be transformed into competent E. coli premodified with pACYC-MlucIM.
2. Mutagenesis of EcoRI
[0218] Initial selection of target amino acid residues resulted from a comparison of EcoRI with its isoschizomer RsrI, which is also known for its star activity.
TABLE-US-00014 EcoRI vs. RsrI . . . . . 4 KKQSNRLTEQHKLSQGVIGIFGDYAKAHDLAVGEVSKLVKKALSNEYPQL 53 | |. || | .| | : ||| |. |||.: ||. | |. ::|| 10 KGQALRLGIQQELGGGPLSIFGAAAQKHDLSIREVTAGVLTKLAEDFPNL 59 . . . . . 54 SFRYRDSIKKTEINEALKKIDPDLGGTLFVSNSSIKPDGGIVEVKDDYGE 103 |. | |: | ||| |: || || ||| ..||:|||||| ||| :| 60 EFQLRTSLTKKAINEKLRSFDPRLGQALFVESASIRPDGGITEVKDRHGN 109 . . . . . 104 WRVVLVAEAKHQGKDIINIRNGLLVGKRGDQDLMAAGNAIERSHKNISEI 153 |||:|| |.|||| |: | |.| || ||| ||||||||| |||: |: 110 WRVILVGESKHQGNDVEKILAGVLQGKAKDQDFMAAGNAIERMHKNVLEL 159 . . . . . 154 ANFMLSESHFPYVLFLEGSNFLTENISITRPDGRVVNLEYNSGILNRLDR 203 |:|| | |||||.||:|||| ||. :|||||||| : :.||.|||:|| 160 RNYMLDEKHFPYVVFLQGSNFATESFEVTRPDGRVVKIVHDSGMLNRIDR 209 . . . . . 204 LTAANYGMPINSNLCINKFVNHKDKSIMLQAASIYTQGDGREWDSKIMFE 253 .||.. || | | | | | | ||:| . | . | | 210 VTASSLSREINQNYCENIVVRAGSFDHMFQIASLYCK..AAPWTAGEMAE 257 254 IMFDISTTSLRVLGRDL 270 (SEQ ID NO: 75) | :. ||||:: || 258 AMLAVAKTSLRIIADDL 274 (SEQ ID NO: 76)
[0219] Except for D91, E111 and K113, which were known active center residues, the 42 charged residues were identical or similar in the two endonucleases. The charged residues were as follows:
[0220] K4, R9, K15, K29, H31, D32, E37, E49, R56, R58, K63, E68, K71, D74, K89, E96, K98, K99, R105, H114, D118, K130, D133, D135, E144, R145, H147, K148, E152, E160, H162, E170, E177, R183, D185, R200, D202, R203, E253, R264, D269.
[0221] All of these charged residues were mutated to Ala (codon GCA, GCT, GCC or GCG) and the mutated genes amplified and cloned as follows:
[0222] The amplification mixture was the same as used in Example 1 (2 .mu.l PCR primers each, 400 mM dNTP, 4 units of Deep Vent DNA polymerase, 10 .mu.l 10.times. Thermopol buffer with additional 0, 2, 6 .mu.l MgSO.sub.4, and the total reaction volume was 100 .mu.l) and was added to 1 .mu.l pUC19-EcoRI).
[0223] The PCR reaction conditions was 94.degree. C. for 5 min, followed by 25 cycles of 94.degree. C. for 30 sec, 55.degree. C. for 30 sec, 72.degree. C. for 3 min and 30 sec and a final extension time at 72.degree. C. for 7 min. After PCR, the product was purified by the standard Qiagen spin column (Qiagen, Valencia, Calif.). 16 .mu.l PCR product was digested by 20 units of DpnI for 1 hour. The digested product was transformed into a methylase protected competent E. coli preparation.
3. Screening EcoRI High Fidelity Mutants
[0224] Three colonies each were picked for each mutation and grown in LB with Ampicillin and Chloramphenicol for overnight. The activity assay was performed on pBR322 and lambda DNA to ensure the mutant had at least similar activity to WT EcoRI. Then these mutants were tested using 3 .mu.l of cell extract in 2-fold serial dilution, 12 .mu.l 50% glycerol, 3 .mu.l of NEB1 buffer, 0.5 .mu.l pBR322 and 11.5 .mu.l water, reacted at 37.degree. C. for one hour. However, none of the mutations improved the performance of star activity.
[0225] From this result, it was concluded that an effective mutation could not always be recognized as a homologous residue between isoschizomers.
4. Repeat Mutagenesis on the Rest of 32 Charged Residues
[0226] All remaining 32 charged residues were mutated into Ala as described in step 2 by targeting amino acid residues 5, 12, 14, 26, 40, 43, 44, 59, 62, 65, 72, 76, 100, 103, 117, 123, 131, 192, 221, 225, 226, 227, 228, 242, 244, 245, 247, 249, 257, 268, 272 and 277.
[0227] The numbers above correspond to amino acid positions in the EcoRI protein sequence (SEQ ID NO:83).
5. Repeat Selection
[0228] Four colonies were picked from each sample containing a different type of mutation and grown in 4 ml LB with CAM. After sonication, cell extracts were tested on lambda DNA substrate in normal glycerol condition in NEB1 buffer. Those extracts with similar activity were tested again on pUC19 substrate by adding 3 .mu.l of cell extract in two-fold serial dilutions, in 3 .mu.l of NEB2 buffer to 0.5 .mu.l of pUC19 and 23.5 .mu.l 50% glycerol to provide a final concentration of 39.2% glycerol in the reaction mixture.
[0229] Among all of these mutants, K62A was found to be the mutation with the least star activity and a high FI. R9A, K15A, R123A, K130A, R131A, R183A mutants all showed partial reduction in star activity. Interestingly, one clone containing the targeted mutation K5A showed a partial improvement. Additionally, a secondary mutation, S2Y was found after sequencing. Separation of these two mutations revealed that the effective mutation for this isolate was S2Y. D135A and R187A EcoRI also had much less star activity. However, the cleavage activity of these mutants was not optimal.
6. Comparison of EcoRI(K62A) with WT EcoRI
[0230] A side-by-side comparison was performed in a 3-fold serial dilution using NEB dilution buffer C, by digesting 0.6 .mu.g of lambda DNA in four different NEB buffers (FIG. 6). EcoRI(K62A) had substantially less star activity than the WT EcoRI.
[0231] A more quantitative comparison was done by determining the Fidelity Index measurement for EcoRI(K62A) and WT EcoRI. The conditions for the fidelity index measurement was the same as for Table 2 using lambda DNA as substrate and, dilution buffer C. The reaction was incubated at 37.degree. C. for 1 hour and the digestion products analyzed on an 0.8% agarose gel.
TABLE-US-00015 TABLE 7 Fidelity Index for EcoRI(K62A) and WT EcoRI EcoRI(K62A) WT EcoRI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 50% 32000 50% 250 128 NEB2 50% 8000 100% 4 2000 NEB3 12.5% 4000 100% 250 16 NEB4 100% 32000 100% 4 8000 EcoRI 12.5% 4000 100% 250 16 buffer
7. Further Mutation of EcoRI
[0232] Though it was not apparent that the EcoRI(K62A) had star activity on lambda DNA substrate, star activity was observed using Litmus28 substrate after a 10 hours digestion. EcoRI(K62A) in NEB4 had significantly reduced star activity compared with WT EcoRI in EcoRI buffer (FIG. 7).
[0233] Further improvements were investigated. EcoRI(K62) was mutated to all other amino acid residues by changing K to the corresponding codons as in the example 1. K62S and K62L were similar as K62A. EcoRI(K62E) had a .gtoreq.100 fold overall fidelity index improvement factor when compared with EcoRI(K62A) as shown in FIG. 6. EcoRI(K62E) was named EcoRI-HF.
8. Comparison of EcoRI-HF and WT EcoRI
[0234] A quantitative comparison was done by the FI measurement on EcoRI-HF and WT EcoRI in diluent C. The conditions for the FI measurement were the same as in Table 2 using lambda DNA as substrate. The reaction conditions were 37.degree. C. for 1 hour and the results analyzed on a 0.8% agarose gel (FIG. 8).
TABLE-US-00016 TABLE 8 Comparison of EcoRI-HF and WT EcoRI EcoRI-HF WT EcoRI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 12.5% 2000 50% 250 8 NEB2 100% 4000 100% 4 1000 NEB3 0.4% 250 100% 250 1 NEB4 100% 16000 100% 4 4000 EcoRI 0.4% 250 100% 250 1 buffer
[0235] The overall fidelity index improvement factor was found to be 64 fold (16000 in NEB4 for EcoRI-HF to 250 of WT EcoRI in NEB3).
Example 3: Engineering a High Fidelity ScaI
1. Expression of ScaI
[0236] The sequence for ScaI restriction endonuclease and methylase are described in REBASE and in GenBank and is presented in FIG. 44 (SEQ ID NO:97). The genes expressing these enzymes were inserted into plasmids to produce pRRS-ScaI and pACYC184-ScaIM respectively. pACYC184-ScaIM was then transformed into competent E. coli host cells. The pRRS vector was derived from pUC19 and differs only in the presence of multiple cloning sites. pRRS-ScaIR was transformed into E. coli (pACYC-ScaIM) to make an expression strain. Plating and cell culture were performed at 30.degree. C.
2. Mutagenesis of ScaI
[0237] ScaI has two sequenced isoschizomers: LlaDI and NmeSI. However, there was no known information on the star activity of LlaDI or NmeSI nor was there any information on the active site of these enzymes. Therefore all 58 charged residues were initially selected for targeted mutation at positions 4, 8, 11, 12, 14, 18, 25, 27, 30, 37, 39, 40, 43, 46, 51, 57, 61, 68, 72, 74, 80, 86, 97, 103, 108, 112, 114, 119, 120, 121, 127, 128, 129, 133, 135, 139, 140, 141, 147, 152, 156, 158, 159, 161, 162, 171, 172, 175, 179, 182, 184, 187, 172, 175, 192, 193, 195, 200, 222, 227 in the protein.
[0238] The numbers above correspond to amino acid positions in the ScaI protein sequence (SEQ ID NO:97).
[0239] The method of primer design and PCR is similar to that described in Example 1 for BamHI and Example 2 for EcoRI. Mutagenesis was achieved by varying annealing temperature and DNA polymerases. The PCR product was digested with DpnI and transformed into competent E. coli (pACYC184-ScaIM).
3. Selection of ScaI High Fidelity Mutants
[0240] Four colonies from each mutant of ScaI mutant were picked and grown in 4 ml LB with 100 .mu.g/ml Amp and 33 .mu.g/ml Cam at 30.degree. C. overnight. Each cell culture was sonicated and the activity tested on lambda DNA in NEB2 buffer. Those that were apparently active were retested in 10, 100, and 1000 fold dilutions. Since ScaI has very significant star activity, the star activity bands were easily compared for the mutants versus the WT restriction endonuclease. Those with reduced star activity were retested with a two-fold serial dilution with NEB dilution buffer A. The FI was measured for each of the mutants. The FI was 1/8 for WT ScaI in NEB 2 buffer. Four mutants with similar activity levels were found to have greatly reduced star activity compared with WT ScaI. Mutant #6-3 ScaI had two fold more activity and the FI was 4 or 32 times better than WT ScaI. #26-2 ScaI has two fold more activity and an FI which was 8 or 64 times better than WT; #28-2 ScaI has 2 fold more activity and FI to be 120 or 1000 times better than WT, #54-3 has same activity as WT and FI to be 250 or 2000 times better than WT.
[0241] Four mutants: #6-3, #26-2, #28-2 and #54-3 ScaI were further tested in the presence of 36.7% glycerol for digestion of lambda DNA substrate. #54-3 showed a greater improvement in reduced star activity than the other three mutants.
[0242] After the plasmid was extracted, #6-3 was sequenced and found to have a mutation at R18A. #26-2 was sequenced and found to have a mutation at R112A. #28-2 was sequenced and found to have a mutation at E119A. These mutations were predicted. However, the #54-3 was found to have a double mutant--H193A/S201F. The S201F was a spontaneously secondary mutation that occurred during PCR, and was located outside the primer region of the H193A mutation.
[0243] To understand which residue was primarily responsible for the reduction in star activity a single mutation (S201F) was introduced into ScaI using the following primers:
TABLE-US-00017 (SEQ ID NO: 77) 5'-GATTGGGTGGCGCAGAAATTTCAAACGGGCCAGCAGTCG-3' (SEQ ID NO: 78) 5'-CGACTGCTGGCCCGTTTGAAATTTCTGCGCCACCCAATC-3'.
[0244] The sequences for ScaI(H193A), ScaI(S201F) and ScaI(H193A/S201F) were confirmed. The three mutants and the WT ScaI were compared at the glycerol level of 5% and 37% (FIG. 9). S201F contributed significantly to the FI in contrast to H193A, which only contributed weakly. However, these two mutations appeared to be additive in improving the FI. S201F did not show star activity in 5% glycerol, but did show some star activity in 37% glycerol. H193A had some star activity in 5% glycerol, and significant star activity in 37% glycerol. However, with the combination of these two mutations, no star activity was detected in either 5% or 37% glycerol. This finding not only shows that the amino acids with hydroxyl group can be major active residue for star activity, but also that the right combination of the mutations can push the improvement in fidelity to a very high level. If mutations of charged residues fail to improve star activity, it is here observed that mutations on Ser, Thr and Tyr can be successful in improving the fidelity index. The ScaI(H193A/S201F) was labelled ScaI-HF.
4. Comparison of ScaI-HF and WT ScaI
[0245] ScaI-HF and WT ScaI were compared at a 2.5 fold serial dilution with NEB dilution buffer A in different NEB buffers on 1 .mu.g lambda DNA in four different NEB buffers, 37.degree. C., 1 hour (FIG. 10).
TABLE-US-00018 TABLE 9 Comparison of Fidelity Index for ScaI-HF and WT ScaI ScaI-HF WT ScaI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 12% 250 6% 1/64 16000 NEB2 100% 120 100% 1/8 2000 NEB3 3% 2000 25% 4 500 NEB4 100% 250 1% 1/32 8000
[0246] ScaI-HF performed best in NEB2 and NEB4 buffers, in which the best FI was 250; WT ScaI performed best in NEB2 buffer, in which the FI was 1/8. The overall FI improvement factor was 250/(1/8)=4000.
Example 4: Engineering of High Fidelity SalI
1. Expression of SalI
[0247] SalI was expressed in E. coli transformed with placzz1-SalIR and pACYC-Hpy166IIM where placzz1 is a pUC19 plasmid which utilizes the lac promoter to express the restriction endonuclease gene that is inserted into an adjacent multi-copy site. Hpy166IIM protects the outside four bases of SalI.
2. Mutagenesis of SalI
[0248] 86 charged residues of SalI were mutated to Ala using the similar PCR methods in the previous examples: 5, 6, 8, 9, 12, 13, 19, 27, 31, 34, 35, 37, 42, 43, 45, 50, 60, 63, 65,67, 73, 82, 83, 84, 90, 93, 97, 100, 101, 103, 107, 109, 111, 114, 116, 119, 126, 129, 131, 134, 140, 143, 145, 147, 148, 156, 157, 164, 168, 172, 173, 174, 180, 181, 186, 190, 191, 193, 210, 218, 226, 232, 235, 237, 238, 244, 246, 250, 256, 257, 258, 259, 260, 261, 264, 266, 271, 275, 297, 300, 304, 305, 306, 308, 309, 311.
[0249] The numbers above correspond to amino acid positions in the SalI protein sequence (SEQ ID NO:94).
[0250] The mutants were grown in LB with Amp and Cam at 30.degree. C. overnight.
3. Selection of SalI-HF
[0251] The selection of SalI-HF was performed as described in the previous examples. The major difference was that the star activity of SalI could not be easily assayed in the crude extract, either in 5% glycerol or high glycerol concentration. Glycerol not only promoted the star activity of SalI, but also greatly inhibited the cognate activity.
[0252] Active mutants were assayed in both 5% glycerol and 37% glycerol on HindIII digested lambda DNA. The mutants #22, #26, #29, #31, #43 and #51 were tested for cleavage activity in all four NEB buffers. After several rounds of comparison in different conditions and substrates, #31, SalI(R107A) was found to be the preferred mutant, retaining high cleavage high activity, but displaying substantially reduced star activity. SalI(R107A) was labeled SalI-HF.
4. Comparison of SalI-HF and WT SalI
[0253] The FI of SalI-HF and WT SalI were determined (FIG. 11). The results are shown as Table 10 (below):
TABLE-US-00019 TABLE 10 Comparison of Sail-HF and WT Sall SalI-HF WT SalI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 50% .gtoreq.1000 0.2% 8 16000 NEB2 100% .gtoreq.2000 6% 1/8 2000 NEB3 25% .gtoreq.500 100% 4 500 NEB4 100% .gtoreq.2000 0.8% 1/32 8000
[0254] SalI-HF performed best in NEB 2 and NEB 4 buffers, in which both FIs are .gtoreq.2000; WT SalI performed best in NEB 3 buffer, in which the FI was 4. The overall FI improvement factor was .gtoreq.2000/4=.gtoreq.500.
Example 5: Engineering of High Fidelity SphI
1. Expression of SphI
[0255] SphI was expressed in E. coli (placzz1-SphIR, pACYC184-CviAIIM). CviAIIM protects the internal four bases of SphI. The transformed cells were grown in LB with Amp and Cam at 37.degree. C. overnight.
2. Mutagenesis of SphI
[0256] All charged residues in SphI were mutated to Ala using the methods described in the Example 1 to Example 4. A total of 71 mutations were made: 3, 5, 12, 18, 21, 24, 25, 30, 31, 35, 43, 46, 51, 54, 57, 58, 60, 61, 72, 75, 77, 78, 87, 90, 91, 95, 100, 104, 107, 108, 110, 113, 120, 123, 124, 125, 129, 130, 131, 139, 140, 142, 146, 147, 155, 157, 159, 164, 170, 172, 173, 175, 178, 184, 186, 190, 194, 196, 197, 198, 206, 207, 209, 212, 215, 221, 227, 230, 231, 232, 235.
[0257] The numbers above correspond to amino acid positions in the SphI protein sequence (SEQ ID NO:98).
3. Selection of SphI-HF
[0258] Four colonies of each mutation were grown up in LB with Amp and Cam at 37.degree. C. overnight. The activity selection was mainly on pBR322 in 5% glycerol and 30% glycerol in NEB2. With the experience of previous examples, the selection of high fidelity SphI was straightforward. SphI mutants D91A, K100A, D139A and D164A were found to significantly reduce star activity in SphI. Among them, K100A was the preferred mutation with the least star activity. SphI(K100A) was named as SphI-HF.
4. Comparison of SphI-HF and WT SphI
[0259] The comparison of SphI-HF and WT SphI was done side by side in their respective preferred buffers. SphI-HF was 2-fold serial diluted with NEB dilution buffer A and reacted in NEB4, and WT SphI was 2-fold serial diluted with NEB dilution buffer B. The digestion on lambda DNA is compared in FIG. 12.
TABLE-US-00020 TABLE 11 FI comparison of SphI-HF and WT SphI SphI-HF WT SphI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 50% .gtoreq.1000 100% 64 .gtoreq.16 NEB2 50% .gtoreq.1000 100% 32 .gtoreq.32 NEB3 3% .gtoreq.120 25% 64 .gtoreq.2 NEB4 100% .gtoreq.2000 50% 16 .gtoreq.64
[0260] SphI-HF performed best in NEB4, in which FI is .gtoreq.2000; WT SphI performed best in NEB1 or NEB2, in which the preferred FI is 64. The overall FI improvement factor was .gtoreq.32.
Example 6: Engineering of High Fidelity PstI
1. Expression of PstI
[0261] PstI was expressed from E. coli (pACYC-HpyCH4VM, pPR594-PstIR). HpyCH4VM protects the internal four bases of PstI. pPR594 is a expression vector with Amp resistance and ptac promoter. The cell was grown in LB with Amp and Cam at 30.degree. C., the culture was then induced by IPTG overnight.
2. Mutagenesis of PstI
[0262] 92 charged residues were mutated to Ala using the method described in the previous examples. These were: 8, 10, 11, 14, 25, 26, 38, 40, 41, 44, 45, 47, 58, 61, 63, 66, 67, 69, 73, 74, 77, 78, 82, 85, 88, 91, 92, 94, 95, 99, 104, 105, 116, 119, 127, 128, 136, 142, 145, 146, 150, 151, 152, 156, 159, 169, 170, 174, 176, 179, 180, 184, 188, 191, 197, 202, 204, 207, 212, 214, 217, 218, 226, 227, 228, 231, 236, 237, 238, 239, 240, 246, 251, 257, 258, 261, 263, 273, 282, 284, 286, 287, 295, 297, 302, 305, 306, 309, 313, 314, 319 and 320.
[0263] The numbers above correspond to amino acid positions in the PstI protein sequence (SEQ ID NO:91).
[0264] After the PCR products were digested with DpnI, the samples were transformed into competent E. co/i(pACYC-HpyCH4VM) and grown on LB plate with Amp and Cam.
3. Selection of PstI-HF
[0265] The selection of PstI-HF was similar to the previous samples. The normal activity enzyme activity was tested on lambda DNA with 5% glycerol, and the star activity was tested on pBR322 substrate in the condition of NEB4 buffer and 20% DMSO. DMSO enhanced the star activity more significantly than the same concentration glycerol. During the selection, #26, #56 and #65 had reduced star activity compared to the WT. When each was sequenced, the mutations were found to be D91A, E204G and K228A/A289V. Mutant #26 PstI(D91A) was labeled PstI-HF.
4. Comparison of PstI-HF and WT PstI
[0266] The FI of PstI-HF and WT PstI were measured separately on lambda DNA substrate in NEB1-4 buffers. The dilution buffer is NEB dilution buffer C. The comparison is shown as in the FIG. 13, and the result is listed in Table 12 (below).
TABLE-US-00021 TABLE 12 Comparison of PstI-HF and WT PstI PstI-HF WT PstI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 12.5% .gtoreq.250 50% 32 .gtoreq.8 NEB2 100% .gtoreq.2000 25% 16 .gtoreq.125 NEB3 25% .gtoreq.500 100% 120 .gtoreq.2 NEB4 100% .gtoreq.2000 50% 8 .gtoreq.250
[0267] PstI-HF performed best in NEB2 and NEB4, in which the preferred FI is .gtoreq.2000; WT PstI performed best in NEB3, in which the FI was 120. The overall FI improvement factor was .gtoreq.2000/120=16 times.
Example 7: Engineering of High Fidelity NcoI
1. Expression of NcoI
[0268] Expression of NcoI was achieved in E. coli (pSYX20-NcoIM, pRRS-NcoIR). pRRS is a pUC19 derivative plasmid, and pSYX20 is a compatible low copy number plasmid with pRRS vector. The cells were grown at 30.degree. C. overnight in the LB with Amp and Kanamycin (Kan).
2. Mutagenesis of NcoI
[0269] All 66 charged residues in NcoI were mutated to Ala. These residues were: 7, 8, 19, 22, 27, 30, 31, 32, 33, 37, 39, 42, 46, 55, 56, 61, 62, 64, 68, 69, 75, 84, 88, 89, 92, 93, 95, 97, 100, 116, 136, 144, 146, 162, 166, 170, 178, 183, 185, 187, 188, 189, 196, 199, 202, 204, 209, 211, 212, 213, 216, 219, 227, 229, 237, 241, 244, 250, 251, 257, 259, 261, 268, 279, 282, 285.
[0270] The numbers above correspond to amino acid positions in the NcoI protein sequence (SEQ ID NO:88).
[0271] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pSYX20-NcoIM).
3. Selection of NcoI-HF
[0272] The selection of NcoI-HF was similar to that of PstI-HF. The activity was assayed as described above using lambda DNA as substrate with 5% glycerol. Star activity was determined using pBR322 or lambda in 19% DMSO. The following mutations were found to improve star activity: A2T/R31A, D56A, H143A, E166A, R212A and D268A. Among these mutants, NcoI(A2T/R31A) was selected as the NcoI-HF.
4. Comparison of NcoI-HF and WT NcoI
[0273] The FIs of NcoI-HF and WT NcoI were determined separately on lambda DNA in NEB1-4 buffers. The comparison is shown in FIG. 14, and the results listed in Table 13 (below).
TABLE-US-00022 TABLE 13 Comparison of NcoI-HF with WT NcoI NcoI-HF WT NcoI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 25% .gtoreq.4000 100% 120 .gtoreq.32 NEB2 25% .gtoreq.4000 100% 32 .gtoreq.125 NEB3 6.3% .gtoreq.1000 25% 120 .gtoreq.8 NEB4 100% .gtoreq.16000 100% 32 .gtoreq.500
[0274] NcoI-HF showed the greatest reduction in star activity in NEB4, in which the preferred FI was .gtoreq.16000; WT NcoI performed best in NEB1, NEB2 and NEB4, in which the preferred FI was 120. The overall FI improvement factor was .gtoreq.16000/120=125.
Example 8: Engineering of High Fidelity NheI
1. Expression of NheI
[0275] NheI was expressed in E. coli transformed with pACYC-NheIM, and placzz1-NheIR. placzz1 is a pUC19 derivative plasmid. The cell was grown at 30.degree. C. for overnight in the LB with Amp and Cam.
2. Mutagenesis of NheI
[0276] All 92 charged residues in NheI were mutated to Ala as the following residues: 5, 6, 7, 14, 17, 19, 22, 25, 28, 31, 38, 39, 42, 47, 49, 52, 56, 58, 59, 60, 64, 74, 75, 76, 77, 80, 91, 93, 104, 105, 110, 112, 116, 117, 123, 126, 130, 131, 133, 135, 137, 147, 149, 152, 159, 160, 165, 167, 170, 171, 174, 179, 183, 195, 202, 205, 207, 209, 210, 211, 214, 216, 218, 221, 225, 231, 241, 243, 244, 250, 252, 256, 257, 259, 264, 266, 267, 281, 285, 287, 288, 289, 291, 297, 300, 307, 313, 315, 318, 321, 324, 325.
[0277] The numbers above correspond to amino acid positions in the NheI protein sequence (SEQ ID NO:89).
[0278] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-NheIM).
3. Selection of NheI-HF
[0279] Selection of NheI-HF was performed according to the previous examples. The standard and star activity assays contained pBR322 as a substrate in NEB4 buffer and 5% glycerol and 39% glycerol, respectively. Only one mutation was found to be significant in improving the NheI. This was E77A. NheI(E77A) was selected as the NheI-HF.
4. Comparison of NheI-HF and WT NheI
[0280] The FIs of NheI-HF and WT NheI were determined separately on pXba, a plasmid substrate containing the XbaI digested piece from Adeno virus in each of NEB1-4 buffers. The comparison is shown in FIG. 15, and the result is listed in Table 14 (below).
TABLE-US-00023 TABLE 14 Comparison of NheI-HF and WT NheI NheI-HF WT NheI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 100% .gtoreq.128000 100% 32 .gtoreq.4000 NEB2 3% .gtoreq.4000 25% 120 .gtoreq.32 NEB3 0.05% .gtoreq.32 12.5% 120 .gtoreq.0.25 NEB4 50% .gtoreq.32000 100% 32 .gtoreq.1000
[0281] NheI-HF showed optimal activity in NEB1 buffer where its FI is .gtoreq.128,000. WT NheI has maximum activity in NEB1 and NEB4 buffers, where its best FI is 32. so, the overall FI improvement factor is .gtoreq.128,000/32=.gtoreq.4000.
Example 9: Engineering of High Fidelity SspI
1. Expression of SspI
[0282] SspI was expressed from E. coli transformed with pACYC-SspIM, and placzz1-SspIR. placzz1 is a pUC19 derivative plasmid. The cells were grown at 30.degree. C. overnight in LB with Amp and Cam.
2. Mutagenesis of SspI
[0283] All 81 charged residues in SspI were mutated to Ala: These were: 3, 8, 12, 13, 18, 19, 20, 35, 40, 42, 44, 47, 52, 60, 62, 65, 68, 69, 72, 74, 76, 77, 78, 79, 83, 85, 88, 89, 90, 96, 100, 108, 109, 118, 119, 127, 128, 129, 131, 132, 137, 144, 153, 154, 155, 156, 158, 165, 168, 170, 172, 177, 178, 179, 181, 185, 186, 187, 191, 194, 195, 197, 202, 204, 215, 222, 229, 237, 240, 246, 250, 256, 257, 259, 260, 264, 265, 267, 268, 269, 274.
[0284] The numbers above correspond to amino acid positions in the SspI protein sequence (SEQ ID NO:99).
[0285] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-SspIM).
3. Selection of SspI High Fidelity Mutants
[0286] The standard cognate and star activity assays of NheI were performed using .phi.X174 substrate in NEB 4 buffer and 5% glycerol and 39% glycerol respectively. Mutants #16(H65A), #20(K74A), #23(E78A), #26(E85A), #28(E89A), #33(K109A), #34(E118A), #52(R177A), #62(K197A), #67(D229A) all showed reduced star activity. K109A showed the greatest reduction in star activity. It was decided to seek further improvements in star activity.
4. Further Mutations
[0287] All residues originally identified as Tyr were mutated to Phe, while other residues Cys, Phe, Met, Asn, Gln, Ser, Thr, and Trp were mutated to Ala. This group included 95 residue mutations at the following positions: 2, 6, 7, 9, 10, 13, 22, 25, 26, 27, 29, 30, 32, 33, 34, 39, 41, 51, 53, 55, 56, 57, 58, 59, 61, 63, 71, 75, 81, 84, 87, 91, 94, 98, 104, 106, 107, 110, 111, 113, 114, 123, 125, 134, 136, 139, 140, 141, 142, 143, 146, 152, 157, 159, 160, 164, 173, 175, 180, 183, 190, 192, 193, 196, 198, 199, 201, 205, 207, 211, 214, 218, 219, 220, 221, 223, 225, 226, 227, 228, 230, 232, 233, 235, 238, 239, 241, 249, 254, 255, 272, 275, 276, 277, 280.
[0288] The numbers above correspond to amino acid positions in the SspI protein sequence (SEQ ID NO:113).
[0289] The PCRs and the selections were done by the same procedure as above. Among these mutants, it was found that Y98F had least star activity, and it was better than SspI(K109A) in this respect. The SspI(Y98F) was labelled SspI-HF and was deposited as the production strain.
5. Comparison of SspI-HF and WT SspI
[0290] The FIs of SspI-HF and WT SspI were determined separately using lambda DNA substrate in NEB1-4 buffers. The diluent was NEBC. The comparison is shown in FIG. 16, and the result is listed in Table 15 (below).
TABLE-US-00024 TABLE 15 Comparison of SspI-HF and WT SspI SspI-HF WT SspI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 50% .gtoreq.4000 100% 64 .gtoreq.64 NEB2 50% 120 100% 16 .gtoreq.8 NEB3 0.6% .gtoreq.32 25% 32 .gtoreq.1 NEB4 100% 500 100% 16 .gtoreq.32
[0291] SspI-HF performed best in NEB4, in which the preferred FI was 500; WT SspI performed best in NEB1, NEB2 and NEB4, in which the preferred FI was 64. The overall FI improvement factor was 500/64=8.
Example 10: Engineering of High Fidelity NotI
1. Expression of NotI
[0292] NotI has significant star activity in NEB4 buffer and less in NEB3 buffer. NotI was engineered to reduce star activity in any NEB buffer. NotI was expressed in competent E. coli transformed with pACYC184-EagIM and placzz2-NotIR. The cells were grown at 37.degree. C. for overnight in the LB with Amp and Cam.
2. Mutagenesis of NotI
[0293] All 97 charged residues in NotI were mutated to Ala as the following residues: 2, 4, 8, 10, 17, 21, 22, 26, 31, 34, 35, 36, 49, 52, 57, 59, 62, 72, 74, 75, 77, 84, 87, 96, 97, 105, 117, 121, 122, 125, 126, 129, 130, 133, 140, 141, 145, 150, 152, 156, 160, 165, 167, 174, 176, 177, 182, 187, 189, 193, 194, 200, 205, 208, 210, 219, 224, 225, 227, 236, 237, 245, 251, 253, 267, 271, 272, 280, 283, 290, 292, 294, 296, 304, 306, 308, 310, 314, 319, 321, 323, 327, 331, 335, 336, 339, 353, 354, 356, 358, 361, 365, 367, 368, 369, 370, 378, 382.
[0294] The numbers above correspond to amino acid positions in the NotI protein sequence (SEQ ID NO:90).
[0295] The method for introducing mutants into the enzyme was the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into
E. coli containing pACYC-EagIM.
3. Selection of NotI-HF
[0296] Selection of NotI-HF was performed as described in the previous examples. The standard cognate and star activity assays used pXba substrate in NEB 4 buffer and 5% glycerol and NEB ExoI buffer (67 mM Glycine-KOH, pH 9.5, 6.7 mM MgCl.sub.2, 10 mM 2-mercaptoethanol) and 37% glycerol respectively. #37(K150A), #44(K176A), #45(R177A), #63(R253A) all showed reduced star activity. K150A was the preferred mutation to reduce star activity. NotI(K150A) was selected as the NotI-HF.
4. Comparison of NotI-HF and WT NotI
[0297] The FIs of NotI-HF and WT NotI were determined separately using pXba substrate in NEB1-4 buffers. The comparison is shown in FIG. 17, and the results are listed in Table 16 (below).
TABLE-US-00025 TABLE 16 Comparison of NotI-HF and WT NotI NotI-HF WT NotI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 6% .gtoreq.8000 6% .gtoreq.8000 ND NEB2 100% .gtoreq.128000 50% 250 .gtoreq.512 NEB3 1.6% .gtoreq.4000 100% 4000 .gtoreq.1 NEB4 50% .gtoreq.64000 12.5% 32 .gtoreq.2000
[0298] ND: Not determinable, for that both FI is an uncertain number over limit.
[0299] NotI-HF performed best in NEB2, in which the preferred FI was .gtoreq.128000; WT NheI performed best in NEB3, in which the preferred FI was 4000. The overall fidelity index improvement factor was .gtoreq.128000/4000=.gtoreq.32. Engineering NotI not only further improved the FI of NotI, but also changed the optimal buffer.
Example 11: Engineering of High Fidelity SacI
1. Expression of SacI
[0300] SacI was expressed in E. coli transformed with pLG-SacIM and pRRS-SacIR. pRRS is a pUC19 derivative plasmid, pLG is a low copy compatible plasmid. The cells were grown at 30.degree. C. overnight in LB with Amp and Kan.
2. Mutagenesis of SacI
[0301] All 101 charged residues in SacI were mutated to Ala as the following residues: 6, 7, 11,15, 16, 19, 24, 25, 29, 30, 39, 40, 42, 45, 58, 61, 62, 63, 65, 67, 70, 71, 72, 74, 75, 76, 81, 85, 94, 98, 104, 105, 114, 116, 120, 123, 127, 129, 133, 134, 141, 143, 144, 145, 146, 150, 151, 154, 169, 170, 172, 181, 187, 196, 197, 200, 201, 211, 216, 220, 221, 224, 227, 228, 232, 238, 240, 246, 248, 250, 258, 270, 271, 277, 281, 288, 289, 295, 296, 297, 299, 303, 306, 313, 314, 321, 322, 324, 332, 336, 337, 340, 342, 344, 345, 347, 349, 350, 353, 357.
[0302] The numbers above correspond to amino acid positions in the SacI protein sequence (SEQ ID NO:93).
[0303] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pLG-SacIM).
3. Selection of SacI-HF
[0304] Selection of SacI-HF was achieved using a method that was similar to the previous examples. The standard activity check used pUC19 with 5% glycerol in NEB4 and the star activity check was on pUC19 in NEB4 buffer with 39% glycerol. #52 SacI (Q117H/R154A/L284P) and #60 SacI (Q117H/R200A) both had reduced star activity, and SacI Q117H/R200A proved to be the preferred mutation. The Q117H was a carry over mutation from the template, which did not affect the activity of SacI. SacI(Q117H/R200A) was selected as the SacI-HF.
4. Comparison of SacI-HF and WT SacI
[0305] The FIs of SacI-HF and WT SacI were determined separately on pXba substrate in NEB1-4 buffers. The comparison is shown in FIG. 18, and the result is listed in Table 17 (below).
TABLE-US-00026 TABLE 17 Comparison of Sad-HF and WT SacI SacI-HF WT SacI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 25% .gtoreq.2000 100% 120 .gtoreq.16 NEB2 12.5% .gtoreq.120 50% 120 .gtoreq.1 NEB3 0.8% .gtoreq.120 3% 120 .gtoreq.1 NEB4 100% 4000 100% 32 120
[0306] SacI-HF performed best in NEB4, in which the FI was 4000; WT SacI performed best in NEB1 and NEB4, in which the preferred FI was 120. The overall FI improvement factor was 4000/120=32.
Example 12: Engineering of High Fidelity PvuII
1. Expression of PvuII
[0307] PvuII was expressed in E. coli transformed with pACYC-PvuIIM and placzz2-PvuIIR. Placzz2 is a pUC19 derivative plasmid; pACYC is a low copy compatible plasmid. The cells were grown at 30.degree. C. overnight in LB with Amp and Cam.
2. Mutagenesis of PvuII
[0308] All 47 charged residues in PvuII were mutated to Ala as the following residues: 3, 5, 8, 11, 15, 18, 21, 25, 26, 30, 34, 38, 54, 55, 58, 61, 66, 68, 70, 75, 78, 83, 84, 85, 93, 95, 105, 110, 114, 116, 118, 119, 121, 125, 126, 128, 129, 130, 134, 136, 137, 138, 143, 147, 151, 152, and 155.
[0309] The numbers above correspond to amino acid positions in the PvuII protein sequence (SEQ ID NO:92).
[0310] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-PvuIIM).
3. Selection of PvuII High Fidelity Mutants
[0311] Selection of PvuII-HF was similar to the previous examples. The standard activity check used lambda DNA substrate with 5% glycerol in NEB4 and the star activity check was on pBR322 in NEB4 buffer with 39% glycerol. None of the mutants were qualified as high fidelity PvuII.
4. Additional Mutagenesis Steps
[0312] An additional mutagenesis step was mutation of all of the Ser, Thr into Ala, and Tyr to Phe in PvuII. The mutated positions were: 2, 19, 46, 49, 67, 71, 77, 81, 82, 94, 104, 113, 123, 124, 132, 133, 148, 154 and 157.
[0313] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-PvuIIM).
[0314] The PvuII(T46A) apped to have less star activity than the WT PvuII, however, further improvement was desired.
[0315] T46 was mutated to all other amino acid residues, by changing the codons to the corresponding amino acids. Among all these mutations, T46H, T46K, T46Y, T46G were all better than T46A. T46G is selected as the PvuII-HF.
5. Comparison of PvuII-HF and WT PvuII
[0316] The FIs of PvuII-HF and WT PvuII were determined separately on pBR322, with diluent A in NEB1-4 buffers. The comparison is shown in FIG. 19, and the result is listed in Table 18 (below).
TABLE-US-00027 TABLE 18 Comparison of PvuII-HF and WT PvuII PvuII-HF WT PvuII Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 3.1% .gtoreq.250 100% 250 .gtoreq.1 NEB2 12.5% .gtoreq.1000 25% 16 .gtoreq.64 NEB3 0.4% .gtoreq.32 3.1% 8 .gtoreq.4 NEB4 100% 500 100% 1/4 2000
[0317] PvuII-HF performed best in NEB4, in which the FI was 500; WT PvuII performed best in NEB1 and NEB4, in which the preferred FI was 250. The overall FI improvement factor was 500/250=2. Though the overall FI improvement factor is not high for PvuII, the FI improved 2000 times in NEB4.
Example 13: Engineering of High Fidelity MfeI
1. Expression of MfeI
[0318] MfeI was expressed in E. coli transformed with pACYC-MIuCIM and pRRS-MfeIR. pRRS is a pUC19 derivative plasmid, pACYC is a low copy compatible plasmid. MIuCIM methylate AATT, which is the inner four nucleic acid sequence of the MfeI. The cells were grown at 37.degree. C. overnight in LB with Amp and Cam.
2. Mutagenesis of MfeI
[0319] The mutagenesis of MfeI was done in three batches. The first batch is all of the charged residues, mutated into Ala as the following amino acid positions: 3,5, 9, 19, 24, 36, 39, 44, 45, 47, 48, 50, 60, 61, 64,65, 72, 83, 87, 90, 92, 93, 98, 100, 101, 103, 107, 109, 110, 115, 119, 120, 121, 124, 132, 135, 142, 143, 144, 153, 155, 158, 159, 161, 162, 164, 165, 171, 172, 175, 181, 184, 187, 188, 192, 195, 196, 198, 199, 200; The second batch is all of the residues with hydroxyl group: Ser, Thr and Tyr, with Ser and Thr changed into Ala and Tyr changed into Phe. The residues are at: 4, 7, 21, 28, 38, 40, 43, 53, 74, 75, 76, 81, 89, 91, 112, 122, 127, 134, 136, 157, 167, 170, 173, 177, 185, and 200. The third batch is the residues of Cys, Phe, Met, Asn, Gln, Trp all changed into Ala, the residues are at: 10, 12, 13, 25, 26, 29, 31, 32, 35, 51, 55, 67, 68, 77, 78, 84, 88, 96, 102, 105, 117, 123, 126, 141, 148, 149, 152, 168, 169, 174, 176, 178, 179, 180, 183, 191, 193, 194.
[0320] The numbers above correspond to amino acid positions in the MfeI protein sequence (SEQ ID NO:5).
[0321] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-MIuCIM).
3. Selection of MfeI-HF
[0322] Selection of MfeI-HF was achieved using a method that was similar to the previous examples. Cleavage activity was determined using .phi.X174 substrate with 5% glycerol in NEB4 and star activity was determined using .phi.X174 substrate in NEB4 buffer with 39% glycerol. A significant difficulty for this enzyme was that many mutations improved cleavage activity of the enzyme with reduced star activity, but required higher glycerol concentrations than the WT enzyme. MfeI(K50A) is one example, having reduced star activity and high cleavage activity in high concentration glycerol, while in lower glycerol concentrations, the activity was low. MfeI(Y173A) also reduced star activity. The preferred mutation was Q13A/F35Y. The mutation of F35Y was from the template, and Q13A was a targeted mutation. MfeI(Q13A/F35Y) was labelled MfeI-HF.
4. Comparison of MfeI-HF and WT MfeI
[0323] The FIs of MfeI-HF and WT MfeI were determined separately on lambda DNA substrate, with the dilution in NEB diluent A in NEB1-4 buffers. The comparison is shown in FIG. 20, and the result is listed in Table 19 (below).
TABLE-US-00028 TABLE 19 Comparison of MfeI-HF and WT MfeI MfeI-HF WT MfeI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 100% .gtoreq.1000 100% 32 .gtoreq.32 NEB2 25% .gtoreq.250 12.5% 16 .gtoreq.16 NEB3 1.3% .gtoreq.16 6.3% 8 .gtoreq.2 NEB4 100% .gtoreq.500 100% 32 .gtoreq.16
[0324] MfeI-HF performed best in NEB1 and NEB4, in which the preferred FI was .gtoreq.1000; WT MfeI performed best in NEB1 and NEB4, in which the preferred FI was 32. The overall FI improvement factor was .gtoreq.1000/32=32 fold.
Example 14: Engineering of high fidelity HindIII
1. Expression of HindIII
[0325] HindIII was expressed in E. coli transformed with pUC19-HindIIIRM, which contains both HindIII endonuclease and methylase genes. The cells were grown at 30.degree. C. overnight in LB with Amp.
2. Mutagenesis of HindIII
[0326] 88 charged residues in HindIII were mutated to Ala. These were: 2, 3, 7, 8, 14, 20, 22, 34, 37, 39, 42, 45, 52, 55, 61, 62, 66, 69, 74, 84, 87, 89, 94, 100, 101, 109, 111, 114, 117, 120, 123, 124, 126, 128, 132, 134, 135, 136, 137, 138, 153, 158, 162, 163, 171, 172, 180, 182, 183, 190, 197, 198, 201, 202, 207, 209, 214, 215, 218, 222, 225, 227, 228, 229, 237, 238, 243, 244, 245, 249, 250, 251, 254, 255, 261, 265, 266, 267, 270, 274, 275, 281, 283, 286, 290, 293, 296, 297.
[0327] All residues Cys, Met, Asn, Gln, Ser, Thr, Trp were changed to Ala while Tyr was changed to Phe at the positions of 4, 11, 15, 17, 18, 19, 21, 23, 26, 27, 30, 31, 36, 38, 46, 57, 58, 59, 60, 63, 64, 76, 77, 80, 82, 83, 88, 91, 99, 102, 103, 104, 112, 113, 116, 118, 121, 122, 125, 131, 133, 139, 143, 146, 147, 148, 149, 151, 152, 154, 155, 157, 159, 160, 164, 168, 169, 170, 178, 184, 185, 187, 188, 189, 191, 193, 194, 195, 199, 200, 203, 204, 206, 210, 211, 212, 213, 216, 217, 219, 220, 221, 224, 230, 232, 233, 236, 240, 241, 246, 252, 253, 256, 258, 262, 263, 264, 277, 278, 279, 280, 284, 287, 288, 294, 295, 299.
[0328] The numbers above correspond to amino acid positions in the HindIII protein sequence (SEQ ID NO:85).
[0329] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli strain ER3081.
3. Selection of HindIII-HF
[0330] Selection of HindIII-HF was achieved using a method that was similar to the previous examples. The standard activity check used lambda DNA with 5% glycerol in NEB4 and star activity was measured using lambda DNA substrate in NEB4 buffer with 39% glycerol. 2 mutants of HindIII were found to have reduced star activity. These were HindIII(K198A) and S188P/E190A. HindIII(K198A) was labelled HindIII-HF.
4. Comparison of HindIII-HF and WT HindIII
[0331] The FIs of HindIII-HF and WT HindIII were determined separately using lambda DNA substrate in each of NEB1-4 buffers with diluent B. The comparison is shown in FIG. 21, and the result is listed in Table 20 (below).
TABLE-US-00029 TABLE 20 Comparison of HindIII-HF and WT HindIII HindIII-HF WT HindIII Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 25% .gtoreq.16000 25% 32 .gtoreq.500 NEB2 100% .gtoreq.64000 100% 250 .gtoreq.250 NEB3 12.5% .gtoreq.16000 25% 4000 .gtoreq.4 NEB4 50% 32000 50% 32 .gtoreq.1000
[0332] HindIII-HF performed best in NEB2, in which the preferred FI was .gtoreq.64000; WT HindIII performed best in NEB2, in which the preferred FI was 250. The overall FI improvement factor was 4000/120=32.
Example 15: Engineering of High Fidelity SbfI
1. Expression of SbfI
[0333] SbfI was expressed in E. coli transformed with pUC19-SbfIRM. The cells were grown at 30.degree. C. overnight in LB with Amp.
2. Mutagenesis of SbfI
[0334] 78 charged residues in SbfI were mutated to Ala. These were: 5, 8, 15, 18, 23, 27, 30, 34, 46, 49, 50, 53, 58, 63, 66, 70, 71, 74, 81, 82, 83, 85, 86, 87, 90, 94, 103, 115, 120, 121, 127, 132, 135, 136, 143, 144, 147, 150, 152, 154, 164, 169, 170, 183, 184, 187, 188, 192, 196, 204, 206, 208, 213, 214, 215, 218, 219, 226, 228, 230, 233, 237, 238, 239, 241, 248, 251, 253, 257, 258, 259, 260, 262, 266, 282, 284, 285, 288, 293, 297, 299, 304, 305, 307, 311, 316, and 322.
[0335] The residues of Ser and Thr in SbfI were also mutated into Ala. Tyr was mutated into Phe. The following positions were targeted: 3, 4, 5, 10, 13, 16, 31, 35, 38, 54, 55, 56, 68, 76, 78, 80, 88, 109, 111, 116, 119, 129, 131, 137, 146, 162, 174, 197, 198, 201, 205, 210, 224, 252, 263, 270, 272, 286, 298, 315, 321.
[0336] Another 55 residues of Cys, Phe, Met, Asn, Gln, Trp were also mutated to Ala at positions of: 2, 24, 26, 29, 32, 51, 62, 65, 67, 72, 84, 91, 92, 95, 97, 101, 104, 106, 110, 112, 114, 117, 124, 134, 140, 157, 160, 171, 178, 179, 185, 189, 193, 212, 217, 225, 231, 243, 245, 247, 256, 265, 268, 277, 279, 280, 281, 283, 287, 289, 290, 296, 301, 313 and 317.
[0337] The numbers above correspond to amino acid positions in the SbfI protein sequence (SEQ ID NO:96).
[0338] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The mutated products were transformed into E. coli strain ER2984.
3. Selection of SbfI-HF
[0339] Selection of SbfI-HF was achieved as described in previous examples. The standard activity check used lambda DNA with 5% glycerol in NEB4 and the star activity check was on lambda DNA in Exonuclease I buffer. SbfI(K251A) was labelled SbfI-HF.
4. Comparison of SbfI-HF and WT SbfI
[0340] The FIs of SbfI-HF and WT SbfI were determined separately on lambda DNA in NEB1-4 buffers with diluent C. The comparison is shown in FIG. 22, and the result is listed in Table 21 (below).
TABLE-US-00030 TABLE 21 Comparison of SbfI-HF and WT SbfI SbfI-HF WT SbfI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 100% 1000 100% 16 64 NEB2 50% 120 50% 32 4 NEB3 3.5% 8 25% 120 1/16 NEB4 100% 250 12.5% 8 32
[0341] SbfI-HF performed best in NEB1 and NEB4, in which the preferred FI was 1000; WT SbfI performed best in NEB1, in which the preferred FI was 8. The overall FI improvement factor was 1000/8=125 fold.
Example 16: Engineering of High Fidelity EagI
1. Expression of EagI
[0342] EagI was expressed in E. coli transformed with pBR322-EagIRM. The cells were grown at 30.degree. C. overnight in LB with 20 .mu.g/ml Tetracycline.
2. Mutagenesis of EagI
[0343] Asp, Glu, His, Lys, Arg, Ser, Thr, Asn and Gln residues were mutated to Ala. Tyr was mutated to Phe. These were the following residues: 2, 3, 4, 5, 6, 9, 13, 14, 17, 19, 21, 23, 27, 35, 36, 37, 40, 42, 43, 44, 45, 46, 49, 51, 53, 55, 56, 58, 60, 66, 67, 69, 71, 72, 73, 74, 75, 77, 78, 80, 82, 86, 87, 92, 93, 94, 95, 98, 99, 100, 102, 103, 104, 105, 112, 113, 114, 116, 117, 119, 122, 125, 127, 132, 134, 135, 137, 139, 140, 141, 145, 147, 148, 150, 152, 154, 155, 156, 157, 160, 162, 163, 164, 166, 169, 172, 173, 176, 177, 178, 179, 182, 185, 187, 188, 189, 193, 196, 197, 201, 202, 203, 204, 205, 206, 208, 209, 212, 217, 220, 221, 222, 224, 225, 230, 235, 236, 237, 238, 239, 240, 241, 243, 245, 246, 247, 248, 251, 255, 257, 258, 259, 260, 263, 264, 265, 266, 270, 272, 273, 275, 276, 277, 279, 280, 283, 286, 288, 289, 291, 295, 296.
[0344] The numbers above correspond to amino acid positions in the EagI protein sequence (SEQ ID NO:82).
[0345] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli strain ER 3081 and grown on the LB agar plate with Tetracycline.
3. Selection of EagI-HF
[0346] Selection of EagI-HF was achieved using a method that was different to the previous examples which used high concentration of glycerol, high concentration of DMSO or high pH. Since the expression was too low to show the star activity in the crude extract, it would be very tedious to purify each of the mutants to check the star activity. From the previous examples, it was deduced that HF endonucleases tended to have increased cleavage activity in NEB4 compared to NEB3. Hence, the activity of EagI in the crude extract was measured in both NEB3 and NEB4; the one with highest ratio of NEB4/NEB3 was selected. EagI(H43A) was labelled EagI-HF.
4. Comparison of EagI-HF and WT EagI
[0347] The FIs of EagI-HF and WT EagI were determined separately on pXba substrate in each of NEB1-4 buffers. The comparison is shown in FIG. 23, and the result is listed in Table 22 (below).
TABLE-US-00031 TABLE 22 Comparison of EagI-HF and WT EagI EagI-HF WT EagI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 50% 250 25% 4 64 NEB2 100% 250 50% 8 32 NEB3 50% 250 100% 250 1 NEB4 100% 500 100% 16 16
[0348] EagI-HF performed best in NEB2 and NEB4, in which the preferred FI was 500; WT EagI performed best in NEB3 and NEB4, in which the preferred FI was 250. The overall FI improvement factor was 500/250=2.
Example 17: Engineering of High Fidelity EcoRV
1. Expression of EcoRV
[0349] EcoRV was expressed in E. coli strain transformed with pACYC-EcoRVM and placzz1-EcoRV. Placzz1 is a pUC19 derivative plasmid and pACYC is a low copy compatible plasmid. The cells were grown at 37.degree. C. overnight in LB with Amp and Cam.
2. Mutagenesis of EcoRV
[0350] Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Gln, Arg, Ser, Thr, and Trp residues were changed to Ala. Tyr was changed to Phe. These were: 2, 4, 5, 6, 9, 12, 13, 14, 15, 16, 17, 18, 19, 21, 25, 27, 29, 31, 35, 36, 37, 38, 41, 42, 44, 45, 47, 48, 49, 53, 54, 57, 58, 59, 61, 64, 65, 67, 68, 69, 70, 71, 72, 74, 75, 76, 78, 79, 81, 82, 84, 85, 86, 90, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 104, 105, 106, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 123, 125, 126, 127, 128, 131, 132, 136, 138, 139, 140, 143, 144, 145, 146, 147, 149, 150, 151, 152, 154, 155, 157, 158, 161, 163, 164, 167, 169, 171, 172, 173, 174, 179, 183, 185, 186, 187, 188, 191, 193, 195, 196, 197, 198, 199, 201, 203, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 226, 227, 228, 229, 230, 231, 232, 234, 235, 236, 237, 238, 239, 241, 242, 244, and 245.
[0351] The numbers above correspond to amino acid positions in the EcoRV protein sequence (SEQ ID NO:84).
[0352] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-EcoRVM).
3. Selection of EcoRV-HF
[0353] Selection of EcoRV-HF was achieved using a method that was similar to the previous examples. The standard activity check used pXba with 5% glycerol in NEB4 and the star activity check was on pXba in Exonuclease I buffer with 39% glycerol. EcoRV(D19A/E27A) was found to have reduced star activity compared with WT EcoRV. This mutant was labeled EcoRV-HF. For this mutant, the E27A was the targeted mutation, and D19A was a spontaneous mutation. The double mutant had greater reduction in star activity than either the D19A and E27A single mutant.
4. Comparison of EcoRV-HF and WT EcoRV
[0354] The FIs of EcoRV-HF and WT EcoRV were determined separately on pXba substrate in each of NEB1-4 buffers. The comparison is shown in FIG. 24, and the result is listed in Table 23 (below).
TABLE-US-00032 TABLE 23 Comparison of EcoRV-HF and WT EcoRV EcoRV-HF WT EcoRV Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 25% .gtoreq.16000 6.3% 32 .gtoreq.500 NEB2 100% .gtoreq.64000 50% 120 .gtoreq.500 NEB3 50% .gtoreq.32000 100% 1000 .gtoreq.32 NEB4 100% .gtoreq.64000 25% 64 .gtoreq.1000
[0355] EcoRV-HF performed best in NEB2 and NEB4, in which the preferred FI was .gtoreq.64000; WT EcoRV performed best in NEB3, in which the preferred FI was 1000. The overall FI improvement factor was .gtoreq.64000/1000=64.
Example 18: Engineering of High Fidelity AvrII
1. Expression of AvrII
[0356] AvrII was expressed in E. coli transformed with pUC19-AvrIIRM. The cells were grown at 30.degree. C. overnight in LB with Amp.
2. Mutagenesis of AvrII
[0357] Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Gln, Arg, Ser, Thr, and Trp residues were mutated to Ala. Tyr was changed to Phe. These were: 2, 3, 4, 6, 8, 9, 10, 12, 15, 17, 19, 20, 22, 23, 27, 29, 30, 31, 32, 34, 36, 40, 41, 42, 43, 44, 46, 47, 48, 50, 51, 53, 55, 56, 57, 58, 59, 60, 65, 68, 70, 72, 74, 75, 76, 77, 79, 80, 82, 83, 84, 86, 87, 88, 94, 95, 96, 97, 100, 104, 105, 106, 107, 108, 110, 112, 113, 116, 117, 119 120, 121, 122, 123, 124, 126, 127, 129, 130, 131, 132, 134, 136, 139, 142, 143, 144, 145, 150, 151, 152, 153, 154, 156, 157, 158, 161, 163, 164, 165, 166, 168, 169, 173, 174, 177, 178, 181, 182, 184, 186, 187, 188, 189, 190, 191, 192, 195, 198, 200, 202, 206, 207, 211, 215, 216, 220, 223, 224, 226, 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 243, 244, 245, 246, 248, 249, 253, 255, 256, 260, 262, 264, 265, 266, 267, 268, 269, 270, 272, 274, 276, 277, 278, 279, 280, 281, 284, 285, 286, 288, 289, 290, 291, 299, 302, 303, 304, 305, 306, 308, 310, 312, 314, 315, 316, 318, 321, 322, 324, 325, 328, 331, 333, 335, 337, 338, 339, 340, 342, 343, 346, 347, 348, 350, 351, 353, 354, 355, 356, 358.
[0358] The numbers above correspond to amino acid positions in the AvrII protein sequence (SEQ ID NO:80).
[0359] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli expression strain ER2984.
3. Selection of AvrII-HF
[0360] Selection of AvrII-HF was achieved using a method that was similar to the previous examples. The cleavage activity was determined using pBC4 with 5% glycerol in NEB4 and the star activity was measured using pBC4 in ExoI buffer with 39% glycerol. Mutants #16 (M29A), #57(E96A), #60(Y104F), #62(K106A), #154(5127A), #170(F142A) all showed improvement. AvrII(Y104F) was labelled AvrII-HF.
4. Comparison of AvrII-HF and WT AvrII
[0361] The FIs of AvrII-HF and WT AvrII were determined separately on T7 DNA substrate with diluent B in each of NEB1-4 buffers. The comparison is shown in FIG. 25, and the result is listed in Table 24 (below).
TABLE-US-00033 TABLE 24 Comparison of AvrII-HF and WT AvrII AvrII-HF WT AvrII Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 100% .gtoreq.500 100% 64 .gtoreq.8 NEB2 50% .gtoreq.500 100% 8 .gtoreq.64 NEB3 3.1% .gtoreq.16 25% 32 .gtoreq.0.5 NEB4 100% 1000 100% 32 32
[0362] AvrII-HF performed best in NEB1 and NEB4, in which the preferred FI was 1000; WT AvrII performed best in NEB1 and NEB4, in which the preferred FI was 64. The overall FI improvement factor was 1000/64=16.
Example 19: Engineering of High Fidelity BstXI
1. Expression of BstXI
[0363] BstXI was expressed in E. coli transformed with pACYCBstXIMS and pUC19-BstXIR. pACYC is a low copy compatible plasmid. The BstXI has to have both Methylase gene and the specificity gene to have a methylase function. The cells were grown at 37.degree. C. overnight in LB with Amp and Cam.
2. Mutagenesis of BstXI
[0364] 237 amino acid mutations were made in BstXI as follows. Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Gln, Arg, Ser, Thr, Trp were mutated to Ala. Try was mutated to Phe. These were: 4, 6, 7, 9, 11, 12, 14, 15, 17, 18, 20, 21, 22, 23, 24, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 42, 43, 46, 48, 50, 53, 54, 57, 58, 59, 60, 62, 63, 64, 65, 66, 71, 72, 73, 75, 76, 78, 80, 81, 82, 83, 84, 86, 89, 91, 93, 94, 95, 96, 97, 98, 103. 105, 106, 108, 110, 111, 112, 114, 117, 118, 120, 123, 124, 125, 126, 127, 128, 129, 130, 131, 137, 138, 139, 141, 142, 144, 145, 146, 148, 151, 152, 153, 154, 155, 156, 159, 162, 163, 166, 168, 169, 171, 172, 173, 174, 175, 176, 177, 178, 182, 185, 188, 189, 191, 193, 194, 195, 196, 198, 199, 201, 204, 208, 209, 210, 211, 212, 214, 215, 216, 217, 218, 219, 220, 221, 223, 228, 229, 230, 233, 235, 236, 238, 239, 240, 244, 245, 248, 249, 250, 253, 254, 255, 258, 259, 260, 261, 263, 264, 265, 267, 268, 269, 272, 276, 277, 278, 279, 280, 282, 285, 286, 287, 288, 289, 291, 293, 294, 295, 300, 301, 302, 304, 305, 306, 308, 309, 312, 314, 317, 318, 319, 320, 323, 324, 325, 326, 330, 331, 333, 334, 335, 337, 343, 344, 345, 346, 347, 349, 353, 355, 356, 357, 358, 359, 360, 362, 363, 364, 365, 367, 369, 371, 373, 374, 376, 377, 378, 379, 380, 381, 382, and 383.
[0365] The numbers above correspond to amino acid positions in the BstXI protein sequence (SEQ ID NO:7).
[0366] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-BstXIMS).
3. Selection of BstXI-HF
[0367] Selection of BstXI-HF was achieved using a method that was similar to the previous examples. The cleavage activity was determined using pBC4 with 5% glycerol in NEB4 and the star activity was determined using pBC4 DNA substrate in NEB4 buffer with 39% glycerol. Mutants #36(Y57F), #44(N65A), #48(E75A), #49(N76A), and #124(K199A) all had reduced star activity. The BstXI(N65A) was labelled BstXI-HF.
4. Comparison of BstXI-HF and WT BstXI
[0368] The FIs of BstXI-HF and WT BstXI were determined separately on lambda DNA substrate with diluent A in each of NEB1-4 buffers. The comparison is shown in FIG. 26, and the result is listed in Table 25 (below).
TABLE-US-00034 TABLE 25 Comparison of BstXI-HF and WT BstXI BstXI-HF WT BstXI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 50% .gtoreq.120 6.3% 4 .gtoreq.32 NEB2 100% .gtoreq.250 100% 32 .gtoreq.8 NEB3 6.3% .gtoreq.16 100% 2 .gtoreq.8 NEB4 100% .gtoreq.250 100% 32 .gtoreq.32
[0369] BstXI-HF performed best in NEB2 and NEB4, in which the preferred FI was .gtoreq.250; WT BstXI performed best in NEB2, NEB3 and NEB4, in which the preferred FI was 32. The overall FI improvement factor was .gtoreq.250/32=8.
Example 20: Engineering of High Fidelity PciI
1. Expression of PciI
[0370] PciI was expressed in E. coli transformed with pACYC-PciIM and placzz1-PciIR. placzz1 is a pUC19 derivative plasmid, pACYC is a low copy compatible plasmid. The cells were grown at 37.degree. C. overnight in LB with Amp and Cam.
2. Mutagenesis of PciI
[0371] 151 amino acid residues in PciI were mutated with Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Gln, Arg, Ser, Thr. Trp was changed to Ala and Tyr to Phe. These were: 2, 3, 4, 6, 8, 9, 10, 11, 12, 14, 17, 18, 19, 21, 24, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 41, 44, 46, 47, 49, 50, 51, 54, 55, 56, 58, 59, 60, 63, 67, 68, 69, 71, 74, 75, 78, 80, 81, 82, 85, 86, 91, 92, 95, 97, 98, 101, 103, 104, 107, 109, 113, 114, 115, 118, 119, 120, 121, 122, 124, 126, 127, 129, 130, 131, 132, 133, 135, 136, 137, 138, 143, 145, 146, 147, 148, 149, 151, 152, 153, 154, 155, 157, 158, 159, 161, 164, 165, 167, 172, 175, 178, 179, 180, 182, 184, 185, 186, 190, 192, 193, 196, 197, 198, 199, 200, 202, 203, 206, 207, 209, 210, 215, 218, 221, 222, 228, 229, 230, 231, 232, 233, 234, 235, 237, 238, 239, 241, 242, 243, 244, 246, 247, 248, 253, 254, 255, 256.
[0372] The numbers above correspond to amino acid positions in the PciI protein sequence (SEQ ID NO:15).
[0373] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-PciIM).
3. Selection of PciI-HF
[0374] Selection of PciI-HF was achieved using a method that was similar to the previous examples. The cleavage activity was determined using SalI-cut pBR322 with 5% glycerol in NEB4 and the star activity was determined using SalI-cut pBR322 in ExoI buffer with 39% glycerol. A double mutant PciI(E78A/S133A) had reduced star activity and strong cleavage activity. This mutant was not one of the targeted mutations described above, but was a fortuitous random event.
4. Comparison of PciI-HF and WT PciI
[0375] The FIs of PciI-HF and WT PciI were determined separately on pXba substrate with diluent A in each of NEB1-4 buffers. The comparison is shown in FIG. 27, and the result is listed in Table 26 (below).
TABLE-US-00035 TABLE 26 Comparison of PciI-HF and WT PciI PciI-HF WT PciI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 NC NC 50% 2000 N/A NEB2 100% .gtoreq.2000 25% 16 .gtoreq.120 NEB3 100% .gtoreq.2000 100% 120 .gtoreq.16 NEB4 100% .gtoreq.1000 12.5% 8 .gtoreq.120
[0376] PciI-HF performed best in NEB2, NEB3 and NEB4, in which the preferred FI was .gtoreq.2000; WT PciI performed best in NEB3, in which the preferred FI was 120. The overall FI improvement factor was .gtoreq.2000/120=16.
Example 21: Engineering of High Fidelity HpaI
1. Expression of HpaI
[0377] HpaI was expressed in E. coli transformed with pACYC-MseIM and placzz1-HpaIR. placzz1 is a pUC19 derivative plasmid, pACYC is a low copy compatible plasmid. The cells were grown at 37.degree. C. overnight in LB with Amp and Cam.
2. Mutagenesis of HpaI
[0378] 156 amino acid residues in HpaI were mutated with Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Gln, Arg, Ser, and Thr. Trp was changed to Ala and Tyr to Phe. These were: 7, 8, 9, 13, 14, 16, 17, 19, 20, 21, 22, 23, 26, 27, 29, 30, 33, 34, 35, 36, 37, 38, 40, 41, 42, 46, 47, 48, 50, 51, 56, 57, 59, 60, 65, 67, 69, 71, 72, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, 86, 89, 91, 93, 94, 95, 99, 100, 104, 105, 106, 108, 109, 110, 113, 115, 117, 119, 121, 122, 123, 124, 127, 128, 130, 131, 133, 135, 136, 137, 138, 139, 141, 142, 146, 147, 149, 150, 152, 156, 158, 159, 160, 162, 164, 165, 166, 167, 168, 169, 170, 172, 173, 176, 177, 180, 181, 182, 184, 185, 187, 188, 190, 191, 192, 193, 195, 196, 197, 202, 204, 206, 208, 209, 211, 212, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 228, 230, 231, 233, 234, 235, 236, 237, 238, 240, 241, 242, 243, 244, 245, 247, 248, 249.
[0379] The numbers above correspond to amino acid positions in the HpaI protein sequence (SEQ ID NO:86).
[0380] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-MseIM).
3. Selection of HpaI-HF
[0381] Selection of HpaI-HF was achieved using a method that was different to the previous examples. The cleavage activity and star activity were determined using lambda DNA substrate in NEB2 buffer. This HpaI has much more star activity in NEB2 than NEB4, and could be clearly observed in 5% glycerol.
[0382] HpaI(Y29F) and HpaI(E56A) were both preferred mutations with reduced star activity. HpaI(E56A) was labelled HpaI-HF.
4. Comparison of HpaI-HF and WT HpaI
[0383] The FIs of HpaI-HF and WT HpaI were determined separately on lambda DNA substrate with diluent A in each of NEB1-4 buffers. The comparison is shown in FIG. 28, and the result is listed in Table 27 (below).
TABLE-US-00036 TABLE 27 Comparison of HpaI-HF and WT HpaI HpaI-HF WT HpaI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 3.1% .gtoreq.32 6.3% 32 .gtoreq.1 NEB2 100% .gtoreq.2000 25% 1 .gtoreq.2000 NEB3 12.5% 2 12.5% 2 1 NEB4 50% .gtoreq.2000 100% 16 .gtoreq.120
[0384] HpaI-HF performed best in NEB2, in which the preferred FI was .gtoreq.2000; WT PciI performed best in NEB4, in which the preferred FI was 16. The overall FI improvement factor was .gtoreq.2000/16=120.
Example 22: Engineering of High Fidelity AgeI
1. Expression of AgeI
[0385] AgeI was expressed in E. coli transformed with pRRS-AgeIRM and psyx20-lacIq. pRRS is a pUC19 derivative plasmid, psyx20-lacIq is a low copy compatible plasmid with lad expressed under lacIq promoter. The cells were grown at 37.degree. C. in LB with Amp and Kan to 200 Klett units, and then induced at 25.degree. C. with 0.5 mM IPTG overnight. The expression of AgeI was extremely difficult to achieve because it was unstable.
2. Mutagenesis of AgeI
[0386] 149 amino acid residues in AgeI were mutated with Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Gln, Arg, Ser, and Thr. Trp was changed to Ala and Tyr to Phe. These were: 2, 4, 6, 7, 9, 14, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 37, 38, 40, 42, 43, 44, 45, 49, 51, 53, 55, 56, 58, 60, 62, 64, 65, 67, 68, 69, 72, 73, 75, 77, 78, 79, 82, 83, 85, 86, 87, 88, 90, 91, 92, 94, 96, 97, 102, 103, 104, 105, 110, 111, 114, 116, 119, 120, 122, 123, 128, 129, 130, 134, 135, 138, 139, 140, 142, 144, 146, 147, 148, 152, 153, 155, 157, 159, 166, 168, 170, 173, 174, 176, 177, 178, 182, 183, 185, 186, 188, 192, 195, 198, 200, 201, 206, 211, 212, 214, 217, 219, 220, 222, 223, 224, 225, 226, 227, 229, 231, 233, 234, 235, 237, 238, 239, 240, 241, 243, 245, 247, 248, 250, 251, 253, 255, 256, 258, 260, 262, 265, 266, 267, 268, 269, 271, 272
[0387] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (psyx20-lacIq).
[0388] The numbers above correspond to amino acid positions in the AgeI protein sequence (SEQ ID NO:79).
3. Selection of AgeI-HF
[0389] Selection of AgeI-HF was achieved using a method that was similar to the previous examples. The standard activity check used pXba with 5% glycerol in NEB4 and the star activity check was on pXba in NEB4 buffer with 39% glycerol. Because of the difficulty of the expression system, this selection was repeated eight times before meaningful mutants were obtained. Two mutants, S201A and R139A had reduced star activity and R139A was labelled AgeI-HF.
4. Comparison of AgeI-HF and WT AgeI
[0390] The FIs of AgeI-HF and WT AgeI were determined separately on pXba substrate with diluent A in each of NEB1-4 buffers. The comparison is shown in FIG. 29, and the result is listed in Table 28 (below).
TABLE-US-00037 TABLE 28 Comparison of AgeI-HF and WT AgeI AgeI-HF WT AgeI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 100% .gtoreq.500 100% 16 .gtoreq.32 NEB2 50% .gtoreq.250 50% 8 .gtoreq.32 NEB3 6.3% .gtoreq.16 12.5% 64 .gtoreq.0.25 NEB4 100% .gtoreq.250 50% 8 .gtoreq.32
[0391] AgeI-HF performed best in NEB1, and NEB4, in which the preferred FI was .gtoreq.500; WT AgeI performed best in NEB3, in which the preferred FI was 16. The overall FI improvement factor was .gtoreq.500/16=32.
Example 23: Engineering of High Fidelity BsmBI
1. Expression of BsmBI
[0392] BsmBI was expressed in E. coli transformed with pACYC-BsmAIM, ptaczz2-BsmBIR and psyx20-lacIq. Ptaczz2 is a pUC19 derivative plasmid which carries a inducible ptac promoter, pACYC is a low copy compatible plasmid. BsmAIM (GTCTC) covers BsmBI (CGTCTC) specificity. The psyx20-lacIq is a low copy vector with strong express of lacI. The cells were grown at 37.degree. C. then induced in LB with Amp, Cam and Kan.
2. Mutagenesis of BsmBI
[0393] 358 amino acid residues in BsmBI were mutated with Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Gln, Arg, Ser, and Thr. Trp was changed to Ala and Tyr to Phe. These were: 8,9, 12, 13, 14, 15, 17, 18, 19, 20, 21, 24, 25, 26, 27, 28, 29, 31, 33, 37, 38, 40, 42, 43, 44, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 72, 76, 78, 79, 80, 81, 82, 83, 84, 88, 91, 93, 95, 96, 98, 99, 101, 103, 104, 105, 106, 109, 110, 111, 113, 114, 115, 117, 118, 119, 120, 121, 122, 124, 126, 127, 128, 130, 131, 132, 133, 134, 135, 138, 141, 143, 144, 145, 147, 149, 150, 154, 155, 157, 158, 160, 162, 163, 164, 165, 166, 167, 168, 169, 172, 174, 175, 176, 177, 179, 180, 181, 182, 184, 185, 186, 188, 189, 191, 194, 195, 197, 200, 201, 203, 205, 206, 207, 208, 211, 212, 213, 214, 215, 216, 217, 220, 221, 222, 223, 224, 225, 226, 228, 229, 230, 231, 232, 233, 235, 236, 237, 238, 239, 240, 242, 243, 247, 250, 251, 252, 257, 258, 260, 262, 263, 264, 265, 266, 268, 269, 271, 273, 274, 279, 280, 282, 283, 284, 287, 288, 289, 292, 294, 295, 296, 297, 299, 300, 301, 302, 303, 304, 305, 306, 307, 309, 310, 313, 314, 315, 316, 317, 318, 320, 321, 324, 325, 326, 328, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 348, 349, 351, 352, 353, 355, 356, 357, 358, 360, 361, 363, 364, 366, 367, 368, 370, 372, 375, 376, 377, 379, 381, 382, 384, 385, 386, 388, 389, 390, 393, 394, 395, 396, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 418, 421, 424, 425, 426, 428, 430, 431, 432, 433, 434, 436, 437, 438, 439, 442, 443, 444, 445, 446, 446, 448, 449, 450, 451, 452, 453, 454, 457, 458, 460, 461, 462, 464, 466, 467, 468, 470, 471, 472, 473, 474, 477, 478, 479, 480, 482, 483, 484, 485, 486, 487, 488, 489, 491, 492, 495, 496, 497, 498, 499, 500, 502, 503, 504, 505, 506, 507, 510, 511, 515, 516, 517, 518, 519, 522, 523.
[0394] The numbers above correspond to amino acid positions in the BsmBI protein sequence (SEQ ID NO:81).
[0395] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-BsmAIM, psyx20-lacIq).
3. Selection of BsmBI-HF
[0396] Selection of BsmBI-HF was achieved using a method that was similar to the previous examples. The cleavage was determined using lambda DNA with 5% glycerol in NEB4 and the star activity was determined using Litmus28i in NEB4 buffer with 39% glycerol. Preferred mutants included H230A, D231A and N185Y/R232A. N185Y/R232A was labeled BsmBI-HF.
4. Comparison of BsmBI-HF and WT BsmBI
[0397] The FIs of BsmBI-HF and WT BsmBI were determined separately on lambda DNA substrate with diluent A in each of NEB1-4 buffers. The comparison is shown in FIG. 30, and the result is listed in Table 29 (below).
TABLE-US-00038 TABLE 29 Comparison of BsmBI-HF and WT BsmBI BsmBI-HF WT BsmBI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 100% 32 12.5% 1 32 NEB2 100% .gtoreq.500 50% 8 .gtoreq.64 NEB3 12.5% .gtoreq.64 100% 120 .gtoreq.0.5 NEB4 100% .gtoreq.500 25% 4 120
[0398] BsmBI-HF performed best in NEB1, NEB2 and NEB4, in which the preferred FI was .gtoreq.500; WT BsmBI performed best in NEB3, in which the preferred FI was 120. The overall FI improvement factor was .gtoreq.500/120=4.
Example 24: Engineering BspQI Variants with Reduced Star Activity
1. Site-Directed Mutagenesis of BspQI Restriction Endonuclease
[0399] E. coli was transformed with pSX33-EarIM1M2 and pZZlacI-PspQI that was Km.sup.R and Amp.sup.R). M.EarI (CTCTTC) also modifies BspQI site (GCTCTTC) and therefore pSX33-earIM1M2 (FIGS. 17 and 18) was used to co-transform and modify the E. coli chromosome. The WT amino acid sequence is shown in FIG. 16.
[0400] 122 charged or non-charged amino acid residues in BspQI (Arg, Lys, His, Glu, Asp, Gln, Asn, Cys) were changed to Ala by site-directed mutagenesis. PCR was carried out under the following conditions: DNA denaturation, 98.degree. C. for 30 sec, 1 cycle; DNA denaturation/primer annealing/extension, 98.degree. C. for 10 sec, 55.degree. C. to 65.degree. C. for 30 sec, 72.degree. C. for 2 min, for PCR 18 cycles; 72.degree. C. for 15 min, 1 cycle. In 100 .mu.l reactions, 2 units of Phusion.TM. DNA polymerase (NEB, Ipswich, Mass.), 1 mM dNTP, 10 ng to 100 ng template DNA, 20 .mu.l 5.times. reaction buffer, 0.04 .mu.M primers, sterile water to 100 .mu.l total volume.
[0401] PCR DNA was digested with DpnI to destroy template DNA (Dam methylated) and co-transformed with pSX33-earIM1M2 into E. coli. Individual transformants were cultured overnight (5 ml LB, 50 .mu.g/ml Km.sup.R and 100 .mu.g/ml Amp.sup.R) and split into two parts. One part (1.5 ml) was harvested by centrifugation and lysed by sonication in a sonication buffer (20 mM Tris-HCl, pH 7.5, 0.1 mM DTT, 50 mM NaCl, 10% glycerol). The cell extract was heated at 50.degree. C. for 1 h and denatured E. coli proteins were removed by centrifugation. The clarified lysate was assayed for restriction activity and star activity on pUC19 DNA.
[0402] BspQI star activity assay condition: 1 .mu.g pUC19 DNA, 5 .mu.l of 10.times.NEB buffer 1, 25% DMSO, 2.5 .mu.l clarified cell extract, sterile deionized water to 50 .mu.l total volume and incubated at 50.degree. C. for 1 h. Digested DNA was resolved by electrophoresis in 0.8 to 1% agarose gel.
[0403] The second part of the cell culture (uninduced) was harvested and plasmid DNA was prepared by Qiagen spin column purification procedure (Qiagen, Valencia, Calif.). The bspQIR alleles were sequenced by Big-dye dideoxy-terminator sequencing method to confirm the desired mutations. After identification of reduced star activity mutants, fresh transformants were obtained and IPTG-induced cultures were made. Restriction and star activity was assayed again to confirm the reduced star activity in comparison with the WT enzyme in all four buffers.
[0404] Among the 122 BspQI mutants constructed by site-directed mutagenesis, two BspQI variants, R388A and K279A, display reduced star activity. The star activity of R388A was reduced approximately 16-fold in buffer 1 and 10% glycerol. However, R388A still displayed star activity at high enzyme concentration. BspQI variant K279A also displayed reduced star activity (>8-fold improvement in reduced star activity).
[0405] To further reduce star activity at high enzyme concentration, R388 and K279 were substituted for Phe, Pro, Tyr, Glu, Asp, or Leu. IPTG-induced cell extracts of various R388X, and K279X mutants were assayed for restriction and star activity. It was found that R388F or K279P displayed the minimal star activity in either cell extracts or purified enzyme. The specific activity was not affected by the amino acid substitutions.
[0406] To still further reduce BspQI star activity, the two amino acid substitutions were combined into one mutant enzyme (double mutant, K279P/R388F) by site-directed mutagenesis. This double mutant lacks star activity in buffer 1 and buffer 2 with 10% glycerol (FIG. 41B).
TABLE-US-00039 TABLE 30 BspQI-HF vs WT BspQI BspQI-HF WT BspQI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 25% .gtoreq.1000 12.5% 2 .gtoreq.1000 NEB2 25% .gtoreq.1000 100% 16 .gtoreq.64 NEB3 1.3% .gtoreq.64 100% 32 .gtoreq.2 NEB4 100% .gtoreq.4000 50% 4 .gtoreq.1000
Example 25: Engineering SapI Variants with Reduced Star Activity
[0407] The conserved K279 and R388 amino acid residues were found in BspQI where corresponding positions are K273 and R380 in SapI. A 6.times.His tagged SapI expression clone was first constructed in pUC19. The SapI expression strain was E. coli transformed with pSX33-earIM1M2 and pUC-SapI that was Km.sup.R and Amp.sup.R. Lys273 to Pro (K273P) and Arg 380 to Phe (R380F) amino acid substitutions were introduced into SapI by site-directed mutagenesis. SapI single mutant R380A was also constructed. Both SapI variants R380A and K273P/R380F showed reduced star activity when restriction activity and star activity reactions were performed (FIG. 42).
[0408] PCR, transformation, plasmid DNA preparation, and enzyme activity assay were carried out as described for BspQI, except that SapI activity was determined at 37.degree. C. The 6.times.His-tagged SapI variant K273P/R380F was purified through Ni-NTA column chromatography and shown to display diminished star activity in the presence of 25% DMSO or 5% glycerol.
Example 26: Engineering KpnI High Fidelity Mutants
[0409] KpnI, which contains two activities, has been changed into a mutant with lower star activity (International Publication No. WO 07/027464). The example below describes novel mutants with improved star activity and similar cleavage activity to the wild-type.
[0410] Charged amino acid residues except the catalytic residues, (Asp, Glu, Arg, Lys and His) or polar amino acids (Ser, Thr, Tyr, Asn, Gln, Phe, Trp, Cys and Met) were individually mutated to Alanine.
[0411] Mutagenesis was carried out by inverse PCR using primers bearing the desired mutations. In general, inverse PCR was performed using 0.4 mM of each of the 4 dNTP, 1.times. ThermoPol Buffer (NEB), 20 ng of template DNA, 40 .mu.mol of each of the primers and 4 U of Vent DNA polymerase (NEB) in a final volume of 100 .mu.L.
[0412] Plasmids pUC19 or pAGR3 containing the KpnIR under the control of Plac or Ptac promoter, respectively, were used as template. PCRs were done with a temperature scheme of 94.degree. C. for 4 minutes followed by 25 cycles of 94.degree. C. for 30 seconds for denaturation, 55.degree. C. for 30 seconds for annealing and 72.degree. C. for 5 minutes for extension. The cycles were followed by incubation at 72.degree. C. for 7 minutes before the reactions were treated by 20 U of DpnI (NEB) at 37.degree. C. for 1 hour to degrade the template DNA. After inactivating DpnI at 80.degree. C. for 20 minutes, 2 .mu.l of the reaction was used to transform 50 .mu.L of chemically competent NEB5alpha (NEB) pre-transformed by pSYX20-KpnIM. The transformed bacteria were plated out onto LB plates containing 100 .mu.g/ml of ampicillin and 30 .mu.g/ml of kanamycin, and incubated at 37.degree. C. for 12 to 15 hours. Three to four colonies of each construct were cultured in 1 ml of LB containing 100 .mu.g/ml of ampicillin and 30 .mu.g/ml of kanamycin at 37.degree. C. for 12 to 15 hours with shaking of 200 rpm. The cultures were spun down and resuspended in 0.2 ml of sonication buffer (20 mM Tris-HCl, pH 8.3, 50 mM NaCl, 1 mM EDTA, 1 mM PMSF.) The resuspended cells were sonicated for 20 seconds, followed by centrifugation at 13,000 rpm at 4.degree. C. for 5 min. Dilutions of the supernatant were made and 5 .mu.L of which were assayed for KpnI cleavage activity.
[0413] For the screening of the mutants, an activity assay reaction was performed using 5 .mu.L of 10- or 100-fold dilutions of the lysate supernatant, 0.5 ug of pXba DNA (NEB) and 1.times.NEBuffer 4 in a total volume of 50 .mu.L. After incubating at 37.degree. C. for 1 hour, the assay reactions were stopped by adding 10 ul of 6.times.DNA loading dye and analyzed by electrophoresis through 0.8% agarose gels in 1.times.TBE. Mutants that showed increased overall cleavage activity compared with the parent enzyme (KpnI D148E) were assayed for reduced star activity in a buffer containing 25% DMSO.
[0414] The assay was performed using 5 .mu.L of dilutions of the enzymes incubated with 1 .mu.g of pXba DNA (NEB) in the presence of 5% glycerol and 0.2 mg/ml of BSA in 1.times. NEBuffer 4 (total volume=50 .mu.L). After incubation at 37.degree. C. for 1 hour, the reactions were treated by 20 .mu.g of proteinase K (NEB) at 37.degree. C. for 15 minutes and then analyzed by electrophoresis through 0.8% agarose gels in 1.times.TBE. Divalent metal-dependent assays were carried out at 37.degree. C. for 1 hour, using 50 U of enzyme and 1 .mu.g of pXba DNA in a buffer containing 20 mM Tris HCl, 50 mM NaCl, pH 7.9, 1 mM DTT and increasing concentration of MgSO4/CaCl2)/MnCl2, followed by electrophoresis through 0.8% agarose gels in 1.times.TBE. The total reaction volume is 50 .mu.L.
[0415] The result of random mutagenesis, was a preferred mutant, KpnI D148E, which showed lower star activity than the wild-type enzyme and KpnI D163I/K165A mutant described previously (International Publication No. WO 07/027464). However, KpnI D148E displayed star activity at high enzyme concentration. Double and triple mutants were constructed in D148E background to reduce this observed star activity. KpnI (D16N/E132A/D148E) was found to have lower star activity and higher specificity activity than mutant D148E. Amino acid substitutions D148 to E and E132 to A were introduced by site-directed mutagenesis. The D16 to N mutation was introduced by PCR. In standard reaction condition, one-hour incubation of purified enzymes with substrate DNA pXba (containing 6 KpnI sites) at 37.degree. C.), resulted in the reduced star activity shown in Table 31.
TABLE-US-00040 TABLE 31 Fidelity indices for KpnI mutants KpnI-HF WT KpnI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 100% .gtoreq.4000 100% 16 .gtoreq.250 NEB2 50% .gtoreq.2000 25% 16 .gtoreq.64 NEB3 6.3% .gtoreq.250 6.3% 8 .gtoreq.32 NEB4 100% .gtoreq.4000 50% 4 .gtoreq.1000
[0416] No star activity of pXba was observed for mutant D16N/E132A/D148E up to 4000 U. Star activity observed for pBR322 substrate, which bears no KpnI site, was also diminished when cleaved with KpnI D148E and D16N/E132A/D148E.
Example 27: Engineering of High Fidelity BsaI
1. Expression of BsaI
[0417] BsaI was expressed in E. coli transformed with pACYC-BsmAIM, pUC19-BsaIR and psyx20-lacIq. pACYC is a low copy compatible plasmid. BsmAIM(GTCTC) covers BsmBI specificity(CGTCTC). The psyx20-lacIq is a low copy vector with strong express of lacI. The cells were grown at 37.degree. C. then induced in LB with Amp, Cam and Kan.
2. Mutagenesis of BsaI
[0418] The amino acid of BsaI is similar to that of BsmBI. 11 amino acids around and at the corresponding previous effective site is mutated as R229A, S230A, Y231F, T232A, T233A, D234A, R235A, R236A, F238A, E239A, Y240F.
[0419] The methods were the same as in the previous examples using inverse PCR followed by DpnI digestion. The treated product was then transformed into E. coli (pACYC-BsmAIM, psyx20-lacIq).
3. Selection of BsaI-HF
[0420] Selection of BsaI-HF was achieved using a method that was similar to the previous examples. The standard activity check used lambda DNA with 5% glycerol in NEB4 and the star activity check was litmus28i in NEB4 buffer with 39% glycerol. One mutant, Y231F, out of the 11 designed ones, reduced star activity and labeled as BsaI-HF.
4. Comparison of BsaI-HF and WT BsaI
[0421] The FIs of BsaI-HF and WT BsaI were determined separately on lambda DNA with diluent A in NEB1-4 buffers. The result is listed in Table 32 (below).
TABLE-US-00041 TABLE 32 Comparison of BsaI-HF and WT BsaI BsaI-HF WT BsaI Fidelity Fidelity Improvement Buffer Activity Index Activity Index Factor NEB1 50% .gtoreq.4000 25% 8 .gtoreq.500 NEB2 100% .gtoreq.8000 100% 120 .gtoreq.64 NEB3 100% 120 25% 16 8 NEB4 100% .gtoreq.8000 100% 32 .gtoreq.250
[0422] BsaI-HF performed best in NEB2, NEB3 and NEB4, in which the best FI was .gtoreq.8000; WT BsaI performed best in NEB2 and NEB4, in which the best FI was 120. So the overall FI improvement factor was .gtoreq.8000/120=.gtoreq.64.
Sequence CWU
1
1
10611089DNAMicrococcus luteus 1atgatcaagt acttgggtag caagcggacg ctcgtgcccg
tcctcggtga catcgcttcg 60gcctctgaag caacagaggc ggttgacctg ttcactggca
cgacgcgtgt ggcgcaagag 120ttcaagcgtc gcgggcttcg agttcttgct aacgacatag
cgacgtactc cgaggtttta 180gcccagtgct atatcgccac caacggccag gaagttgacc
gccgtgcgct cgaggccgct 240ctggcggagc tgaacgcctt gcccggcgaa cctggatact
tcacggaaac cttctgtgag 300gcttctcgct acttccagcc caagaacggg gctcgggtgg
atgcaatcag gaatgcgatc 360gacgaccggt acgcggactc atggatgcga ccgatcctcc
tcacgagctt gatgcttgcg 420gccgaccgcg tcgactccac taccggagtg cagatggctt
acctgaagca gtgggccgcg 480cgtgcgcaca atgatctaga gttgcggctt ccagacctaa
tcgcaggtga cggtgacgct 540gctcgtgagg atgcggtgac tctcgcacaa gagctgcctc
gcgtccagct gatgtacctt 600gatcctccct ataaccagca caggtacttc accaactacc
atatttggga gaccctgatt 660cgttgggatg cccctgagag ttatgggatc gcctgtaagc
gcattgactc tcgagatgat 720gccaccaaga gcccctataa tatgaagcgg cgaatgcccg
acgagatgcg tcgcctgctg 780atgaccatca aggcggacct cgcggttgta tcttacaaca
atgagtcgtg gattgatccg 840gagacgatga tgtcgaccct gcgcgatgcg ggatatgagg
acgtgcgtct gctcgctttc 900gactataagc gctacgttgg ggctcaaatc gggatctaca
atccctccgg ggaaaaggtc 960ggtcgtgtga gtcacctccg aaacatcgag tatctctttc
ttgcgggacc aacggagcgc 1020gttgaggtgt gcgccgcgag tgttgaacac cgagcactac
ccaaggaacc ggaactcacc 1080gcgttctag
10892362PRTMicrococcus luteus 2Met Ile Lys Tyr Leu
Gly Ser Lys Arg Thr Leu Val Pro Val Leu Gly1 5
10 15Asp Ile Ala Ser Ala Ser Glu Ala Thr Glu Ala
Val Asp Leu Phe Thr 20 25
30Gly Thr Thr Arg Val Ala Gln Glu Phe Lys Arg Arg Gly Leu Arg Val
35 40 45Leu Ala Asn Asp Ile Ala Thr Tyr
Ser Glu Val Leu Ala Gln Cys Tyr 50 55
60Ile Ala Thr Asn Gly Gln Glu Val Asp Arg Arg Ala Leu Glu Ala Ala65
70 75 80Leu Ala Glu Leu Asn
Ala Leu Pro Gly Glu Pro Gly Tyr Phe Thr Glu 85
90 95Thr Phe Cys Glu Ala Ser Arg Tyr Phe Gln Pro
Lys Asn Gly Ala Arg 100 105
110Val Asp Ala Ile Arg Asn Ala Ile Asp Asp Arg Tyr Ala Asp Ser Trp
115 120 125Met Arg Pro Ile Leu Leu Thr
Ser Leu Met Leu Ala Ala Asp Arg Val 130 135
140Asp Ser Thr Thr Gly Val Gln Met Ala Tyr Leu Lys Gln Trp Ala
Ala145 150 155 160Arg Ala
His Asn Asp Leu Glu Leu Arg Leu Pro Asp Leu Ile Ala Gly
165 170 175Asp Gly Asp Ala Ala Arg Glu
Asp Ala Val Thr Leu Ala Gln Glu Leu 180 185
190Pro Arg Val Gln Leu Met Tyr Leu Asp Pro Pro Tyr Asn Gln
His Arg 195 200 205Tyr Phe Thr Asn
Tyr His Ile Trp Glu Thr Leu Ile Arg Trp Asp Ala 210
215 220Pro Glu Ser Tyr Gly Ile Ala Cys Lys Arg Ile Asp
Ser Arg Asp Asp225 230 235
240Ala Thr Lys Ser Pro Tyr Asn Met Lys Arg Arg Met Pro Asp Glu Met
245 250 255Arg Arg Leu Leu Met
Thr Ile Lys Ala Asp Leu Ala Val Val Ser Tyr 260
265 270Asn Asn Glu Ser Trp Ile Asp Pro Glu Thr Met Met
Ser Thr Leu Arg 275 280 285Asp Ala
Gly Tyr Glu Asp Val Arg Leu Leu Ala Phe Asp Tyr Lys Arg 290
295 300Tyr Val Gly Ala Gln Ile Gly Ile Tyr Asn Pro
Ser Gly Glu Lys Val305 310 315
320Gly Arg Val Ser His Leu Arg Asn Ile Glu Tyr Leu Phe Leu Ala Gly
325 330 335Pro Thr Glu Arg
Val Glu Val Cys Ala Ala Ser Val Glu His Arg Ala 340
345 350Leu Pro Lys Glu Pro Glu Leu Thr Ala Phe
355 36031146DNAHelicobacter pylori J166 3ttggagaatt
ttttgaataa tttagatatt aaaaccttag ggcaggtttt cacccctaaa 60aagatagtgg
atttcatgct cactctcaag cacaatcatg ggagtgtttt agagccaagc 120gcgggcgatg
ggagtttttt aaagcgctta aaaaaggctg tagggattga aatcgatcct 180aaaatctgcc
ctaaaaatgc cctttgcatg gacttttttg actacccttt agaaaatcaa 240tttgacacga
ttattggcaa tccgccctat gtcaagcaca aggatattgc gccaagcacg 300aaagaaaaac
tccattacag cctttttgat gaaaggagta atctatactt gtttttcata 360gaaaaagcga
tcaagcattt aaagcctaaa ggcgaattga ttttcatcac cccaagggat 420tttttaaaat
ccacttctag cgtgaaatta aacgaatgga tttacaaaga aggcacgata 480acgcattttt
ttgaattagg cgatcaaaag attttcccaa acgccatgcc taattgcgtg 540atttttcgtt
tttgtaaagg tgatttcagt agaatcacca acgatggttt gcaatttgtg 600tgcaaaaaag
gcattttgta tttcctcaac caatcttaca cgcaaaaatt aagcgaggtt 660tttaaggtta
aggtgggggc agtgagcggg tgcgataaga tttttaaaaa tgaaacatac 720gggaatttag
aatttgtcac ctcaatcacc aaaagaacca atgttttaga aaaaatggtt 780tttgtcaata
aacctaatga ttatttactc cagcataaag acagcttgat gcaaagaaag 840attaaaaaat
tcaatgaaag taattggttt gaatggggga ggatgcatca catatcccct 900aaaaaacgca
tttatgttaa cgccaaaacg cgccaaaaaa accccttttt catccaccaa 960tgccctaatt
atgacggctc tattttagcg ctattccctt ataaccaaaa tttggattta 1020caaaacctct
gcgataaact caacgctatc aactggcaag aattaggctt tgtgtgcggc 1080gggcgttttt
tgttttcgca gcgctcttta gaaaacgccc ttttgcctaa agacttttta 1140aattag
11464609DNAMycoplasma fermentans 4atgggtaaat ctgaattaag tggaagatta
aattggcaag cattggctgg attaaaagct 60agtggtgctg aacaaaactt atataacgtg
tttaacgctg tttttgaagg aactaaatac 120gttttatacg agaagccaaa gcaccttaaa
aatctatacg ctcaagtagt cttacctgat 180gatgttatta aagaaatttt taatccttta
attgatttat caactactca atggggtgtt 240tctccagatt tcgcaataga aaatacagaa
acgcataaaa ttctttttgg tgaaattaaa 300agacaagatg gatgggtaga aggtaaagat
cctagtgctg gcaggggtaa tgcacatgag 360agatcttgta aattatttac tcctggatta
ttaaaagctt atagaacaat tggtggaatt 420aacgatgaag agatattgcc attctgggtt
gtattcgaag gtgatataac acgagatccc 480aaaagagtaa gagaaattac tttctggtat
gaccactatc aagataatta tttcatgtgg 540cgaccaaatg aatcaggcga aaaattagtt
caacacttca atgaaaaatt aaaaaaatat 600ttagattaa
6095202PRTMycoplasma fermentans 5Met
Gly Lys Ser Glu Leu Ser Gly Arg Leu Asn Trp Gln Ala Leu Ala1
5 10 15Gly Leu Lys Ala Ser Gly Ala
Glu Gln Asn Leu Tyr Asn Val Phe Asn 20 25
30Ala Val Phe Glu Gly Thr Lys Tyr Val Leu Tyr Glu Lys Pro
Lys His 35 40 45Leu Lys Asn Leu
Tyr Ala Gln Val Val Leu Pro Asp Asp Val Ile Lys 50 55
60Glu Ile Phe Asn Pro Leu Ile Asp Leu Ser Thr Thr Gln
Trp Gly Val65 70 75
80Ser Pro Asp Phe Ala Ile Glu Asn Thr Glu Thr His Lys Ile Leu Phe
85 90 95Gly Glu Ile Lys Arg Gln
Asp Gly Trp Val Glu Gly Lys Asp Pro Ser 100
105 110Ala Gly Arg Gly Asn Ala His Glu Arg Ser Cys Lys
Leu Phe Thr Pro 115 120 125Gly Leu
Leu Lys Ala Tyr Arg Thr Ile Gly Gly Ile Asn Asp Glu Glu 130
135 140Ile Leu Pro Phe Trp Val Val Phe Glu Gly Asp
Ile Thr Arg Asp Pro145 150 155
160Lys Arg Val Arg Glu Ile Thr Phe Trp Tyr Asp His Tyr Gln Asp Asn
165 170 175Tyr Phe Met Trp
Arg Pro Asn Glu Ser Gly Glu Lys Leu Val Gln His 180
185 190Phe Asn Glu Lys Leu Lys Lys Tyr Leu Asp
195 20061152DNABacillus stearothermophilus X1
6atggctatta cattatgtga cataaatggt tgtagacttg agagaggaca tactggtaaa
60cataataaat ttcctgaatt tgtatggact tctcaattta ataaaaaaga tattgataag
120gtcaataaag caggatatgc aacaccaaga ggtggggaca aaggagccta tcagaaccat
180gtttacagaa ataataaagt aattattcct tttgaaaggt tggaaaatgt taatttaaat
240aactatcaag atggatatgt tattaggtta ttccctaatc agtactttga atcagccggg
300gtagttaagc cggaattctt acaaccaaat tcatttgtta aagttgggga caatgcattt
360attttatatc gcacacattc atcttttgag gaattacctc ctctaccaga ctgggaggtt
420agacatctaa aaaagaacgg taatatagtt accagaagaa gtaaggacgt aatcgatgct
480ggacattatg tcttacgatt atcatcaatt agtaacaaaa aagaaagaaa agagggccct
540cctcaaggta tttttgcacc tgaatatgca aatgcagaga ctaattatct gtcaaaagca
600tttttagcct ggttaattat taaaactcaa aatagtccgt ataatgaaga acaattccaa
660cacttaagag cgatcttaat tagtcataat ctcatcaata tttctcaact tgaagaaaag
720gctattctaa agaatggtat cacatgctgc cctttatgcg agcaaattat tttttacgaa
780cagctacacg aaatggtttc ttttgaaggt gcgtctggcc ttgcgaattc acaagaacag
840gttgagggtg caactaggtc aacatcagtt aatttattcc atatggtacc attagtatat
900gaaaccttgg aacacaaacc tgatcaaata gcatggggcc atgccatttg taatactaga
960cttggtcaaa gagagtgcct gcctcttagt agactaaaac aagaaggtac gcccgttggt
1020cttcttgatg aagattcgaa tcttgaagta ttaggatgga ttagtaaaga taagcaattt
1080attcgtacag aaaatgggga agtttggatt aaaattacag atattgaatt taacgatgac
1140tttgaagaat aa
11527383PRTBacillus stearothermophilus X1 7Met Ala Ile Thr Leu Cys Asp
Ile Asn Gly Cys Arg Leu Glu Arg Gly1 5 10
15His Thr Gly Lys His Asn Lys Phe Pro Glu Phe Val Trp
Thr Ser Gln 20 25 30Phe Asn
Lys Lys Asp Ile Asp Lys Val Asn Lys Ala Gly Tyr Ala Thr 35
40 45Pro Arg Gly Gly Asp Lys Gly Ala Tyr Gln
Asn His Val Tyr Arg Asn 50 55 60Asn
Lys Val Ile Ile Pro Phe Glu Arg Leu Glu Asn Val Asn Leu Asn65
70 75 80Asn Tyr Gln Asp Gly Tyr
Val Ile Arg Leu Phe Pro Asn Gln Tyr Phe 85
90 95Glu Ser Ala Gly Val Val Lys Pro Glu Phe Leu Gln
Pro Asn Ser Phe 100 105 110Val
Lys Val Gly Asp Asn Ala Phe Ile Leu Tyr Arg Thr His Ser Ser 115
120 125Phe Glu Glu Leu Pro Pro Leu Pro Asp
Trp Glu Val Arg His Leu Lys 130 135
140Lys Asn Gly Asn Ile Val Thr Arg Arg Ser Lys Asp Val Ile Asp Ala145
150 155 160Gly His Tyr Val
Leu Arg Leu Ser Ser Ile Ser Asn Lys Lys Glu Arg 165
170 175Lys Glu Gly Pro Pro Gln Gly Ile Phe Ala
Pro Glu Tyr Ala Asn Ala 180 185
190Glu Thr Asn Tyr Leu Ser Lys Ala Phe Leu Ala Trp Leu Ile Ile Lys
195 200 205Thr Gln Asn Ser Pro Tyr Asn
Glu Glu Gln Phe Gln His Leu Arg Ala 210 215
220Ile Leu Ile Ser His Asn Leu Ile Asn Ile Ser Gln Leu Glu Glu
Lys225 230 235 240Ala Ile
Leu Lys Asn Gly Ile Thr Cys Cys Pro Leu Cys Glu Gln Ile
245 250 255Ile Phe Tyr Glu Gln Leu His
Glu Met Val Ser Phe Glu Gly Ala Ser 260 265
270Gly Leu Ala Asn Ser Gln Glu Gln Val Glu Gly Ala Thr Arg
Ser Thr 275 280 285Ser Val Asn Leu
Phe His Met Val Pro Leu Val Tyr Glu Thr Leu Glu 290
295 300His Lys Pro Asp Gln Ile Ala Trp Gly His Ala Ile
Cys Asn Thr Arg305 310 315
320Leu Gly Gln Arg Glu Cys Leu Pro Leu Ser Arg Leu Lys Gln Glu Gly
325 330 335Thr Pro Val Gly Leu
Leu Asp Glu Asp Ser Asn Leu Glu Val Leu Gly 340
345 350Trp Ile Ser Lys Asp Lys Gln Phe Ile Arg Thr Glu
Asn Gly Glu Val 355 360 365Trp Ile
Lys Ile Thr Asp Ile Glu Phe Asn Asp Asp Phe Glu Glu 370
375 38081536DNABacillus stearothermophilus
X1misc_featureDNA sequence of M.BstXI 8atgatttttg ctgatattga atttgaaaaa
gaactttttt cagctgctaa taaattaagg 60ggaaaaattg ctccaagtga gtataagcat
tatgttttgc ctttgatatt ccttagatat 120ttatctctta aataccaaca aagaaggaat
gaaattcaac aacagataaa tgattcaagg 180gatcacaaga aaaatcaaga tgaagtgtta
aagatattgg aagacaggac tgaatacacc 240aaagtaaatg ttttctatat tcctgaaaaa
gctagttggg aatacttatt gaaaaattcc 300gaaaatgata aaattaaaga aatgatagat
tcagctatgg aaatactgga aaatgaatat 360gacgagttaa aaggtgtttt gccaaagata
tataaaaact caaatatacc gaatgaagtt 420attagtgatt tactaaaact attttctcaa
gaagtatttt cagcacatga tggaagaaat 480gttgatttat tggggagagt ttatgaatac
tttataagta attttgctac tacagaaggt 540actagaggtg gtgaatattt tacaccgtct
tcaatcgtaa aattattggt agcaatgcta 600gagcccatta aaggtacagt ttatgatccg
gcctgtggga caggaggaat gtttattcag 660tctaataaat atagagaaaa taatcataac
ttgtgttttg taggccagga acaaaacgag 720cttactatca aattggctaa aatgaatgga
attctacatg gaataaatcc tgaaattaga 780caaggtgatt cattattaaa tgaccgttat
ccagaattga aagctgaaat tgtaatatct 840aatccaccgt ttaatatgaa ggattgggga
gctgaacgcc tgccacttaa tgataagcga 900ttaataggac cggtaacaaa cagtaatgca
aattacatgt ggatacagca ttttctatac 960catttaaaag atggtggttt agcaggattt
gttattgcta atggagcttt gactagtaat 1020ctggctgctg aaaaaattgt aaggaaacac
ttaatagaca atgattatgt agattgtgtt 1080gttcaattac ctgaaaaaat gttctttggt
actggcattc caagtgcttt agtgttttta 1140agtaagaatc gaaatggaag taacggccat
gccaaaagag aaaaagaggt tctatttatt 1200gatgcaagcg ataagggaac attagtgggt
aaaaagaata aaatattttt agatgatgaa 1260ataaaagaaa ttgcagattt atatcattca
tttaaatttt taaatgataa tgattataac 1320catagtggtt tttacaaaaa ggttaacatt
gaaaaaatcg tggaaaatga ttataaatta 1380actccaactc tctatgtagg tgtaaaggaa
gagactgaaa tggagaagcc atttagagaa 1440atgataatag aatataaagc gatattagag
caacaatttg aagaatcaaa caaactacag 1500cagaaaatat taaagaattt agagggatta
ttatga 15369511PRTBacillus stearothermophilus
X1misc_featureprotein sequence for M.BstXI 9Met Ile Phe Ala Asp Ile Glu
Phe Glu Lys Glu Leu Phe Ser Ala Ala1 5 10
15Asn Lys Leu Arg Gly Lys Ile Ala Pro Ser Glu Tyr Lys
His Tyr Val 20 25 30Leu Pro
Leu Ile Phe Leu Arg Tyr Leu Ser Leu Lys Tyr Gln Gln Arg 35
40 45Arg Asn Glu Ile Gln Gln Gln Ile Asn Asp
Ser Arg Asp His Lys Lys 50 55 60Asn
Gln Asp Glu Val Leu Lys Ile Leu Glu Asp Arg Thr Glu Tyr Thr65
70 75 80Lys Val Asn Val Phe Tyr
Ile Pro Glu Lys Ala Ser Trp Glu Tyr Leu 85
90 95Leu Lys Asn Ser Glu Asn Asp Lys Ile Lys Glu Met
Ile Asp Ser Ala 100 105 110Met
Glu Ile Leu Glu Asn Glu Tyr Asp Glu Leu Lys Gly Val Leu Pro 115
120 125Lys Ile Tyr Lys Asn Ser Asn Ile Pro
Asn Glu Val Ile Ser Asp Leu 130 135
140Leu Lys Leu Phe Ser Gln Glu Val Phe Ser Ala His Asp Gly Arg Asn145
150 155 160Val Asp Leu Leu
Gly Arg Val Tyr Glu Tyr Phe Ile Ser Asn Phe Ala 165
170 175Thr Thr Glu Gly Thr Arg Gly Gly Glu Tyr
Phe Thr Pro Ser Ser Ile 180 185
190Val Lys Leu Leu Val Ala Met Leu Glu Pro Ile Lys Gly Thr Val Tyr
195 200 205Asp Pro Ala Cys Gly Thr Gly
Gly Met Phe Ile Gln Ser Asn Lys Tyr 210 215
220Arg Glu Asn Asn His Asn Leu Cys Phe Val Gly Gln Glu Gln Asn
Glu225 230 235 240Leu Thr
Ile Lys Leu Ala Lys Met Asn Gly Ile Leu His Gly Ile Asn
245 250 255Pro Glu Ile Arg Gln Gly Asp
Ser Leu Leu Asn Asp Arg Tyr Pro Glu 260 265
270Leu Lys Ala Glu Ile Val Ile Ser Asn Pro Pro Phe Asn Met
Lys Asp 275 280 285Trp Gly Ala Glu
Arg Leu Pro Leu Asn Asp Lys Arg Leu Ile Gly Pro 290
295 300Val Thr Asn Ser Asn Ala Asn Tyr Met Trp Ile Gln
His Phe Leu Tyr305 310 315
320His Leu Lys Asp Gly Gly Leu Ala Gly Phe Val Ile Ala Asn Gly Ala
325 330 335Leu Thr Ser Asn Leu
Ala Ala Glu Lys Ile Val Arg Lys His Leu Ile 340
345 350Asp Asn Asp Tyr Val Asp Cys Val Val Gln Leu Pro
Glu Lys Met Phe 355 360 365Phe Gly
Thr Gly Ile Pro Ser Ala Leu Val Phe Leu Ser Lys Asn Arg 370
375 380Asn Gly Ser Asn Gly His Ala Lys Arg Glu Lys
Glu Val Leu Phe Ile385 390 395
400Asp Ala Ser Asp Lys Gly Thr Leu Val Gly Lys Lys Asn Lys Ile Phe
405 410 415Leu Asp Asp Glu
Ile Lys Glu Ile Ala Asp Leu Tyr His Ser Phe Lys 420
425 430Phe Leu Asn Asp Asn Asp Tyr Asn His Ser Gly
Phe Tyr Lys Lys Val 435 440 445Asn
Ile Glu Lys Ile Val Glu Asn Asp Tyr Lys Leu Thr Pro Thr Leu 450
455 460Tyr Val Gly Val Lys Glu Glu Thr Glu Met
Glu Lys Pro Phe Arg Glu465 470 475
480Met Ile Ile Glu Tyr Lys Ala Ile Leu Glu Gln Gln Phe Glu Glu
Ser 485 490 495Asn Lys Leu
Gln Gln Lys Ile Leu Lys Asn Leu Glu Gly Leu Leu 500
505 510101179DNABacillus stearothermophilus
X1misc_featureDNA sequence of S.BstXI 10atgaaaagta ctttgaagga atataaattg
ggtgatatta ccgaagtcgt taatggtgcc 60actccttcaa ctaaaaagcc tgagtactat
gaaaatggta caattccatg gattactcct 120aaagatttat caggctatta ctttaaatat
atatctcatg gtgaacgtaa tataacagag 180cttggtctaa gaaatagttc agctaagttg
ttaccaaaag gaactgtatt attttcctca 240agagccccaa taggatacgt agcaatagct
gataattggt taactacgaa ccagggattt 300aaaagtttta tatgtaatga ggagattatt
tacaatgaat acctttatta ttttcttatt 360gctaaaaggg attttattga aacatttgcg
aatgggagta cgtttaaaga gctttcatca 420acttctgcaa agaatatacc aatcaatctt
cctagtttag aagagcaaaa gaagattgtg 480acaattttag gggatttgga tagaaagata
gaattaaatt ataaaattat tgaaagctta 540gaaaaaatag cagaaagaac atataaatat
tggtttgtcg atgaattaaa tcaagatgaa 600cagcacatcc gtaatggatg ggaaactgct
aaaattggcg atgtggtgga acttttggga 660gggggaaccc ctaaaacttc ggaaagtaag
tattgggaag atggagatat taattggttt 720actccttcag atttaacaaa aactagacag
ctttttgtac gtgattctca aagaaaaata 780acaattgatg gacttaataa cagtgcagcg
aaattaattc ccccttattc cttgttaatg 840tcaagtagag ctacaattgg cgagttggca
attaatcaag aatctgctac tacaaatcaa 900gggtttattg tattaatacc aaatgaaaaa
atttctattt accaattata cttttgggct 960aaacttaata agagcaaaat tatttcaatg
gcaaatggta gtacttttaa agaaattagt 1020aagcgggatt ttaaatcttt ggagataata
ttaccaaaaa atatagacac ttttaattca 1080attatgcaag attattttag gaaaattgag
gagttaattg atgaaataaa aatcttaaaa 1140accgcaagag ataatttaat tccaaaactt
ataaaatga 117911392PRTBacillus
stearothermophilus X1misc_featureprotein sequence for S.BstXI 11Met Lys
Ser Thr Leu Lys Glu Tyr Lys Leu Gly Asp Ile Thr Glu Val1 5
10 15Val Asn Gly Ala Thr Pro Ser Thr
Lys Lys Pro Glu Tyr Tyr Glu Asn 20 25
30Gly Thr Ile Pro Trp Ile Thr Pro Lys Asp Leu Ser Gly Tyr Tyr
Phe 35 40 45Lys Tyr Ile Ser His
Gly Glu Arg Asn Ile Thr Glu Leu Gly Leu Arg 50 55
60Asn Ser Ser Ala Lys Leu Leu Pro Lys Gly Thr Val Leu Phe
Ser Ser65 70 75 80Arg
Ala Pro Ile Gly Tyr Val Ala Ile Ala Asp Asn Trp Leu Thr Thr
85 90 95Asn Gln Gly Phe Lys Ser Phe
Ile Cys Asn Glu Glu Ile Ile Tyr Asn 100 105
110Glu Tyr Leu Tyr Tyr Phe Leu Ile Ala Lys Arg Asp Phe Ile
Glu Thr 115 120 125Phe Ala Asn Gly
Ser Thr Phe Lys Glu Leu Ser Ser Thr Ser Ala Lys 130
135 140Asn Ile Pro Ile Asn Leu Pro Ser Leu Glu Glu Gln
Lys Lys Ile Val145 150 155
160Thr Ile Leu Gly Asp Leu Asp Arg Lys Ile Glu Leu Asn Tyr Lys Ile
165 170 175Ile Glu Ser Leu Glu
Lys Ile Ala Glu Arg Thr Tyr Lys Tyr Trp Phe 180
185 190Val Asp Glu Leu Asn Gln Asp Glu Gln His Ile Arg
Asn Gly Trp Glu 195 200 205Thr Ala
Lys Ile Gly Asp Val Val Glu Leu Leu Gly Gly Gly Thr Pro 210
215 220Lys Thr Ser Glu Ser Lys Tyr Trp Glu Asp Gly
Asp Ile Asn Trp Phe225 230 235
240Thr Pro Ser Asp Leu Thr Lys Thr Arg Gln Leu Phe Val Arg Asp Ser
245 250 255Gln Arg Lys Ile
Thr Ile Asp Gly Leu Asn Asn Ser Ala Ala Lys Leu 260
265 270Ile Pro Pro Tyr Ser Leu Leu Met Ser Ser Arg
Ala Thr Ile Gly Glu 275 280 285Leu
Ala Ile Asn Gln Glu Ser Ala Thr Thr Asn Gln Gly Phe Ile Val 290
295 300Leu Ile Pro Asn Glu Lys Ile Ser Ile Tyr
Gln Leu Tyr Phe Trp Ala305 310 315
320Lys Leu Asn Lys Ser Lys Ile Ile Ser Met Ala Asn Gly Ser Thr
Phe 325 330 335Lys Glu Ile
Ser Lys Arg Asp Phe Lys Ser Leu Glu Ile Ile Leu Pro 340
345 350Lys Asn Ile Asp Thr Phe Asn Ser Ile Met
Gln Asp Tyr Phe Arg Lys 355 360
365Ile Glu Glu Leu Ile Asp Glu Ile Lys Ile Leu Lys Thr Ala Arg Asp 370
375 380Asn Leu Ile Pro Lys Leu Ile Lys385
39012770DNAplanococcus citreus SE-F45 12atgaaacagt
ttgcagatcc ttttgaaaga agattccttg atgcaattga acatcatctt 60gatggaattt
ctgagaaaat aaaaaaagac tttacacaca aaaacttttt aaaagaattg 120aatggcctta
aaggtgataa agtctatcat gacttaggct ttgataccgc tgaatatact 180ctggtacgtc
ttataggaag aatgagcata agcgttggga gaaggctggg ggagatatac 240gataaagtcc
ctcgttatgt tgctgccgcg cgatttggtc ttcaaccaaa tcaaattgca 300gaagtatttg
atggtcttga gttagatata gctttgcgca atagcctttt gtcagatgat 360gataaaattc
acataaaaaa aataactgaa aagatgtcag gcgaaacata ctcgggaatc 420ggaatcgaaa
ttcgttataa ctttaatcca aatgacagtt cccgtttaag aaaagacgtc 480gatgtagctt
ctaaattgtc ggccgcgggg ttatttcctg tttatttaat atttagctct 540ctcagtccta
ggaatgatgc aatagcccgt cttaaaagag ggggatggag ctttaaacag 600gggcaggaag
ccttagactt ccttaccgaa cttttaggag tggatattgg gtctgtttta 660tctgacccaa
taatagccgc agaaactagg gagaaaacat caaaaattat gaagtctata 720tttgaatcag
aggcattcca atctgttata ccgggagagt ggagtaaact
77013257PRTplanococcus citreus SE-F45 13Met Lys Gln Phe Ala Asp Pro Phe
Glu Arg Arg Phe Leu Asp Ala Ile1 5 10
15Glu His His Leu Asp Gly Ile Ser Glu Lys Ile Lys Lys Asp
Phe Thr 20 25 30His Lys Asn
Phe Leu Lys Glu Leu Asn Gly Leu Lys Gly Asp Lys Val 35
40 45Tyr His Asp Leu Gly Phe Asp Thr Ala Glu Tyr
Thr Leu Val Arg Leu 50 55 60Ile Gly
Arg Met Ser Ile Ser Val Gly Arg Arg Leu Gly Glu Ile Tyr65
70 75 80Asp Lys Val Pro Arg Tyr Val
Ala Ala Ala Arg Phe Gly Leu Gln Pro 85 90
95Asn Gln Ile Ala Glu Val Phe Asp Gly Leu Glu Leu Asp
Ile Ala Leu 100 105 110Arg Asn
Ser Leu Leu Ser Asp Asp Asp Lys Ile His Ile Lys Lys Ile 115
120 125Thr Glu Lys Met Ser Gly Glu Thr Tyr Ser
Gly Ile Gly Ile Glu Ile 130 135 140Arg
Tyr Asn Phe Asn Pro Asn Asp Ser Ser Arg Leu Arg Lys Asp Val145
150 155 160Asp Val Ala Ser Lys Leu
Ser Ala Ala Gly Leu Phe Pro Val Tyr Leu 165
170 175Ile Phe Ser Ser Leu Ser Pro Arg Asn Asp Ala Ile
Ala Arg Leu Lys 180 185 190Arg
Gly Gly Trp Ser Phe Lys Gln Gly Gln Glu Ala Leu Asp Phe Leu 195
200 205Thr Glu Leu Leu Gly Val Asp Ile Gly
Ser Val Leu Ser Asp Pro Ile 210 215
220Ile Ala Ala Glu Thr Arg Glu Lys Thr Ser Lys Ile Met Lys Ser Ile225
230 235 240Phe Glu Ser Glu
Ala Phe Gln Ser Val Ile Pro Gly Glu Trp Ser Lys 245
250 255Leu141347DNAplanococcus citreus
SE-F45misc_featureDNA sequence for M.PciI 14atgacaaatt tttcgcactc
agctctaacg agctacgatc ttctcgggca tgaaattgtc 60caagattctg aagctgttag
ctcgggtcca tatctggtca gctatgaccc gatccctgta 120cgtcggtcta cattcctagc
tggactgtca gagaacgttc actcgtggtt tcgtctcaca 180ccaagtttcg gaccggatct
agttcgaaca atcatcaaac agatgaatct tgcgccgcac 240tcacacatcc atgacccttt
ctcaggagcc gggactaccg cgattgaggc ttcgttagag 300ggctatgaag caagctgcgt
agaagttaat ccgtttctct acttcgtggg gaaaacatcc 360atagattggt ctatcaatgc
tgatgatgct gcagcgcagc tagaaagcat taaaaataaa 420tattatagca tgtctgcaac
cgctactttg gataacatag ccgacctagg aatagatata 480ccaaaaatac acaatattca
tcggtggtgg agaaacgatg ttcttaaaga tatattagtc 540ctaaaatctt ctatcagatc
ttgcacacaa gataagtatt gttccttttt tgagctagcc 600ctagctgcag ttctcgttcc
agatttgaca aatgtaacgc taggaaaact acaactgcac 660tttgtaaaca aagacgataa
agagataaac gtctggccta catatgaatc tcatgcaaaa 720aaaatgattc acgacttgtc
attaattaat aagcaaaatt tcgaattttt gcccaagatt 780atttatggtg attcaactca
aaaatcaaca tttagcgagg tggcagggat agatgctata 840ataacatccc ctccgtaccc
taataggtac agctatattt ggaatactcg ccctcacctg 900tacattcttg atatgatttc
cgaagcaaaa gaggcttcgc aaatagatcg tagaacgatt 960ggtggaacat gggggacagc
aacttccgaa ttaggaaagg gtatattttc tccaatcaat 1020gctgtagtca aagacgcgct
tgaaggggtt cacgaaagaa tcgccggttc cgatcaactc 1080atggcaaact atgtaactca
ttattttaat cggctctttt tacatataga agctataaaa 1140ccatcactta atccaaaagc
aaagcttgct tatgttgttg ggaactcttg gattaagggc 1200gaatatgtag ccactgacgt
aatcttagca aaaattatcg aaggggcttt gccaggctca 1260tcaattgatg gtcttcatcg
tttccgtcgc cggaacagtg gaaagaatct ctttgaaact 1320atagtttact ccactctccc
ggtataa 134715448PRTplanococcus
citreus SE-F45misc_featureProtein sequence for M.PciI 15Met Thr Asn Phe
Ser His Ser Ala Leu Thr Ser Tyr Asp Leu Leu Gly1 5
10 15His Glu Ile Val Gln Asp Ser Glu Ala Val
Ser Ser Gly Pro Tyr Leu 20 25
30Val Ser Tyr Asp Pro Ile Pro Val Arg Arg Ser Thr Phe Leu Ala Gly
35 40 45Leu Ser Glu Asn Val His Ser Trp
Phe Arg Leu Thr Pro Ser Phe Gly 50 55
60Pro Asp Leu Val Arg Thr Ile Ile Lys Gln Met Asn Leu Ala Pro His65
70 75 80Ser His Ile His Asp
Pro Phe Ser Gly Ala Gly Thr Thr Ala Ile Glu 85
90 95Ala Ser Leu Glu Gly Tyr Glu Ala Ser Cys Val
Glu Val Asn Pro Phe 100 105
110Leu Tyr Phe Val Gly Lys Thr Ser Ile Asp Trp Ser Ile Asn Ala Asp
115 120 125Asp Ala Ala Ala Gln Leu Glu
Ser Ile Lys Asn Lys Tyr Tyr Ser Met 130 135
140Ser Ala Thr Ala Thr Leu Asp Asn Ile Ala Asp Leu Gly Ile Asp
Ile145 150 155 160Pro Lys
Ile His Asn Ile His Arg Trp Trp Arg Asn Asp Val Leu Lys
165 170 175Asp Ile Leu Val Leu Lys Ser
Ser Ile Arg Ser Cys Thr Gln Asp Lys 180 185
190Tyr Cys Ser Phe Phe Glu Leu Ala Leu Ala Ala Val Leu Val
Pro Asp 195 200 205Leu Thr Asn Val
Thr Leu Gly Lys Leu Gln Leu His Phe Val Asn Lys 210
215 220Asp Asp Lys Glu Ile Asn Val Trp Pro Thr Tyr Glu
Ser His Ala Lys225 230 235
240Lys Met Ile His Asp Leu Ser Leu Ile Asn Lys Gln Asn Phe Glu Phe
245 250 255Leu Pro Lys Ile Ile
Tyr Gly Asp Ser Thr Gln Lys Ser Thr Phe Ser 260
265 270Glu Val Ala Gly Ile Asp Ala Ile Ile Thr Ser Pro
Pro Tyr Pro Asn 275 280 285Arg Tyr
Ser Tyr Ile Trp Asn Thr Arg Pro His Leu Tyr Ile Leu Asp 290
295 300Met Ile Ser Glu Ala Lys Glu Ala Ser Gln Ile
Asp Arg Arg Thr Ile305 310 315
320Gly Gly Thr Trp Gly Thr Ala Thr Ser Glu Leu Gly Lys Gly Ile Phe
325 330 335Ser Pro Ile Asn
Ala Val Val Lys Asp Ala Leu Glu Gly Val His Glu 340
345 350Arg Ile Ala Gly Ser Asp Gln Leu Met Ala Asn
Tyr Val Thr His Tyr 355 360 365Phe
Asn Arg Leu Phe Leu His Ile Glu Ala Ile Lys Pro Ser Leu Asn 370
375 380Pro Lys Ala Lys Leu Ala Tyr Val Val Gly
Asn Ser Trp Ile Lys Gly385 390 395
400Glu Tyr Val Ala Thr Asp Val Ile Leu Ala Lys Ile Ile Glu Gly
Ala 405 410 415Leu Pro Gly
Ser Ser Ile Asp Gly Leu His Arg Phe Arg Arg Arg Asn 420
425 430Ser Gly Lys Asn Leu Phe Glu Thr Ile Val
Tyr Ser Thr Leu Pro Val 435 440
44516430PRTBacillus sphaericus 16Met Arg Arg Leu Ala Lys Asn Ser Arg Asn
Asp Ser Tyr Leu Ser Asn1 5 10
15Arg Asp Tyr Gln Glu Ile Val Arg Glu Asn Thr Thr Thr Ile Ser Phe
20 25 30Pro Leu Lys Glu Lys His
Thr Leu Thr Leu Thr Lys Lys Ile Gly Leu 35 40
45Asn Gln Thr Ala Gly Phe Gly Gly Trp Phe Phe Pro Asp Ser
Pro Cys 50 55 60Leu Leu Thr Val Thr
Val Leu Ser Ser Phe Gly Thr Lys Val Thr Ser65 70
75 80Lys Thr Phe Ser Leu Ser Lys Asp Trp Asn
Arg Val Gly Leu Ala Trp 85 90
95Ile Asn Glu His Ser Ser Asp Thr Met Ser Ile Val Leu Glu Phe Ser
100 105 110Asp Val Glu Ile Val
His Thr Trp Gly Leu Thr Cys Asp Val Phe Asn 115
120 125Val His Glu Leu Ile Ile Asp Ala Ile Glu Asp Gln
Asn Lys Leu Ile 130 135 140Asp Val Leu
Asn Gln Glu His Leu Ser Pro Glu Thr Tyr Tyr Leu Asn145
150 155 160His Asp Ser Asp Thr Asp Leu
Ile Glu Asn Leu Glu Ser Thr Glu Glu 165
170 175Ile Lys Ile Val Asn Gln Ser Gln Lys Gln Ile Ser
Leu Lys Lys Cys 180 185 190Cys
Tyr Cys Gln Arg Tyr Met Pro Val Asn Ile Leu Val Arg Ser Asn 195
200 205Ser Ser Phe His Lys His Lys Ser Lys
Lys Thr Gly Phe Gln Asn Glu 210 215
220Cys Arg Ala Cys Lys Lys Trp Arg Ile Asn Asn Ser Phe Asn Pro Val225
230 235 240Arg Thr Lys Asp
Gln Leu His Glu Ser Ala Val Ile Thr Arg Glu Lys 245
250 255Lys Ile Leu Leu Lys Glu Pro Glu Ile Leu
Gln Lys Ile Lys Asn Arg 260 265
270Asn Asn Gly Glu Gly Leu Lys Ser Ile Ile Trp Lys Lys Phe Asp Lys
275 280 285Lys Cys Phe Asn Cys Glu Lys
Glu Leu Thr Ile Glu Glu Val Arg Leu 290 295
300Asp His Thr Arg Pro Leu Ala Tyr Leu Trp Pro Ile Asp Glu His
Ala305 310 315 320Thr Cys
Leu Cys Glu Lys Cys Asn Asn Thr Lys His Asp Met Phe Pro
325 330 335Ile Asp Phe Tyr Gln Gly Asp
Glu Asp Lys Leu Arg Arg Leu Ala Arg 340 345
350Ile Thr Gly Leu Asp Tyr Glu Ser Leu Val Lys Arg Asp Val
Asn Glu 355 360 365Val Glu Leu Ala
Arg Ile Ile Asn Asn Ile Glu Asp Phe Ala Thr Asn 370
375 380Val Glu Ala Arg Thr Phe Arg Ser Ile Arg Asn Lys
Val Lys Glu Val385 390 395
400Arg Pro Asp Thr Asp Leu Phe Glu Ile Leu Lys Ser Lys Asn Ile Asn
405 410 415Leu Tyr Asn Glu Leu
Gln Tyr Glu Leu Leu Thr Arg Lys Asp 420 425
430171245DNAEnterobacter aerogenes 17gtgaatcaga aaaatgaaaa
atcatttatg cgtttgcaat caacctttag cggtggcaaa 60ggtagtccaa tgcatgattg
gtacccatat ttagagggtt attctcccga atttgtgaaa 120tgcttgattt cacgatttgc
tcctaaagcc aaaacaattt tagatccatt ttgtggctct 180ggaacaacag ccattgtttc
cgttttagag ggtttaaata attactattg cgaagtaaac 240cctttatgcc aatatattat
tgaaactaaa ctaatagctt taacattaag cgaagaagaa 300aaaacaaaat tagtaaatga
actttattct atttctaatg aaataactaa tgtactcaaa 360ccttctgcaa ccgagacaga
tctagagaaa tcatttaaat ccgtttttgg taatacgaaa 420ttttttgagg atcacatatt
taaagatata cttagttatc aatgttacat tagctctatc 480gaagatgaaa atcttaagag
acttctgaca atagcaggga ttagatcgtt aatcccttcc 540tcgttattgg taagacgagg
tgatttacga ttcaagacac aaaaagaatt agagaaaggc 600aaccagggct ttcgctttca
tgtacaaaaa agcttagaat taattgccag tgatttatta 660gacattacgg aaggtagtgg
tttagctacc ttcttatgtg atgatgccaa agaaatatct 720gggaataacc tgattgatgc
tgtaataaca agcccgccat atttaaatgg cacaaattat 780tttagaaata ctaaaattga
actttggttt atagggaaat taaagaccaa atcagatcta 840agacattata gggatttagc
tattaccagt ggtattaacg atgtaactaa aggtaaaagc 900ttatcttcaa ataatactat
tatctcagaa ataccattat tatctgaatg tattaaagaa 960ctaagcataa aagagtatga
tagtcgtatt tcaatgatgg ttgaaaacta cttttgggac 1020atgttcaaat tcttatcaaa
actcccaaaa ttactaacta atgatgcgac tatctgtata 1080gatttaggtg attctgttta
ttgtaacgtc tacatcccta cacaagatat tttgaaagaa 1140atgatgtcaa agttaggttt
tgaagagaac gaaagggtca ttcttcgtga acgaaaatcc 1200cgcaatggaa caaagttagt
ccagactgtt caggttttta aatga 1245181140DNAEnterobacter
aerogenes 18atgaaaaata aatattttag taaaaaatgg gagcaattca agaaagaatt
accccatcaa 60tcaggtgaaa tggtaaagag aaattggggc cataactggc actctatgtg
ttcataccaa 120gggaaactta aaccatcaat agctagatct ttaattgata cattcatgcc
atcaagtaag 180ggacgtatat tagatgtctt ctcaggtgtt ggcaccattc ctttcgaagc
aagattactt 240ggtcatactg catatggatt tgatattagt ccagcagcag ttaatatttc
acgcgcaaaa 300ctagaagtta taagtaaaaa tgaaatccaa gaggtaatta ataaattatc
tgattttatt 360gagcaaaaca aaaattcaat agattataac gaacataatt taataaggtt
taatggttca 420attgaatcct attttcatcc tgaaactttt aaggaaatac tgtgtgctcg
taaattcttt 480ttaataaaag gtgaattaaa tgcatctgaa tcgttagtac agtcatgttt
attacatatt 540ttacatggta atcgtccgta tgcattgagt agaaagtccc atcctattac
acctttcgcg 600cctactggag attttatata cagtaattta gttataaagt taatcaaaaa
agttgaaaga 660gtcttgcaaa attctgatgg tatcccagat actggcagca aagtatttta
tcaggactct 720acaaaaagtt ggcctgaaga agtaaataat ttagatgcaa ttataacatc
acctccattt 780tatgatagta cccgtttcta ttcagcaaat tggatgcgat tatggttttc
tggttgggaa 840aaagatgact tccaaacgaa gccaaaagat tttgtggacg aaactcagaa
aaaaagcttt 900gaaatatatg ataatatatt caaacaatct caacaatgct taaaaaaaga
tggcgttttt 960ttaatgcacg ttggcaaaag taaaaaaagt gatatggcag gacaaattgc
taaaattggt 1020agtaattatc ttagccttat agatatattt gacgaaagtg ttgaacattg
cgaaagtcac 1080ggaattaaag acaaaggcac gacaacccat catcagtacc ttgtctttac
gaaagattag 114019213PRTBacillus amyloliquefaciens H 19Met Glu Val Glu
Lys Glu Phe Ile Thr Asp Glu Ala Lys Glu Leu Leu1 5
10 15Ser Lys Asp Lys Leu Ile Gln Gln Ala Tyr
Asn Glu Val Lys Thr Ser 20 25
30Ile Cys Ser Pro Ile Trp Pro Ala Thr Ser Lys Thr Phe Thr Ile Asn
35 40 45Asn Thr Glu Lys Asn Cys Asn Gly
Val Val Pro Ile Lys Glu Leu Cys 50 55
60Tyr Thr Leu Leu Glu Asp Thr Tyr Asn Trp Tyr Arg Glu Lys Pro Leu65
70 75 80Asp Ile Leu Lys Leu
Glu Lys Lys Lys Gly Gly Pro Ile Asp Val Tyr 85
90 95Lys Glu Phe Ile Glu Asn Ser Glu Leu Lys Arg
Val Gly Met Glu Phe 100 105
110Glu Thr Gly Asn Ile Ser Ser Ala His Arg Ser Met Asn Lys Leu Leu
115 120 125Leu Gly Leu Lys His Gly Glu
Ile Asp Leu Ala Ile Ile Leu Met Pro 130 135
140Ile Lys Gln Leu Ala Tyr Tyr Leu Thr Asp Arg Val Thr Asn Phe
Glu145 150 155 160Glu Leu
Glu Pro Tyr Phe Glu Leu Thr Glu Gly Gln Pro Phe Ile Phe
165 170 175Ile Gly Phe Asn Ala Glu Ala
Tyr Asn Ser Asn Val Pro Leu Ile Pro 180 185
190Lys Gly Ser Asp Gly Met Ser Lys Arg Ser Ile Lys Lys Trp
Lys Asp 195 200 205Lys Val Glu Asn
Lys 21020194PRTOceanospirillum kriegii 20Met Lys Ile Lys Arg Ile Glu
Val Leu Ile Asn Asn Gly Ser Val Pro1 5 10
15Gly Ile Pro Met Ile Leu Asn Glu Ile Gln Asp Ala Ile
Lys Thr Val 20 25 30Ser Trp
Pro Glu Gly Asn Asn Ser Phe Val Ile Asn Pro Val Arg Lys 35
40 45Gly Asn Gly Val Lys Pro Ile Lys Asn Ser
Cys Met Arg His Leu His 50 55 60Gln
Lys Gly Trp Ala Leu Glu His Pro Val Arg Ile Lys Ala Glu Met65
70 75 80Arg Pro Gly Pro Leu Asp
Ala Val Lys Met Ile Gly Gly Lys Ala Phe 85
90 95Ala Leu Glu Trp Glu Thr Gly Asn Ile Ser Ser Ser
His Arg Ala Ile 100 105 110Asn
Lys Met Val Met Gly Met Leu Glu Arg Val Ile Ile Gly Gly Val 115
120 125Leu Ile Leu Pro Ser Arg Asp Met Tyr
Asn Tyr Leu Thr Asp Arg Val 130 135
140Gly Asn Phe Arg Glu Leu Glu Pro Tyr Phe Ser Val Trp Arg Gln Phe145
150 155 160Asn Leu Lys Asp
Ala Tyr Leu Ala Ile Val Glu Ile Glu His Asp Ser 165
170 175Val Asp Ala Gln Val Ser Leu Ile Pro Lys
Gly Thr Asp Gly Arg Ala 180 185
190Ile Arg21180PRTBacillus amyloliquefaciens
HMISC_FEATURE(1)..(180)Residues 1-180 correspond to residues 22-201
of the protein sequence of BamHI (seq id no. 19) 21Ile Gln Gln Ala Tyr
Asn Glu Val Lys Thr Ser Ile Cys Ser Pro Ile1 5
10 15Trp Pro Ala Thr Ser Lys Thr Phe Thr Ile Asn
Asn Thr Glu Lys Asn 20 25
30Cys Asn Gly Val Val Pro Ile Lys Glu Leu Cys Tyr Thr Leu Leu Glu
35 40 45Asp Thr Tyr Asn Trp Tyr Arg Glu
Lys Pro Leu Asp Ile Leu Lys Leu 50 55
60Glu Lys Lys Lys Gly Gly Pro Ile Asp Val Tyr Lys Glu Phe Ile Glu65
70 75 80Asn Ser Glu Leu Lys
Arg Val Gly Met Glu Phe Glu Thr Gly Asn Ile 85
90 95Ser Ser Ala His Arg Ser Met Asn Lys Leu Leu
Leu Gly Leu Lys His 100 105
110Gly Glu Ile Asp Leu Ala Ile Ile Leu Met Pro Ile Lys Gln Leu Ala
115 120 125Tyr Tyr Leu Thr Asp Arg Val
Thr Asn Phe Glu Glu Leu Glu Pro Tyr 130 135
140Phe Glu Leu Thr Glu Gly Gln Pro Phe Ile Phe Ile Gly Phe Asn
Ala145 150 155 160Glu Ala
Tyr Asn Ser Asn Val Pro Leu Ile Pro Lys Gly Ser Asp Gly
165 170 175Met Ser Lys Arg
18022177PRTOceanospirillum kriegiiMISC_FEATURE(1)..(177)Residues 1-177
correspond to residues 18-194 of the protein sequence of OkrAI (seq
id no. 20) 22Ile Pro Met Ile Leu Asn Glu Ile Gln Asp Ala Ile Lys Thr Val
Ser1 5 10 15Trp Pro Glu
Gly Asn Asn Ser Phe Val Ile Asn Pro Val Arg Lys Gly 20
25 30Asn Gly Val Lys Pro Ile Lys Asn Ser Cys
Met Arg His Leu His Gln 35 40
45Lys Gly Trp Ala Leu Glu His Pro Val Arg Ile Lys Ala Glu Met Arg 50
55 60Pro Gly Pro Leu Asp Ala Val Lys Met
Ile Gly Gly Lys Ala Phe Ala65 70 75
80Leu Glu Trp Glu Thr Gly Asn Ile Ser Ser Ser His Arg Ala
Ile Asn 85 90 95Lys Met
Val Met Gly Met Leu Glu Arg Val Ile Ile Gly Gly Val Leu 100
105 110Ile Leu Pro Ser Arg Asp Met Tyr Asn
Tyr Leu Thr Asp Arg Val Gly 115 120
125Asn Phe Arg Glu Leu Glu Pro Tyr Phe Ser Val Trp Arg Gln Phe Asn
130 135 140Leu Lys Asp Ala Tyr Leu Ala
Ile Val Glu Ile Glu His Asp Ser Val145 150
155 160Asp Ala Gln Val Ser Leu Ile Pro Lys Gly Thr Asp
Gly Arg Ala Ile 165 170
175Arg2338DNAartificialprimer 23attcaacaag catacaatgc agttaaaaca tctattgt
382439DNAartificialprimer 24acaaatagat
gttttaactg cattgtatgc ttgttgaat
392539DNAartificialprimer 25caagcataca atgaagttgc aacatctatt tgttcacct
392639DNAartificialprimer 26aggtgaacaa atagatgttg
caacttcatt gtatgcttg 392739DNAartificialprimer
27acgattaaca acaccgaagc aaattgtaac ggtgtagta
392840DNAartificialprimer 28acgattaaca acaccgaagc aaattgtaac ggtgtagtat
402939DNAartificialprimer 29aacggtgtag taccaattgc
agaactatgt tacacctta 393039DNAartificialprimer
30taaggtgtaa catagttctg caattggtac tacaccgtt
393139DNAartificialprimer 31aacccccttg atatacttgc acttgaaaag aaaaaaggt
393239DNAartificialprimer 32accttttttc ttttcaagtg
caagtatatc aaggggttt 393336DNAartificialprimer
33gatatactta aacttgcaaa gaaaaaaggt ggtccg
363439DNAartificialprimer 34cggaccacct tttttctttg caagtttaag tatatcaag
393539DNAartificialprimer 35atacttaaac ttgaaaaggc
aaaaggtggt ccgattgat 393640DNAartificialprimer
36atcaatcgga ccaccttttg cctttttcaa gtttaagtat
403739DNAartificialprimer 37ggtccgattg atgtttatgc agagttcata gaaaacagt
393839DNAartificialprimer 38actgttttct atgaactctg
cataaacatc aatcggacc 393937DNAartificialprimer
39atagaaaaac agtgaacttg cacgtgtagg tatggaa
374039DNAartificialprimer 40aaattccata cctacacgtg caagttcact gttttctat
394139DNAartificialprimer 41ggaaatatta gttctgccgc
acgttcaatg aacaaactt 394239DNAartificialprimer
42aagtttgttc attgaaacgt gcggcagaac taatattcc
394339DNAartificialprimer 43aatattagtt ctgcccacgc atcaatgaac aaacttcta
394439DNAartificialprimer 44tagaagtttg ttcattgatg
cgtgggcaga actaatatt 394542DNAartificialprimer
45gcccaccgtt caatgaacgc acttctatta ggattaaaac at
424642DNAartificialprimer 46atgttttaat cctaatagaa gtgcggtcat tgaacggtgg
gc 424739DNAartificialprimer 47attatcctta
tgcctattgc acaattggcc tattatctt
394839DNAartificialprimer 48aagataatag gccaattgtg caataggcat aaggataat
394939DNAartificialprimer 49ttggcctatt atcttacagc
acgtgttacc aatttcgag 395039DNAartificialprimer
50ctcgaaattg gtaacacgtg ctgtaagata ataggccaa
395139DNAartificialprimer 51gcctattatc ttacagatgc agttaccaat ttcgaggaa
395239DNAartificialprimer 52ttcctcgaaa ttggtaactg
catctgtaag ataataggc 395339DNAartificialprimer
53cgtgttacca atttcgaggc attagaacct tattttgaa
395439DNAartificialprimer 54ttcaaaataa ggttctaatg cctcgaaatt ggtaacacg
395539DNAartificialprimer 55accaatttcg aggaattagc
accttatttt gaacttact 395639DNAartificialprimer
56agtaagttca aaataaggtg ctaattcctc gaaattggt
395742DNAartificialprimer 57ccttattttg aacttactgc aggacaacca tttattttta
tt 425842DNAartificialprimer 58aataaaaata
aatggttgtg ctgcagtaag ttcaaaataa gg
425945DNAartificialprimer 59tttattttta ttggatttaa tgctgcagct tataattcta
atgtc 456045DNAartificialprimer 60gacattagaa
ttataagctg cagcattaaa tccaataaaa ataaa
456139DNAartificialprimer 61aatgtccctt taattcccgc aggttctgac ggtatgtca
396239DNAartificialprimer 62tgacataccg tcagaacctg
cgggaattaa agggacatt 396339DNAartificialprimer
63ttaattccca aaggttctgc aggtatgtca aaacgctca
396439DNAartificialprimer 64tgagcgtttt gacatacctg cagaaccttt gggaattaa
396539DNAartificialprimer 65tctgacggta tgtcaaaagc
atcaattaag aaatggaaa 396639DNAartificialprimer
66tttccatttc ttaattgatg cttttgacat accgtcaga
396754DNAartificialprimer 67ggtggtgcat gcggaggtaa ataaatggaa gtagaaaaag
agtttattac tgat 546851DNAartificialprimer 68ggtggtggta
ccctatttgt tttcaacttt atctttccat ttcttaattg a
516939DNAartificialprimermisc_feature(19)..(21)n=a, c, g or t
69caagcataca atgaagttnn nacatctatt tgttcacct
397039DNAartificialprimermisc_feature(19)..(21)n=a. c. g or t
70aggtgaacaa atagatgtnn naacttcatt gtatgcttg
397137DNAartificialprimermisc_feature(16)..(18)misc_feature(16)..(18)n=a,
c, g, or t 71gatatactta aacttnnnaa gaaaaaaagg tggtccg
377239DNAartificialprimermisc_feature(19)..(21)misc_feature(19).-
.(21)n=a, c, g, or t 72cggaccacct tttttcttnn naagtttaag tatatcaag
397354DNAartificialprimer 73ggtggtgcat gcggaggtaa
ataaatgtct aataaaaaac agtcaaatag gcta 547439DNAartificialprimer
74ggtggtggta cctcacttag atctaagctg ttcaaacaa
3975267PRTEscherichia coli RY13MISC_FEATURE(1)..(267)Amino acid residues
1-267 correspond to residues 4-270 of the protein sequence of EcoRI
75Lys Lys Gln Ser Asn Arg Leu Thr Glu Gln His Lys Leu Ser Gln Gly1
5 10 15Val Ile Gly Ile Phe Gly
Asp Tyr Ala Lys Ala His Asp Leu Ala Val 20 25
30Gly Glu Val Ser Lys Leu Val Lys Lys Ala Leu Ser Asn
Glu Tyr Pro 35 40 45Gln Leu Ser
Phe Arg Tyr Arg Asp Ser Ile Lys Lys Thr Glu Ile Asn 50
55 60Glu Ala Leu Lys Lys Ile Asp Pro Asp Leu Gly Gly
Thr Leu Phe Val65 70 75
80Ser Asn Ser Ser Ile Lys Pro Asp Gly Gly Ile Val Glu Val Lys Asp
85 90 95Asp Tyr Gly Glu Trp Arg
Val Val Leu Val Ala Glu Ala Lys His Gln 100
105 110Gly Lys Asp Ile Ile Asn Ile Arg Asn Gly Leu Leu
Val Gly Lys Arg 115 120 125Gly Asp
Gln Asp Leu Met Ala Ala Gly Asn Ala Ile Glu Arg Ser His 130
135 140Lys Asn Ile Ser Glu Ile Ala Asn Phe Met Leu
Ser Glu Ser His Phe145 150 155
160Pro Tyr Val Leu Phe Leu Glu Gly Ser Asn Phe Leu Thr Glu Asn Ile
165 170 175Ser Ile Thr Arg
Pro Asp Gly Arg Val Val Asn Leu Glu Tyr Asn Ser 180
185 190Gly Ile Leu Asn Arg Leu Asp Arg Leu Thr Ala
Ala Asn Tyr Gly Met 195 200 205Pro
Ile Asn Ser Asn Leu Cys Ile Asn Lys Phe Val Asn His Lys Asp 210
215 220Lys Ser Ile Met Leu Gln Ala Ala Ser Ile
Tyr Thr Gln Gly Asp Gly225 230 235
240Arg Glu Trp Asp Ser Lys Ile Met Phe Glu Ile Met Phe Asp Ile
Ser 245 250 255Thr Thr Ser
Leu Arg Val Leu Gly Arg Asp Leu 260
26576265PRTRhodopseudomonas sphaeroidesMISC_FEATURE(1)..(265)amino acid
residues 1-265 correspond to residues 10-274 of the protein sequence
of RsrI 76Lys Gly Gln Ala Leu Arg Leu Gly Ile Gln Gln Glu Leu Gly Gly
Gly1 5 10 15Pro Leu Ser
Ile Phe Gly Ala Ala Ala Gln Lys His Asp Leu Ser Ile 20
25 30Arg Glu Val Thr Ala Gly Val Leu Thr Lys
Leu Ala Glu Asp Phe Pro 35 40
45Asn Leu Glu Phe Gln Leu Arg Thr Ser Leu Thr Lys Lys Ala Ile Asn 50
55 60Glu Lys Leu Arg Ser Phe Asp Pro Arg
Leu Gly Gln Ala Leu Phe Val65 70 75
80Glu Ser Ala Ser Ile Arg Pro Asp Gly Gly Ile Thr Glu Val
Lys Asp 85 90 95Arg His
Gly Asn Trp Arg Val Ile Leu Val Gly Glu Ser Lys His Gln 100
105 110Gly Asn Asp Val Glu Lys Ile Leu Ala
Gly Val Leu Gln Gly Lys Ala 115 120
125Lys Asp Gln Asp Phe Met Ala Ala Gly Asn Ala Ile Glu Arg Met His
130 135 140Lys Asn Val Leu Glu Leu Arg
Asn Tyr Met Leu Asp Glu Lys His Phe145 150
155 160Pro Tyr Val Val Phe Leu Gln Gly Ser Asn Phe Ala
Thr Glu Ser Phe 165 170
175Glu Val Thr Arg Pro Asp Gly Arg Val Val Lys Ile Val His Asp Ser
180 185 190Gly Met Leu Asn Arg Ile
Asp Arg Val Thr Ala Ser Ser Leu Ser Arg 195 200
205Glu Ile Asn Gln Asn Tyr Cys Glu Asn Ile Val Val Arg Ala
Gly Ser 210 215 220Phe Asp His Met Phe
Gln Ile Ala Ser Leu Tyr Cys Lys Ala Ala Pro225 230
235 240Trp Thr Ala Gly Glu Met Ala Glu Ala Met
Leu Ala Val Ala Lys Thr 245 250
255Ser Leu Arg Ile Ile Ala Asp Asp Leu 260
2657739DNAartificialprimer 77gattgggtgg cgcagaaatt tcaaacgggc cagcagtcg
397839DNAartificialprimer 78cgactgctgg
cccgtttgaa atttctgcgc cacccaatc
3979272PRTAgrobacterium gelatinovorum 79Met Arg Leu Asp Leu Asp Phe Gly
Arg Gly Leu Val Ala His Val Met1 5 10
15Leu Asp Asn Val Ser Glu Glu Gln Tyr Gln Gln Ile Ser Asp
Tyr Phe 20 25 30Val Pro Leu
Val Asn Lys Pro Lys Leu Lys Ser Arg Asp Ala Ile Gly 35
40 45Gln Ala Phe Val Met Ala Thr Glu Val Cys Pro
Asp Ala Asn Pro Ser 50 55 60Asp Leu
Trp His His Val Leu Tyr Arg Ile Tyr Ile Arg Glu Lys Ile65
70 75 80Gly Thr Asp Pro Ser Gln Ser
Trp Val Arg Thr Ser Gly Glu Ala Phe 85 90
95Glu Val Ala Leu Val Glu Arg Tyr Asn Pro Val Leu Ala
Arg His Gly 100 105 110Ile Arg
Leu Thr Ala Leu Phe Lys Gly Gln Lys Gly Leu Ala Leu Thr 115
120 125Arg Met Gly Val Ala Asp Arg Val Gly Ser
Arg Lys Val Asp Val Met 130 135 140Ile
Glu Lys Gln Gly Gly Gly Arg Ser Pro Asp Ala Glu Gly Phe Gly145
150 155 160Val Val Gly Gly Ile His
Ala Lys Val Ser Leu Ala Glu Arg Val Ser 165
170 175Asp Asp Ile Pro Ala Ser Arg Ile Met Met Gly Glu
Gly Leu Leu Ser 180 185 190Val
Leu Ser Thr Leu Asp Val Lys Ser Phe Pro Pro Pro His Gly Asp 195
200 205Leu Val Asn Arg Gly Glu Leu Gly Thr
Pro Asp Arg Pro Ser Asp Lys 210 215
220Arg Asn Tyr Ile Glu Gly His Gly Asp Phe Ser Ala Cys Phe Ser Tyr225
230 235 240Asn Leu Arg Thr
Pro Pro Ser Asn Ala Thr Thr Pro Ser Gly Arg His 245
250 255Ile Tyr Val Ser Ala Ser Leu Val Arg Thr
Thr Ser Ser Pro Thr Thr 260 265
27080358PRTAnabaena variabilis 80Met Glu Glu Asp Leu Asp Leu Ser Glu Asn
Ile Glu Ala Ala Ser Ala1 5 10
15Glu Leu Thr Thr Leu Tyr Gln Val Ala Ala Asp Ala Met Lys Asp Tyr
20 25 30Ile Glu Ile Tyr Leu Ala
Leu Ser Lys Gln Ser Asp Gly Phe Ser Asn 35 40
45Ile Asn Asn Leu Asp Leu Thr Ser Arg Asn Arg Arg Leu Val
Val Ile 50 55 60His Gly Leu Ser Leu
Glu Leu Asp Pro Asp Thr Ser Thr Pro Glu Glu65 70
75 80Ile Lys Arg Glu Ala Glu Arg Met Leu Ala
Ile Ala Leu Asp Thr Glu 85 90
95Ser Ala Ile Thr Ala Gly Val Tyr Glu Lys Met Arg Leu Phe Ala Ser
100 105 110Ser Leu Val Asp Gln
Leu Phe Glu Gln Thr Asp Glu Leu Asn Ser Leu 115
120 125Ser Ser Glu Tyr Leu Ser Ala Asn Pro Gly Phe Leu
Pro Phe Phe Gln 130 135 140Gln Leu Ala
Gly Leu Arg Ser Lys Ser Glu Leu Lys Arg Glu Val Gly145
150 155 160Asn Ala Ser Asp Asn Ser Ile
Ser Lys Ala Val Ala Glu Arg Ile Leu 165
170 175Glu Arg Ile Ile Arg Asn Leu Arg Ile Arg Thr Phe
Ser Lys Glu Lys 180 185 190Leu
Leu Gln Ala Val Glu Pro Thr Leu Glu Gly Ile Val Arg Asp Leu 195
200 205Val Gly Lys Val Leu Leu Glu Asn Ile
Val Ala Asp Ala Leu Ser Asp 210 215
220Leu Gln Val Pro Phe Met Arg Glu Ser Glu Tyr Gln Ser Leu Lys Gly225
230 235 240Val Ile Tyr Asp
Phe Arg Ala Asp Phe Val Ile Pro Asp Ala Gln Asn 245
250 255Pro Ile Ala Phe Ile Glu Val Arg Lys Ser
Ser Thr Arg His Ala Ser 260 265
270Leu Tyr Ala Lys Asp Lys Met Phe Ser Ala Ile Asn Trp Lys Gly Lys
275 280 285Asn Lys Arg Leu Leu Gly Ile
Leu Val Val Glu Gly Pro Trp Thr Arg 290 295
300Glu Thr Leu Arg Val Met Ala Asn Val Phe Asp Tyr Val Thr Pro
Leu305 310 315 320Thr Arg
Val Ser Gln Val Ala Glu Ala Ile Arg Ala Tyr Leu Asp Gly
325 330 335Asp Lys Thr Arg Leu Lys Trp
Leu Val Asn Phe Ser Ile Glu Glu Ala 340 345
350Asp His Asp Asn Ile Thr 35581530PRTBacillus
stearothermophilus B61 81Met Ala Lys Tyr Gly Arg Gly Lys Phe Leu Pro His
Gln Asn Tyr Ile1 5 10
15Asp Tyr Met His Phe Ile Val Asn His Lys Asn Tyr Ser Gly Met Pro
20 25 30Asn Ala Ile Gly Glu Asp Gly
Arg Ile Asn Trp Gln Val Ser Ser Gly 35 40
45Lys Thr Thr Ser Phe Tyr Glu Tyr Tyr Gln Ala Arg Phe Glu Trp
Trp 50 55 60Glu Lys Lys Ala Asp Glu
Leu Asn Leu Pro Gly Thr Gly Asn Ser Asn65 70
75 80Lys Arg Phe Ser Leu Ala Ala Arg Leu Ile His
Pro Thr Gly Gln Arg 85 90
95Pro Cys Arg Leu Cys Gly Lys Tyr Gln Tyr Val Gly Tyr Met Tyr Val
100 105 110Ser His Asn Leu Tyr Lys
Arg Trp Ser Lys Ile Thr Gly Arg Glu Asp 115 120
125Leu Phe Phe Lys Lys Gln Asn Ile Ile Glu Ala Ala Asn Ile
Phe Lys 130 135 140Ser Ile Met Gly Glu
Gln Ala Leu Ile Asn Glu Leu Thr Thr Ile Phe145 150
155 160Pro Glu Arg Lys Asp Tyr Phe Asn Arg Leu
Pro Asn Ile Glu Asp Phe 165 170
175Phe Val Ser Ser Ser His Ile Lys Asn Asn Gly Asn Tyr Ile Ser Pro
180 185 190Gly Phe Met Ala Asn
Pro Pro Asp Arg Leu Asp Gly Phe His Asp Tyr 195
200 205Gly Ile Cys Cys Arg Lys Glu Lys Asp Pro Gly Arg
His Asp Asp Asn 210 215 220Met Arg Leu
Tyr Asn His Asp Arg Arg Ala Phe Met Trp Trp Ser Glu225
230 235 240Gly Asp Trp Ala Leu Ala Asp
Ala Leu Tyr Asn Lys Ala Gly Ala Gly 245
250 255Lys Cys Ala Asp Pro Asp Cys Gln Lys Glu Val Glu
Lys Ile Ser Pro 260 265 270Asp
His Val Gly Pro Ile Ser Cys Gly Phe Lys Gln Ile Pro Phe Phe 275
280 285Lys Pro Leu Cys Ala Ser Cys Asn Ser
Ala Lys Asn Arg Arg Phe Ser 290 295
300Tyr Gln Asp Val Lys Glu Leu Leu Lys Tyr Glu Asn Tyr Thr Gly Asp305
310 315 320Ser Val Ala Ser
Trp Gln Val Arg Ala Leu Trp Asp Asn Cys Lys His 325
330 335Leu Val Lys Asn Asp Asp Asp Ser Lys Leu
Leu Ser Asn Leu Met Arg 340 345
350Ser Leu Gln Asp Tyr Tyr Leu Arg Ser Leu Tyr Lys Leu Phe Ser Asn
355 360 365Gly Phe Ala His Leu Leu Ser
Tyr Phe Leu Thr Pro Glu Tyr Ala His 370 375
380Tyr Lys Ile Thr Phe Glu Gly Leu Asn Thr Ser Thr Leu Glu Tyr
Glu385 390 395 400Arg Tyr
Tyr Lys Thr Phe Lys Lys Thr Lys Ser Thr Ser Ser Leu Ala
405 410 415Ala Arg Ile Val Arg Ile Ala
Phe Glu Glu Leu Glu Ile Tyr Asn Ser 420 425
430Lys Asp Ile Asn Glu Arg Lys Leu Ile Lys Phe Asp Thr Ser
Ser Trp 435 440 445Glu Lys Asp Phe
Glu Asn Ile Ile Ser Tyr Ala Thr Lys Asn Leu Ser 450
455 460Leu Asp Glu Glu Ala Ser Lys Trp Asn Lys Val Leu
Thr Asp Lys Asn465 470 475
480Leu Ser Ser Thr Glu Lys Asp Lys Lys Ile Ser Ser Leu Leu Glu Asp
485 490 495Lys Asn Tyr Glu Val
Tyr Lys Lys Gln Phe Tyr Ile Leu Lys Asp Leu 500
505 510Leu Val Glu His Phe Asn Lys Ile Gly Glu Gln Ile
Ala Lys Asp Tyr 515 520 525Met Lys
53082301PRTEnterobacter agglomerans 82Met Lys Lys Arg Arg Asp Leu Val
Glu Val Phe Gly Tyr Asn Pro Met1 5 10
15Asp Leu Ser Pro Glu Val Arg Ala Leu Trp Asn Leu Gly Ala
Cys Pro 20 25 30Phe Leu Asn
Lys Glu Cys Ile Lys Ile Asn His Asp Gln Thr Ile Ile 35
40 45Tyr Gly Thr Cys Ser Val Thr Ser Pro Tyr Gly
Asp Val Ile Ile Cys 50 55 60Pro Asn
Arg Leu Tyr Ala Asn Asp Tyr Glu Thr Leu His Lys Val Ser65
70 75 80Arg Asp Ala Phe Gly Asp Asp
Val Pro Phe Leu Thr Tyr Ser Asn Phe 85 90
95Ile Lys Tyr Arg Ala Thr Tyr Lys Asp Cys Ile Val Ala
Leu Gly Lys 100 105 110Asn Ser
Gly Lys Glu Val Gln Val Gly Arg Ala Leu Ser Met Asp Trp 115
120 125Val Leu Val Arg Ile Thr Asp Gly Glu Leu
Lys Glu Tyr Val Gly Val 130 135 140Glu
Ile Gln Ser Ile Asp Ile Thr Gly Asn Tyr Arg Asp Ala Trp His145
150 155 160Ala Tyr Lys Asn Leu Lys
Pro Ile Asp Ile Ile Asp Asn Leu Pro Thr 165
170 175Ser Gln His Gly Leu Asn Trp Ala Asn Val His Lys
Arg Leu Ile Pro 180 185 190Gln
Ile Ile Arg Lys Gly Val Val Tyr Ser Arg Ser Asn Tyr Val Lys 195
200 205Lys Gly Leu Tyr Phe Ile Leu Pro Glu
Ile Val Tyr Asn Lys Phe Glu 210 215
220Asp Val Ile Gly Ala Asp Ile Pro Leu Leu Lys Thr Gln Thr Asn Lys225
230 235 240Ser Ile Thr Val
His Thr Tyr Ser Leu Gly Glu Pro Ala Ala Asn Gly 245
250 255Glu Gln Arg Lys Leu Ile Ser Glu Arg Glu
Ile Ile Phe Asp Leu Asp 260 265
270Glu Phe Ser Lys Arg Phe Thr Thr Gly Pro Asn Leu Pro Lys Gly Asp
275 280 285Asp Leu Asp Ala Val Ile Lys
Lys Ala Leu Gly Met Met 290 295
30083277PRTEscherichia coli RY13 83Met Ser Asn Lys Lys Gln Ser Asn Arg
Leu Thr Glu Gln His Lys Leu1 5 10
15Ser Gln Gly Val Ile Gly Ile Phe Gly Asp Tyr Ala Lys Ala His
Asp 20 25 30Leu Ala Val Gly
Glu Val Ser Lys Leu Val Lys Lys Ala Leu Ser Asn 35
40 45Glu Tyr Pro Gln Leu Ser Phe Arg Tyr Arg Asp Ser
Ile Lys Lys Thr 50 55 60Glu Ile Asn
Glu Ala Leu Lys Lys Ile Asp Pro Asp Leu Gly Gly Thr65 70
75 80Leu Phe Val Ser Asn Ser Ser Ile
Lys Pro Asp Gly Gly Ile Val Glu 85 90
95Val Lys Asp Asp Tyr Gly Glu Trp Arg Val Val Leu Val Ala
Glu Ala 100 105 110Lys His Gln
Gly Lys Asp Ile Ile Asn Ile Arg Asn Gly Leu Leu Val 115
120 125Gly Lys Arg Gly Asp Gln Asp Leu Met Ala Ala
Gly Asn Ala Ile Glu 130 135 140Arg Ser
His Lys Asn Ile Ser Glu Ile Ala Asn Phe Met Leu Ser Glu145
150 155 160Ser His Phe Pro Tyr Val Leu
Phe Leu Glu Gly Ser Asn Phe Leu Thr 165
170 175Glu Asn Ile Ser Ile Thr Arg Pro Asp Gly Arg Val
Val Asn Leu Glu 180 185 190Tyr
Asn Ser Gly Ile Leu Asn Arg Leu Asp Arg Leu Thr Ala Ala Asn 195
200 205Tyr Gly Met Pro Ile Asn Ser Asn Leu
Cys Ile Asn Lys Phe Val Asn 210 215
220His Lys Asp Lys Ser Ile Met Leu Gln Ala Ala Ser Ile Tyr Thr Gln225
230 235 240Gly Asp Gly Arg
Glu Trp Asp Ser Lys Ile Met Phe Glu Ile Met Phe 245
250 255Asp Ile Ser Thr Thr Ser Leu Arg Val Leu
Gly Arg Asp Leu Phe Glu 260 265
270Gln Leu Thr Ser Lys 27584245PRTEscherichia coli J62 pLG74
84Met Ser Leu Arg Ser Asp Leu Ile Asn Ala Leu Tyr Asp Glu Asn Gln1
5 10 15Lys Tyr Asp Val Cys Gly
Ile Ile Ser Ala Glu Gly Lys Ile Tyr Pro 20 25
30Leu Gly Ser Asp Thr Lys Val Leu Ser Thr Ile Phe Glu
Leu Phe Ser 35 40 45Arg Pro Ile
Ile Asn Lys Ile Ala Glu Lys His Gly Tyr Ile Val Glu 50
55 60Glu Pro Lys Gln Gln Asn His Tyr Pro Asp Phe Thr
Leu Tyr Lys Pro65 70 75
80Ser Glu Pro Asn Lys Lys Ile Ala Ile Asp Ile Lys Thr Thr Tyr Thr
85 90 95Asn Lys Glu Asn Glu Lys
Ile Lys Phe Thr Leu Gly Gly Tyr Thr Ser 100
105 110Phe Ile Arg Asn Asn Thr Lys Asn Ile Val Tyr Pro
Phe Asp Gln Tyr 115 120 125Ile Ala
His Trp Ile Ile Gly Tyr Val Tyr Thr Arg Val Ala Thr Arg 130
135 140Lys Ser Ser Leu Lys Thr Tyr Asn Ile Asn Glu
Leu Asn Glu Ile Pro145 150 155
160Lys Pro Tyr Lys Gly Val Lys Val Phe Leu Gln Asp Lys Trp Val Ile
165 170 175Ala Gly Asp Leu
Ala Gly Ser Gly Asn Thr Thr Asn Ile Gly Ser Ile 180
185 190His Ala His Tyr Lys Asp Phe Val Glu Gly Lys
Gly Ile Phe Asp Ser 195 200 205Glu
Asp Glu Phe Leu Asp Tyr Trp Arg Asn Tyr Glu Arg Thr Ser Gln 210
215 220Leu Arg Asn Asp Lys Tyr Asn Asn Ile Ser
Glu Tyr Arg Asn Trp Ile225 230 235
240Tyr Arg Gly Arg Lys 24585300PRTHaemophilus
influenzae Rd (exo-mutant) 85Met Lys Lys Ser Ala Leu Glu Lys Leu Leu Ser
Leu Ile Glu Asn Leu1 5 10
15Thr Asn Gln Glu Phe Lys Gln Ala Thr Asn Ser Leu Ile Ser Phe Ile
20 25 30Tyr Lys Leu Asn Arg Asn Glu
Val Ile Glu Leu Val Arg Ser Ile Gly 35 40
45Ile Leu Pro Glu Ala Ile Lys Pro Ser Ser Thr Gln Glu Lys Leu
Phe 50 55 60Ser Lys Ala Gly Asp Ile
Val Leu Ala Lys Ala Phe Gln Leu Leu Asn65 70
75 80Leu Asn Ser Lys Pro Leu Glu Gln Arg Gly Asn
Ala Gly Asp Val Ile 85 90
95Ala Leu Ser Lys Glu Phe Asn Tyr Gly Leu Val Ala Asp Ala Lys Ser
100 105 110Phe Arg Leu Ser Arg Thr
Ala Lys Asn Gln Lys Asp Phe Lys Val Lys 115 120
125Ala Leu Ser Glu Trp Arg Glu Asp Lys Asp Tyr Ala Val Leu
Thr Ala 130 135 140Pro Phe Phe Gln Tyr
Pro Thr Thr Lys Ser Gln Ile Phe Lys Gln Ser145 150
155 160Leu Asp Glu Asn Val Leu Leu Phe Ser Trp
Glu His Leu Ala Ile Leu 165 170
175Leu Gln Leu Asp Leu Glu Glu Thr Asn Ile Phe Pro Phe Glu Gln Leu
180 185 190Trp Asn Phe Pro Lys
Lys Gln Ser Lys Lys Thr Ser Val Ser Asp Ala 195
200 205Glu Asn Asn Phe Met Arg Asp Phe Asn Lys Tyr Phe
Met Asp Leu Phe 210 215 220Lys Ile Asp
Lys Asp Thr Leu Asn Gln Leu Leu Gln Lys Glu Ile Asn225
230 235 240Phe Ile Glu Glu Arg Ser Leu
Ile Glu Lys Glu Tyr Trp Lys Lys Gln 245
250 255Ile Asn Ile Ile Lys Asn Phe Thr Arg Glu Glu Ala
Ile Glu Ala Leu 260 265 270Leu
Lys Asp Ile Asn Met Ser Ser Lys Ile Glu Thr Ile Asp Ser Phe 275
280 285Ile Lys Gly Ile Lys Ser Asn Asp Arg
Leu Tyr Leu 290 295
30086254PRTHaemophilus parainfluenzae 86Met Lys Tyr Glu Glu Ile Asn Phe
Lys Val Pro Val Glu Ser Pro Tyr1 5 10
15Tyr Pro Asn Tyr Ser Gln Cys Val Ile Glu Arg Ile Tyr Ser
Ile Leu 20 25 30Arg Asn Gln
Lys Asp Met Gly Asp Asp Arg Ile Ile Ile Asn Thr Asn 35
40 45Leu Lys Lys Gly Leu Pro Leu Glu Asn Ile Asn
Lys Ile Ala Gly Pro 50 55 60Met Ile
Glu Ala Trp Ala Glu Glu Val Phe Ser Gly Ile Arg Asp Asn65
70 75 80Arg Asp Asn Gln Tyr Asn Leu
Ile Asn Val Glu Ala Gln Glu Arg Leu 85 90
95Gly Ile Ser Asp Ile Ile Leu Gln Phe Gln Val Asn Asn
Asn Val Ile 100 105 110Thr Gly
Asn Val Asp Val Lys Ala Thr Ser Asn Asp Ile Pro Asp Ser 115
120 125Gly Lys Ser Pro Asn Ile Thr Ser Phe Ser
Arg Ile Arg Thr Ala Tyr 130 135 140Val
Lys Asp Pro Asn Phe Ile Phe Ile Ile Leu Ser Ile Lys His Ser145
150 155 160Val Tyr Val Lys Arg Asn
Glu Tyr Thr Asn Leu Met Asp Gly Ile Met 165
170 175Gln Ile Ile Asp Phe Asn Val Tyr Asp Leu Lys Tyr
Ile Ser Asp Ser 180 185 190Asp
Ile Ser Tyr Asn Pro Ala Leu Gly Thr Gly Gln Ile Gln Ile Lys 195
200 205Asp Ile His Tyr Val Ser Ser Gln Lys
Arg Thr Thr Trp Gln Met Cys 210 215
220Gln Leu Leu Asp Leu Lys Tyr Leu Arg Ser Lys Lys Arg Thr Ile Glu225
230 235 240Gln Phe Tyr Asn
Glu Ala Lys Arg Asn Lys Trp Ile Lys Asp 245
25087218PRTKlebsiella pnermoniae OK8 87Met Asp Val Phe Asp Lys Val Tyr
Ser Asp Asp Asn Asn Ser Tyr Asp1 5 10
15Gln Lys Thr Val Ser Gln Arg Ile Glu Ala Leu Phe Leu Asn
Asn Leu 20 25 30Gly Lys Val
Val Thr Arg Gln Gln Ile Ile Arg Ala Ala Thr Asp Pro 35
40 45Lys Thr Gly Lys Gln Pro Glu Asn Trp His Gln
Arg Leu Ser Glu Leu 50 55 60Arg Thr
Asp Lys Gly Tyr Thr Ile Leu Ser Trp Arg Asp Met Lys Val65
70 75 80Leu Ala Pro Gln Glu Tyr Ile
Met Pro His Ala Thr Arg Arg Pro Lys 85 90
95Ala Ala Lys Arg Val Leu Pro Thr Lys Glu Thr Trp Glu
Gln Val Leu 100 105 110Asp Arg
Ala Asn Tyr Ser Cys Glu Trp Gln Glu Asp Gly Gln His Cys 115
120 125Gly Leu Val Glu Gly Asp Ile Asp Pro Ile
Gly Gly Gly Thr Val Lys 130 135 140Leu
Thr Pro Asp His Met Thr Pro His Ser Ile Asp Pro Ala Thr Asp145
150 155 160Val Asn Asp Pro Lys Met
Trp Gln Ala Leu Cys Gly Arg His Gln Val 165
170 175Met Lys Lys Asn Tyr Trp Asp Ser Asn Asn Gly Lys
Ile Asn Val Ile 180 185 190Gly
Ile Leu Gln Ser Val Asn Glu Lys Gln Lys Asn Asp Ala Leu Glu 195
200 205Phe Leu Leu Asn Tyr Tyr Gly Leu Lys
Arg 210 21588288PRTNocardia corallina 88Met Ala Thr
Ala Pro Gly His Leu Leu Gly Gln Ile Ile Gly Asn Val1 5
10 15Met Glu Glu Ala Leu Lys Pro Val Leu
Gln Glu Met Ala Asp Arg His 20 25
30Asp Leu Tyr Leu Asp Ser Lys Gly Leu Arg Pro Gly Val Arg Ser Gly
35 40 45Ala Leu Val Thr Trp Thr Asp
Asp Leu Gly Asn Asn His Asp Leu Asp 50 55
60Phe Val Leu Glu Arg Gly Gly Ser Ala Thr Lys Ala Gly Asn Pro Ala65
70 75 80Ala Phe Ile Glu
Ala Ala Trp Arg Arg Tyr Thr Lys His Ser Lys Ala 85
90 95Lys Ala Gln Glu Ile Gln Gly Ala Val Leu
Pro Val Leu Ala Ala Trp 100 105
110Asn Asn Val Lys Pro Thr Pro Ala Ala Val Val Ala Gly Gln Trp Thr
115 120 125Ala Pro Ser Leu Gln Gln Met
Arg Ser Asn Gly Phe Val Val Leu His 130 135
140Leu His Phe Pro Thr Thr Ala Gln Val Phe Gly Gly Asn Gly Ile
Asn145 150 155 160Ile Glu
Gly Thr Gly Glu Gly Thr Pro Asp Ala Phe Trp Gln Gln Gln
165 170 175Cys Asp Ala Tyr Thr Ser Lys
Ser Glu Ala Asp Lys Asp Ser Leu Ala 180 185
190Thr Ala Leu Arg Thr Ala His Ala Gln Glu Phe Arg Thr Phe
Val Ala 195 200 205Glu Leu Glu Arg
Arg Val Val Arg Ala Ile Asp Tyr Val Val Val Thr 210
215 220Pro Leu His Gly His Gly Ser Gln Tyr Thr Ser Ile
Glu Asn Ala Ile225 230 235
240Glu Ala Val Arg Thr Tyr Ser Cys Gly Glu Glu Ser Ala Pro Phe Leu
245 250 255Arg Phe Glu Ile Arg
Ile Ser Tyr Thr Asn Gly Asp Val Ile Gln Ala 260
265 270Thr Phe Gly Ser Ser Ser Asp Ala Ile Glu Phe Leu
Asp Thr Phe Asn 275 280
28589328PRTNeisseria mucosa 89Met Ser Ser Tyr His Asp Asp Leu Asn Ile Leu
Asn Val Asp Phe Asn1 5 10
15His Leu Arg Leu Thr Glu Leu Ile Lys Leu Ala Asp Gln Ala Glu Pro
20 25 30Phe Tyr Leu Trp Val Glu Lys
Ile Phe Arg Gln Val Ser Gly Arg Ala 35 40
45Asp Ser Leu Glu Thr Ile Ile Glu Val Glu Glu Arg Val Val Leu
Lys 50 55 60Met Ala Ile Leu Thr Cys
Phe Thr Ser Asp Glu Lys Glu Leu Pro Lys65 70
75 80Leu Phe Asn Gly Val Gly Val Pro Tyr Pro His
Ile Lys Ala Cys Tyr 85 90
95Phe Phe Phe Ala Trp Leu Val Arg Asp Ala Ala Thr Gln Arg Leu Asp
100 105 110Pro Leu Ile Arg Glu Ala
Phe Thr Gln Leu Lys Ser Ile His Pro Gln 115 120
125Met Lys Lys Thr Glu Leu Glu Ser Glu Ile Phe Ser Gln Leu
Leu Val 130 135 140Asn Tyr Arg Asn Glu
Leu Ile His Phe Ser Trp Pro Val Ile Arg Glu145 150
155 160Val Leu Ile Ser Arg Leu Glu Gly Ser Arg
Arg Ala Ala Arg Gly Ser 165 170
175Tyr Leu Glu Leu Phe Val Arg Thr Ala Leu Ala Gln Ser Ile Thr Tyr
180 185 190Phe Tyr Lys Ile Tyr
Gly Asn Tyr Gly Lys Phe Leu Asp Val Lys Ile 195
200 205His Asp Lys Pro Leu Lys Val Lys Asn Arg Thr Tyr
Asp Val Val Ala 210 215 220Glu Leu Ile
Gly Asn Asn His Asn Thr Gln Tyr Leu Ile Leu Pro Val225
230 235 240Lys Thr Arg Glu Thr Gln Gly
Gly Gly His Ala His Leu Phe Thr Arg 245
250 255Asp Ile Glu Gln Ser Asn Asn Asp Ile Arg Glu Leu
Tyr Pro Asn Ala 260 265 270Val
Ile Ala Pro Val Ile Ile Ala Glu Asn Trp Ser Asp Thr Glu Lys 275
280 285Asp Leu Glu Asn Val Gly Tyr Asn Asp
Ile Phe His Phe Ser Val Asn 290 295
300Pro Asn Arg Phe Ala Gly Phe Ser Asp Val Glu Gln Ile Arg Leu Asn305
310 315 320Arg Leu Val Glu
Arg Ile Leu Leu 32590383PRTNocardia otitidis-caviarum
90Met Arg Ser Asp Thr Ser Val Glu Pro Glu Gly Ala Asn Phe Ile Ala1
5 10 15Glu Phe Phe Gly His Arg
Val Tyr Pro Glu Val Val Ser Thr Glu Ala 20 25
30Ala Arg Asn Asp Gln Ala Thr Gly Thr Cys Pro Phe Leu
Thr Ala Ala 35 40 45Lys Leu Val
Glu Thr Ser Cys Val Lys Ala Glu Thr Ser Arg Gly Val 50
55 60Cys Val Val Asn Thr Ala Val Asp Asn Glu Arg Tyr
Asp Trp Leu Val65 70 75
80Cys Pro Asn Arg Ala Leu Asp Pro Leu Phe Met Ser Ala Ala Ser Arg
85 90 95Lys Leu Phe Gly Tyr Gly
Pro Thr Glu Pro Leu Gln Phe Ile Ala Ala 100
105 110Pro Thr Leu Ala Asp Gln Ala Val Arg Asp Gly Ile
Arg Glu Trp Leu 115 120 125Asp Arg
Gly Val His Val Val Ala Tyr Phe Gln Glu Lys Leu Gly Gly 130
135 140Glu Leu Ser Ile Ser Lys Thr Asp Ser Ser Pro
Glu Phe Ser Phe Asp145 150 155
160Trp Thr Leu Ala Glu Val Glu Ser Ile Tyr Pro Val Pro Lys Ile Lys
165 170 175Arg Tyr Gly Val
Leu Glu Ile Gln Thr Met Asp Phe His Gly Ser Tyr 180
185 190Lys His Ala Val Gly Ala Ile Asp Ile Ala Leu
Val Glu Gly Ile Asp 195 200 205Phe
His Gly Trp Leu Pro Thr Pro Ala Gly Arg Ala Ala Leu Ser Lys 210
215 220Lys Met Glu Gly Pro Asn Leu Ser Asn Val
Phe Lys Arg Thr Phe Tyr225 230 235
240Gln Met Ala Tyr Lys Phe Ala Leu Ser Gly His Gln Arg Cys Ala
Gly 245 250 255Thr Gly Phe
Ala Ile Pro Gln Ser Val Trp Lys Ser Trp Leu Arg His 260
265 270Leu Ala Asn Pro Thr Leu Ile Asp Asn Gly
Asp Gly Thr Phe Ser Leu 275 280
285Gly Asp Thr Arg Asn Asp Ser Glu Asn Ala Trp Ile Phe Val Phe Glu 290
295 300Leu Asp Pro Asp Thr Asp Ala Ser
Pro Arg Pro Leu Ala Pro His Leu305 310
315 320Glu Ile Arg Val Asn Val Asp Thr Leu Ile Asp Leu
Ala Leu Arg Glu 325 330
335Ser Pro Arg Ala Ala Leu Gly Pro Ser Gly Pro Val Ala Thr Phe Thr
340 345 350Asp Lys Val Glu Ala Arg
Met Leu Arg Phe Trp Pro Lys Thr Arg Arg 355 360
365Arg Arg Ser Thr Thr Pro Gly Gly Gln Arg Gly Leu Phe Asp
Ala 370 375 38091326PRTProvidencia
stuartii 164 91Met Lys Glu Leu Lys Leu Lys Glu Ala Lys Glu Ile Leu Lys
Ala Leu1 5 10 15Gly Leu
Pro Pro Gln Gln Tyr Asn Asp Arg Ser Gly Trp Val Leu Leu 20
25 30Ala Leu Ala Asn Ile Lys Pro Glu Asp
Ser Trp Lys Glu Ala Lys Ala 35 40
45Pro Leu Leu Pro Thr Val Ser Ile Met Glu Phe Ile Arg Thr Glu Tyr 50
55 60Gly Lys Asp Tyr Lys Pro Asn Ser Arg
Glu Thr Ile Arg Arg Gln Thr65 70 75
80Leu His Gln Phe Glu Gln Ala Arg Ile Val Asp Arg Asn Arg
Asp Leu 85 90 95Pro Ser
Arg Ala Thr Asn Ser Lys Asp Asn Asn Tyr Ser Leu Asn Gln 100
105 110Val Ile Ile Asp Ile Leu His Asn Tyr
Pro Asn Gly Asn Trp Lys Glu 115 120
125Leu Ile Gln Gln Phe Leu Thr His Val Pro Ser Leu Gln Glu Leu Tyr
130 135 140Glu Arg Ala Leu Ala Arg Asp
Arg Ile Pro Ile Lys Leu Leu Asp Gly145 150
155 160Thr Gln Ile Ser Leu Ser Pro Gly Glu His Asn Gln
Leu His Ala Asp 165 170
175Ile Val His Glu Phe Cys Pro Arg Phe Val Gly Asp Met Gly Lys Ile
180 185 190Leu Tyr Ile Gly Asp Thr
Ala Ser Ser Arg Asn Glu Gly Gly Lys Leu 195 200
205Met Val Leu Asp Ser Glu Tyr Leu Lys Lys Leu Gly Val Pro
Pro Met 210 215 220Ser His Asp Lys Leu
Pro Asp Val Val Val Tyr Asp Glu Lys Arg Lys225 230
235 240Trp Leu Phe Leu Ile Glu Ala Val Thr Ser
His Gly Pro Ile Ser Pro 245 250
255Lys Arg Trp Leu Glu Leu Glu Ala Ala Leu Ser Ser Cys Thr Val Gly
260 265 270Lys Val Tyr Val Thr
Ala Phe Pro Thr Arg Thr Glu Phe Arg Lys Asn 275
280 285Ala Ala Asn Ile Ala Trp Glu Thr Glu Val Trp Ile
Ala Asp Asn Pro 290 295 300Asp His Met
Val His Phe Asn Gly Asp Arg Phe Leu Gly Pro His Asp305
310 315 320Lys Lys Pro Glu Leu Ser
32592157PRTProteus vulgaris 92Met Ser His Pro Asp Leu Asn Lys Leu
Leu Glu Leu Trp Pro His Ile1 5 10
15Gln Glu Tyr Gln Asp Leu Ala Leu Lys His Gly Ile Asn Asp Ile
Phe 20 25 30Gln Asp Asn Gly
Gly Lys Leu Leu Gln Val Leu Leu Ile Thr Gly Leu 35
40 45Thr Val Leu Pro Gly Arg Glu Gly Asn Asp Ala Val
Asp Asn Ala Gly 50 55 60Gln Glu Tyr
Glu Leu Lys Ser Ile Asn Ile Asp Leu Thr Lys Gly Phe65 70
75 80Ser Thr His His His Met Asn Pro
Val Ile Ile Ala Lys Tyr Arg Gln 85 90
95Val Pro Trp Ile Phe Ala Ile Tyr Arg Gly Ile Ala Ile Glu
Ala Ile 100 105 110Tyr Arg Leu
Glu Pro Lys Asp Leu Glu Phe Tyr Tyr Asp Lys Trp Glu 115
120 125Arg Lys Trp Tyr Ser Asp Gly His Lys Asp Ile
Asn Asn Pro Lys Ile 130 135 140Pro Val
Lys Tyr Val Met Glu His Gly Thr Lys Ile Tyr145 150
15593358PRTStreptomyces achromogenes 93Met Gly Ile Thr Ile Lys
Lys Ser Thr Ala Glu Gln Val Leu Arg Lys1 5
10 15Ala Tyr Glu Ala Ala Ala Ser Asp Asp Val Phe Leu
Glu Asp Trp Ile 20 25 30Phe
Leu Ala Thr Ser Leu Arg Glu Val Asp Ala Pro Arg Thr Tyr Thr 35
40 45Ala Ala Leu Val Thr Ala Leu Leu Ala
Arg Ala Cys Asp Asp Arg Val 50 55
60Asp Pro Arg Ser Ile Lys Glu Lys Tyr Asp Asp Arg Ala Phe Ser Leu65
70 75 80Arg Thr Leu Cys His
Gly Val Val Val Pro Met Ser Val Glu Leu Gly 85
90 95Phe Asp Leu Gly Ala Thr Gly Arg Glu Pro Ile
Asn Asn Gln Pro Phe 100 105
110Phe Arg Tyr Asp Gln Tyr Ser Glu Ile Val Arg Val Gln Thr Lys Ala
115 120 125Arg Pro Tyr Leu Asp Arg Val
Ser Ser Ala Leu Ala Arg Val Asp Glu 130 135
140Glu Asp Tyr Ser Thr Glu Glu Ser Phe Arg Ala Leu Val Ala Val
Leu145 150 155 160Ala Val
Cys Ile Ser Val Ala Asn Lys Lys Gln Arg Val Ala Val Gly
165 170 175Ser Ala Ile Val Glu Ala Ser
Leu Ile Ala Glu Thr Gln Ser Phe Val 180 185
190Val Ser Gly His Asp Val Pro Arg Lys Leu Gln Ala Cys Val
Ala Ala 195 200 205Gly Leu Asp Met
Val Tyr Ser Glu Val Val Ser Arg Arg Ile Asn Asp 210
215 220Pro Ser Arg Asp Phe Pro Gly Asp Val Gln Val Ile
Leu Asp Gly Asp225 230 235
240Pro Leu Leu Thr Val Glu Val Arg Gly Lys Ser Val Ser Trp Glu Gly
245 250 255Leu Glu Gln Phe Val
Ser Ser Ala Thr Tyr Ala Gly Phe Arg Arg Val 260
265 270Ala Leu Met Val Asp Ala Ala Ser His Val Ser Leu
Met Ser Ala Asp 275 280 285Asp Leu
Thr Ser Ala Leu Glu Arg Lys Tyr Glu Cys Ile Val Lys Val 290
295 300Asn Glu Ser Val Ser Ser Phe Leu Arg Asp Val
Phe Val Trp Ser Pro305 310 315
320Arg Asp Val His Ser Ile Leu Ser Ala Phe Pro Glu Ala Met Tyr Arg
325 330 335Arg Met Ile Glu
Ile Glu Val Arg Glu Pro Glu Leu Asp Arg Trp Ala 340
345 350Glu Ile Phe Pro Glu Thr
35594315PRTStreptomyces albus G 94Met Ile Asn Ala Asp Lys Pro His Arg Trp
Asn Asp Asp Val Gln Ala1 5 10
15Ser Val Arg Leu Tyr Asn Gln Trp Phe Leu Asp Ala Ala Pro Lys Ala
20 25 30Tyr Arg Asp Thr Arg Gln
Leu Thr Ile Asp Glu Val Glu Gln Ala Phe 35 40
45Gln Arg Thr Ala Asn Met Thr Ser Ile Thr Pro Glu Val Leu
Lys Ala 50 55 60His Pro Lys Thr Leu
Ala Thr Leu Arg Met Ser Thr Ala Pro Pro Ile65 70
75 80Ala Arg Asp Arg Leu Val Gly Leu Ser His
Gly Ser Lys Ser Leu Leu 85 90
95Asp Thr Met Glu Lys Gly Lys Leu Pro Pro Arg Met Lys Gly Asp Val
100 105 110Leu Asp Thr His Leu
Ala Lys Met Cys Ala Val Leu Thr Asp Leu Leu 115
120 125Asp Leu Asp Leu Phe His Trp Tyr Pro Thr Gly Glu
Pro Ala Glu Pro 130 135 140Arg Gln Arg
Glu Leu Ala Ala Thr Val Val Ala Asp Arg Leu Cys Gly145
150 155 160Ala Ile Ala Asp Pro Ile Val
Arg Asn Ala Gln Glu Arg Arg Gln Leu 165
170 175Ala Leu Ile Glu Glu Trp Leu Leu Ala Arg Gly Tyr
Thr Lys Lys Thr 180 185 190His
Ser Ala Ser Leu Pro Leu Asn Thr Met Gln Pro Gly Thr Phe Ser 195
200 205Phe Arg Gln Asn Val Val Val Gly Ser
Asp Leu Pro Val Asn Ile Pro 210 215
220Val Asp Ala Val Ile Gln Pro His Thr Pro His Ser His Lys Leu Pro225
230 235 240Ile Leu Ile Glu
Ala Lys Ser Ala Gly Asp Phe Thr Asn Thr Asn Lys 245
250 255Arg Arg Lys Glu Glu Ala Thr Lys Ile His
Gln Leu Gln Leu Lys Tyr 260 265
270Gly Asn Glu Ile Ser Leu Thr Leu Phe Leu Cys Gly Tyr Phe Asn Thr
275 280 285Gly Tyr Leu Gly Tyr Ser Ala
Ala Glu Gly Leu Asp Trp Val Trp Glu 290 295
300His Arg Ile Asp Asp Leu Glu Ala Ala Gly Ala305
310 31595432PRTSaccharopolyspora sp. 95Met Arg Arg Leu
Ala Thr Gln Arg Arg Glu Asp Ala Tyr Lys Ser Asn1 5
10 15Arg Asp Tyr Gln Thr Val His Glu Ala Gln
Ser Leu Arg Val Asn Ser 20 25
30Thr Asp Asp Asp Asn Leu Ser Leu Phe Leu Leu Lys Asp Ile Ser Pro
35 40 45Arg Glu Asp Ser Lys Asn Ile Val
Gly Phe Gly Gly Phe Val Lys Pro 50 55
60Glu Ile Ala Thr Thr Met Ala Leu Thr Leu Thr Thr Asp Ile Asp Lys65
70 75 80Gln Ile Lys Ser Val
Pro Leu Ser Ser Asn Trp Asn Arg Ile Ser Ile 85
90 95Val Ala Lys Phe Ala Ser Asn Pro Ser Val Ser
Ile Thr Leu Gly Phe 100 105
110Asp Gln Thr Pro Trp Val Asp Phe Trp Gly Ile Asn Ser Asp Asp Ile
115 120 125Gly Leu Ser Phe Val Ser Asp
Ala Val Pro Leu Glu Met Ser Met Ile 130 135
140Asp Ser Ile His Ile Ala Pro Glu Thr Leu Tyr Leu Asp His Ser
Ser145 150 155 160Ala Cys
Leu Leu Asp Ile Asp Pro Val Glu Ser Thr Arg Phe Lys Thr
165 170 175Gly His Gly Asp Pro Leu Ser
Leu Lys Lys Cys Ser Tyr Cys Gly Arg 180 185
190Leu Leu Pro Ile Asp Leu Glu Arg Pro Gly Lys Leu Ser Phe
His Lys 195 200 205His Arg Ala Lys
Ile Thr Asn His Gln Asn Glu Cys Arg Ser Cys Lys 210
215 220Lys Trp Arg Ile Asn Asn Ser Phe Asn Pro Met Arg
Thr Ile Asp Gln225 230 235
240Leu Asn Glu Ser Ala Leu Ile Thr Arg Glu Arg Lys Ile Phe Leu Gln
245 250 255Glu Pro Glu Ile Leu
Gln Glu Ile Lys Asp Arg Thr Gly Ala Gly Leu 260
265 270Lys Ser Gln Val Trp Glu Arg Phe His Arg Lys Cys
Phe Asn Cys Arg 275 280 285Lys Asp
Leu Lys Leu Ser Glu Val Gln Leu Asp His Thr Arg Pro Leu 290
295 300Ala Tyr Leu Trp Pro Ile Asp Glu His Ala Thr
Cys Leu Cys Ala Gln305 310 315
320Cys Asn Asn Thr Lys Lys Asp Arg Phe Pro Val Asp Phe Tyr Ser Glu
325 330 335Gln Gln Ile Arg
Glu Leu Ser Asp Ile Cys Gly Leu Pro Tyr Gln Asp 340
345 350Leu Cys Ala Arg Ser Leu Asn Leu Asp Gln Leu
Asp Arg Ile Glu Arg 355 360 365Asn
Ile Ala Glu Phe Ser Lys Glu Trp Asp Val Arg Thr Phe Ala Ser 370
375 380Thr Ala Arg Arg Ile Ser Glu Val Tyr Pro
Ala Arg Asp Leu Phe Glu385 390 395
400Thr Leu Lys Lys Glu Ser Glu Ser Ala Tyr Asn Lys Ile Ile Glu
Lys 405 410 415Leu Lys Glu
Arg Pro Asp Ala Leu Leu Asp Glu Ala Leu Pro Leu Asp 420
425 43096323PRTStreptomyces species Bf-61 96Met
Asn Ser Ser Asp Gly Ile Asp Gly Thr Val Ala Ser Ile Asp Thr1
5 10 15Ala Arg Ala Leu Leu Lys Arg
Phe Gly Phe Asp Ala Gln Arg Tyr Asn 20 25
30Val Arg Ser Ala Val Thr Leu Leu Ala Leu Ala Gly Leu Lys
Pro Gly 35 40 45Asp Arg Trp Val
Asp Ser Thr Thr Pro Arg Leu Gly Val Gln Lys Ile 50 55
60Met Asp Trp Ser Gly Glu His Trp Ala Lys Pro Tyr Ala
Thr Gly Ser65 70 75
80Arg Glu Asp Phe Arg Lys Lys Thr Leu Arg Gln Trp Val Asp Asn Gly
85 90 95Phe Ala Val Leu Asn Ala
Asp Asn Leu Asn Ile Ala Thr Asn Ser Gln 100
105 110Leu Asn Glu Tyr Cys Leu Ser Asp Glu Ala Leu Gln
Ala Leu Arg Ala 115 120 125Tyr Gly
Thr Glu Gly Phe Glu Glu Ser Leu Val Val Phe Leu Asp Glu 130
135 140Ala Ser Lys Ala Val Lys Ala Arg Ala Glu Ala
Leu Gln Ala Ala Met145 150 155
160Ile Ser Val Asp Leu Pro Gly Gly Glu Glu Phe Leu Leu Ser Pro Ala
165 170 175Gly Gln Asn Pro
Leu Leu Lys Lys Met Val Glu Glu Phe Val Pro Arg 180
185 190Phe Ala Pro Arg Ser Thr Val Leu Tyr Leu Gly
Asp Thr Arg Gly Lys 195 200 205His
Ser Leu Phe Glu Arg Glu Ile Phe Glu Glu Val Leu Gly Leu Thr 210
215 220Phe Asp Pro His Gly Arg Met Pro Asp Leu
Ile Leu His Asp Glu Val225 230 235
240Arg Gly Trp Leu Phe Leu Met Glu Ala Val Lys Ser Lys Gly Pro
Phe 245 250 255Asp Glu Glu
Arg His Arg Ser Leu Gln Glu Leu Phe Val Thr Pro Ser 260
265 270Ala Gly Leu Ile Phe Val Asn Cys Phe Glu
Asn Arg Glu Ser Met Arg 275 280
285Gln Trp Leu Pro Glu Leu Ala Trp Glu Thr Glu Ala Trp Val Ala Glu 290
295 300Asp Pro Asp His Leu Ile His Leu
Asn Gly Ser Arg Phe Leu Gly Pro305 310
315 320Tyr Glu Arg97227PRTStreptomyces caespitosus 97Met
Ile Asn Asp Gln Leu Pro Arg Trp Val Arg Glu Ala Arg Val Gly1
5 10 15Thr Arg Thr Gly Gly Pro Ala
Met Arg Pro Lys Thr Ser Asp Ser Pro 20 25
30Tyr Phe Gly Trp Asp Ser Glu Asp Trp Pro Glu Val Thr Arg
Gln Leu 35 40 45Leu Ser Glu Gln
Pro Leu Ser Gly Asp Thr Leu Val Asp Ala Val Leu 50 55
60Ala Ser Trp Glu Ser Ile Phe Glu Ser Arg Leu Gly Ser
Gly Phe His65 70 75
80Ile Gly Thr Gln Ile Arg Pro Thr Pro Gln Ile Met Gly Phe Leu Leu
85 90 95His Ala Leu Ile Pro Leu
Glu Leu Ala Asn Gly Asp Pro Ser Trp Arg 100
105 110Ala Asp Leu Asn Ser Ser Glu Lys Asp Leu Val Tyr
Gln Pro Asp His 115 120 125Lys Tyr
Ser Ile Glu Met Lys Thr Ser Ser His Lys Asp Gln Ile Phe 130
135 140Gly Asn Arg Ser Phe Gly Val Glu Asn Pro Gly
Lys Gly Lys Lys Ala145 150 155
160Lys Asp Gly Tyr Tyr Val Ala Val Asn Phe Glu Lys Trp Ser Asp Ala
165 170 175Pro Gly Arg Leu
Pro Arg Ile Arg Thr Ile Arg Tyr Gly Trp Leu Asp 180
185 190His Thr Asp Trp Val Ala Gln Lys Ser Gln Thr
Gly Gln Gln Ser Ser 195 200 205Leu
Pro Ala Val Val Ser Asn Thr Gln Leu Leu Ala Ile His Thr Gly 210
215 220Gly Gln Arg22598235PRTStreptomyces
phaeochromogenes 98Met Thr Ser Lys Asp Pro Ile Val Leu Ser Ala Asp Gln
Ile Ala Trp1 5 10 15Leu
Arg Gln Leu Lys Met Ser Lys Arg Ala Ala Leu Val Arg Asp Tyr 20
25 30Ile Leu Glu Tyr Gly Ala Val Thr
Thr Gly Lys Leu Ala Glu Leu Gly 35 40
45Tyr Ser His Pro Pro Arg Ala Ala Arg Asp Leu Lys Asp Ala Gly Ala
50 55 60Gly Val Val Thr Ile Met Val Lys
Gly Pro Asp Gly Arg Arg Met Ala65 70 75
80Ser Tyr Ala Phe Asn Gly Lys Ala Asn Glu Asp Gly Ala
Gly Arg Val 85 90 95Val
Ile Pro Lys Ala Phe Gly Glu Ala Leu Lys Arg Ala His Gly Gly
100 105 110Lys Cys Ala Val Cys Tyr Gly
Asp Phe Ser Glu Arg Glu Leu Gln Cys 115 120
125Asp His Arg Val Pro Phe Ala Ile Ala Gly Asp Lys Pro Lys Leu
Val 130 135 140Gln Glu Asp Phe Met Pro
Leu Cys Ala Ser Asp Asn Arg Ala Lys Ser145 150
155 160Trp Ser Cys Glu Asn Cys Pro Asn Trp Glu Leu
Lys Asp Glu Asp Thr 165 170
175Cys Arg Ser Cys Phe Trp Ala Ser Pro Glu Asn Tyr Thr His Val Ser
180 185 190Thr Arg Pro Glu Arg Arg
Ile Asn Leu Leu Phe Gln Gly Asp Glu Val 195 200
205Glu Ile Phe Asp Ala Leu Lys Asn Ala Ala Ala Asn Glu Gly
Val Ser 210 215 220Leu Thr Glu Ala Thr
Lys Arg Lys Leu Ala Asp225 230
23599281PRTSphaerotilus species 99Met Ser Lys Ala Ala Tyr Gln Asp Phe Thr
Lys Arg Phe Ser Leu Leu1 5 10
15Ile Lys Lys His Pro Asn Leu Ile Thr Met Thr Leu Ser Asn Ile Phe
20 25 30Thr Met Arg Leu Ile Gly
Asn Lys Thr His Gly Asp Leu Ala Glu Ile 35 40
45Ala Ile Ser Glu Phe Ile Asn Gln Tyr Met Tyr Asp Phe Lys
Ser Ile 50 55 60His Val Gly Lys Asp
Leu Tyr Arg Ala Lys Ser Lys Glu Glu Asp Ile65 70
75 80Thr Val Glu Asn Glu Ile Thr Lys Glu Lys
Phe Pro Ile Ser Leu Lys 85 90
95Ala Tyr Gly Asp Gly Pro Leu Gln Leu Ser Thr Asp Lys Asn Phe Leu
100 105 110Met Tyr Pro Leu Leu
Glu Glu Ile Gly Ala Phe Ile Asn Ala Lys Glu 115
120 125Lys Ile Glu Glu Ile Phe Ala Asn Glu Ala Phe Ser
Cys Phe Ser Glu 130 135 140Ile Asn Val
Leu Pro Leu Ile Tyr Asp Glu Lys Arg Gln Arg Cys Asn145
150 155 160Ile Leu Val Phe Asp Ala Ala
Arg Ala Arg Ala Glu Thr Ala Tyr Ile 165
170 175Arg Lys Glu Thr Glu Gly Ser Gly Arg Lys His Pro
Ala Tyr Arg Phe 180 185 190Phe
Asp Lys Asn Lys Asn Tyr Ile Cys Glu Val Arg Tyr Gly Asn Ala 195
200 205Ala Ala Asn Ala Leu Gln Arg Gly Leu
Trp Thr Asn Thr Lys Asn Ala 210 215
220Thr Ser Phe Phe Asp Ser Val Thr Asn Gly Trp Val Asp Tyr Ser His225
230 235 240Asn Leu Val Leu
Val Lys Leu Leu Ser His Ala Leu Val Ser Ser Arg 245
250 255Lys Gly His Glu Ala Ala Leu Glu Glu Ile
Lys Lys Asp Ile Leu Gln 260 265
270Leu Lys Gln Thr Asn Gly Ile Asn Val 275
2801001077DNAAnabaena variabilis 100atggaagaag accttgattt atctgaaaat
atcgaagctg catctgcgga gcttacgact 60ctttatcagg tagctgctga tgctatgaaa
gattatattg aaatctatct tgcgctgagt 120aaacagtctg atgggttttc aaatattaac
aatcttgact taacttctcg taacaggcgt 180ttggtagtta tacatggact ttcgttagag
ttagatccag atacttcgac tccagaggaa 240attaaacgtg aagctgaacg aatgctagcg
atagctcttg atacagagtc agcaattacg 300gcaggagtat atgaaaaaat gcgtctcttc
gcaagctctt tagtagatca gctatttgaa 360caaacggatg aacttaattc attatcatcg
gaatatttgt cagcaaatcc aggatttttg 420ccgtttttcc agcagttggc ggggcttaga
agtaaatcag agttaaagag agaagtagga 480aatgcctctg acaatagtat ttctaaagcg
gttgcagaga gaatattaga gcgcattata 540cgtaacttga gaattcgcac tttttccaaa
gagaaactat tacaagctgt tgagcctact 600ttagaaggaa tagtcaggga tctcgtagga
aaagtgttat tggaaaatat agttgctgat 660gctttatctg atttacaagt tcctttcatg
cgtgaatcag agtatcaaag ccttaaagga 720gtgatttatg atttccgcgc tgattttgtg
ataccagacg cacaaaatcc aattgctttt 780atcgaggtgc gaaaaagctc tacacgacat
gcgtcactct atgccaagga taagatgttt 840tcagcgatta attggaaagg aaaaaataaa
aggcttttgg gtattttggt tgtggaagga 900ccttggacaa gagaaactct tcgcgtcatg
gcaaatgtgt ttgattacgt tacaccttta 960actcgtgttt cccaagttgc agaagctatc
agagcatatc tagatgggga taaaacgaga 1020ctgaagtggt tagttaattt cagtattgaa
gaagcagacc acgacaacat aacctaa 10771011293DNABacillus sphaericus
101atgagacgat tagcaaaaaa ttcacggaac gacagttatt taagtaatag ggattaccag
60gaaatcgtga gggaaaatac cactacaata tcgtttccct taaaagaaaa acatactctg
120actttaacga aaaaaatagg gctaaatcag actgctggat tcggaggatg gtttttccct
180gattcaccat gtttattaac agtaactgta ctatcctctt tcggtacaaa ggtaacttct
240aaaaccttta gcctttctaa agattggaat cgtgttgggc ttgcttggat taacgagcat
300tcgagtgaca ccataagcat tgtcctagag tttagtgatg tggaaatagt tcatacatgg
360ggacttacat gtgatgtttt taatgtccat gaattaatta ttgatgctat agaagatcaa
420aataaactaa tagacgtgct aaatcaagaa catttatctc ctgaaacata ttatttaaac
480catgactctg atactgattt aattgagaat ttggaatcta cagaagagat aaagatagtt
540aaccaaagcc aaaagcaaat ctctttaaaa aaatgctgtt attgtcaacg ttatatgcct
600gtgaacatat tagttcgttc aaattcatca tttcataaac acaagagtaa gaaaactggt
660tttcaaaatg aatgtcgggc ttgtaagaag tggagaataa ataattcatt caatccagtc
720agaacaaaag accaactaca tgaatcagca gttattacac gtgaaaaaaa aatattactt
780aaagaacctg aaatattaca gaaaatcaaa aatagaaata acggtgaggg cttaaaaagt
840attatatgga aaaaatttga taaaaaatgc tttaattgtg aaaaagaatt aaccattgaa
900gaggtacgcc tagaccatac aagaccactt gcttatctgt ggcctatcga tgaacacgca
960acttgtttat gtgaaaaatg caacaataca aaacatgata tgtttcctat cgatttttat
1020caaggggacg aagacaaatt aagacgttta gctagaatta cggggttaga ttatgaatct
1080ctagttaaga gggacgtaaa tgaagttgaa cttgcaagaa taatcaataa cattgaagac
1140tttgcaacta atgtagaggc acgtactttt cgctcaataa gaaataaagt aaaagaagta
1200cgtcccgata ctgacctatt tgaaattctt aaatctaaaa atattaattt atataatgaa
1260cttcaatatg aacttcttac ccgtaaggat taa
12931022189DNAartificialplasmid placzz2 102ctggcgaaag ggggatgtgc
tgcaaggcga ttaagttggg taacgccagg gttttcccag 60tcacgacgtt gtaaaacgac
ggccagtgaa ttcgagctcg gtacccgggg gcgcgccgga 120tccttaatta agtctagagt
cgactgttta aacctgcagg catgcaagct tggcgtaatc 180atggtcatat gttaacctcc
ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240tatccgctca caattccaca
caacatacga gccggaagca taaagtgtaa agcctggggt 300gcctaatgag tgagctaact
cacattaatt gcgttgcgct cactgcccgc tttccagtcg 360ggaaacctgt cgtgccagca
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 420ggccgcgttg ctggcgtttt
tccataggct ccgcccccct gacgagcatc acaaaaatcg 480acgctcaagt cagaggtggc
gaaacccgac aggactataa agataccagg cgtttccccc 540tggaagctcc ctcgtgcgct
ctcctgttcc gaccctgccg cttaccggat acctgtccgc 600ctttctccct tcgggaagcg
tggcgctttc tcatagctca cgctgtaggt atctcagttc 660ggtgtaggtc gttcgctcca
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 720ctgcgcctta tccggtaact
atcgtcttga gtccaacccg gtaagacacg acttatcgcc 780actggcagca gccactggta
acaggattag cagagcgagg tatgtaggcg gtgctacaga 840gttcttgaag tggtggccta
actacggcta cactagaagg acagtatttg gtatctgcgc 900tctgctgaag ccagttacct
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 960caccgctggt agcggtggtt
tttttgtttg caagcagcag attacgcgca gaaaaaaagg 1020atctcaagaa gatcctttga
tcttttctac ggggtctgac gctcagtgga acgaaaactc 1080acgttaaggg attttggtca
tgagattatc aaaaaggatc ttcacctaga tccttttaaa 1140ttaaaaatga agttttaaat
caatctaaag tatatatgag taaacttggt ctgacagtta 1200ccaatgctta atcagtgagg
cacctatctc agcgatctgt ctatttcgtt catccatagt 1260tgcctgactc cccgtcgtgt
agataactac gatacgggag ggcttaccat ctggccccag 1320tgctgcaatg ataccgcgag
acccacgctc accggctcca gatttatcag caataaacca 1380gccagccgga agggccgagc
gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 1440tattaattgt tgccgggaag
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 1500tgttgccatt gctacaggca
tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 1560ctccggttcc caacgatcaa
ggcgagttac atgatccccc atgttgtgca aaaaagcggt 1620tagctccttc ggtcctccga
tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 1680ggttatggca gcactgcata
attctcttac tgtcatgcca tccgtaagat gcttttctgt 1740gactggtgag tactcaacca
agtcattctg agaatagtgt atgcggcgac cgagttgctc 1800ttgcccggcg tcaatacggg
ataataccgc gccacatagc agaactttaa aagtgctcat 1860cattggaaaa cgttcttcgg
ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 1920ttcgatgtaa cccactcgtg
cacccaactg atcttcagca tcttttactt tcaccagcgt 1980ttctgggtga gcaaaaacag
gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 2040gaaatgttga atactcatac
tcttcctttt tcaatattat tgaagcattt atcagggtta 2100ttgtctcatg agcggataca
tatttgaatg tatttagaaa aataaacaaa taggggttcc 2160gcgcacattt ccccgaaaag
tgccacctg
218910310673DNAartificialplasmid pBC4 103tcgcgcgttt cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca
gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg
cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat
gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg
aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg
ccagtgaatt cgagctcggt acccggggat 420cctctagagt cgaaccccgg atccggccgt
ccgccgtgat ccatgcggtt accgcccgcg 480tgtcgaaccc aggtgtgcga cgtcagacaa
cgggggagcg ctccttttgg cttccttcca 540ggcgcggcgg ctgctgcgct agcttttttg
gccactggcc gcgcgcggcg taagcggtta 600ggctggaaag cgaaagcatt aagtggctcg
ctccctgtag ccggagggtt attttccaag 660ggttgagtcg caggaccccc ggttcgagtc
tcgggccggc cggactgcgg cgaacggggg 720tttgcctccc cgtcatgcaa gaccccgctt
gcaaattcct ccggaaacag ggacgagccc 780cttttttgct tttcccagat gcatccggtg
ctgcggcaga tgcgcccccc tcctcagcag 840cggcaagagc aagagcagcg gcagacatgc
agggcaccct ccccttctcc taccgcgtca 900ggaggggcaa catccgcggc tgacgcggcg
gcagatggtg attacgaacc cccgcggcgc 960cgggcccggc actacctgga cttggaggag
ggcgagggcc tggcgcggct aggagcgccc 1020tctcctgagc gacacccaag ggtgcagctg
aagcgtgaca cgcgcgaggc gtacgtgccg 1080cggcagaacc tgtttcgcga ccgcgaggga
gaggagcccg aggagatgcg ggatcgaaag 1140ttccacgcag ggcgcgagtt gcggcatggc
ctgaaccgcg agcggttgct gcgcgaggag 1200gactttgagc ccgacgcgcg gaccgggatt
agtcccgcgc gcgcacacgt ggcggccgcc 1260gacctggtaa ccgcgtacga gcagacggtg
aaccaggaga ttaactttca aaaaagcttt 1320aacaaccacg tgcgcacgct tgtggcgcgc
gaggaggtgg ctataggact gatgcatctg 1380tgggactttg taagcgcgct ggagcaaaac
ccaaatagca agccgctcat ggcgcagctg 1440ttccttatag tgcagcacag cagggacaac
gaggcattca gggatgcgct gctaaacata 1500gtagagcccg agggccgctg gctgctcgat
ttgataaaca ttctgcagag catagtggtg 1560caggagcgca gcttgagcct ggctgacaag
gtggccgcca ttaactattc catgctcagt 1620ctgggcaagt tttacgcccg caagatatac
catacccctt acgttcccat agacaaggag 1680gtaaagatcg aggggttcta catgcgcatg
gcgttgaagg tgcttacctt gagcgacgac 1740ctgggcgttt atcgcaacga gcgcatccac
aaggccgtga gcgtgagccg gcggcgcgag 1800ctcagcgacc gcgagctgat gcacagcctg
caaagggccc tggctggcac gggcagcggc 1860gatagagagg ccgagtccta ctttgacgcg
ggcgctgacc tgcgctgggc cccaagccga 1920cgcgccctgg aggcagctgg ggccggacct
gggctggcgg tggcacccgc gcgcgctggc 1980aacgtcggcg gcgtggagga atatgacgag
gacgatgagt acgagccaga ggacggcgag 2040tactaagcgg tgatgtttct gatcagatga
tgcaagacgc aacggacccg gcggtgcggg 2100cggcgctgca gagccagccg tccggcctta
actccacgga cgactggcgc caggtcatgg 2160accgcatcat gtcgctgact gcgcgtaacc
ctgacgcgtt ccggcagcag ccgcaggcca 2220accggctctc cgcaattctg gaagcggtgg
tcccggcgcg cgcaaacccc acgcacgaga 2280aggtgctggc gatcgtaaac gcgctggccg
aaaacagggc catccggccc gatgaggccg 2340gcctggtcta cgacgcgctg cttcagcgcg
tggctcgtta caacagcggc aacgtgcaga 2400ccaacctgga ccggctggtg ggggatgtgc
gcgaggccgt ggcgcagcgt gagcgcgcgc 2460agcagcaggg caacctgggc tccatggttg
cactaaacgc cttcctgagt acacagcccg 2520ccaacgtgcc gcggggacag gaggactaca
ccaactttgt gagcgcactg cggctaatgg 2580tgactgagac accgcaaagt gaggtgtacc
agtccgggcc agactatttt ttccagacca 2640gtagacaagg cctgcagacc gtaaacctga
gccaggcttt caagaacttg caggggctgt 2700ggggggtgcg ggctcccaca ggcgaccgcg
cgaccgtgtc tagcttgctg acgcccaact 2760cgcgcctgtt gctgctgcta atagcgccct
tcacggacag tggcagcgtg tcccgggaca 2820catacctagg tcacttgctg acactgtacc
gcgaggccat aggtcaggcg catgtggacg 2880agcatacttt ccaggagatt acaagtgtca
gccgcgcgct ggggcaggag gacacgggca 2940gcctggaggc aaccctgaac tacctgctga
ccaaccggcg gcagaagatc ccctcgttgc 3000acagtttaaa cagcgaggag gagcgcatct
tgcgctatgt gcagcagagc gtgagcctta 3060acctgatgcg cgacggggta acgcccagcg
tggcgctgga catgaccgcg cgcaacatgg 3120aaccgggcat gtatgcctca aaccggccgt
ttatcaatcg cctaatggac tacttgcatc 3180gcgcggccgc cgtgaacccc gagtatttca
ccaatgccat cttgaacccg cactggctac 3240cgccccctgg tttctacacc gggggatttg
aggtgcccga gggtaacgat ggattcctct 3300gggacgacat agacgacagc gtgttttccc
cgcaaccgca gaccctgcta gagttgcaac 3360agcgcgagca ggcagaggcg gcgctgcgaa
aggaaagctt ccgcaggcca agcagcttgt 3420ccgatctagg cgctgcggcc ccgcggtcag
atgcgagtag cccatttcca agcttgatag 3480ggtcttttac cagcactcgc accacccgcc
cgcgcctgct gggcgaggag gagtacctaa 3540acaactcgct gctgcagccg cagcgcgaaa
agaacctgcc tccggcattt cccaacaacg 3600ggatagagag cctagtggac aagatgagta
gatggaagac gtatgcgcag gagcacaggg 3660atgtgcccgg cccgcgcccg cccacccgtc
gtcaaaggca cgaccgtcag cggggtctgg 3720tgtgggagga cgatgactcg gcagacgaca
gcagcgtcct ggatttggga gggagtggca 3780acccgtttgc gcaccttcgc cccaggctgg
ggagaatgtt ttaaaaaaaa aaaaaaaaag 3840catgatgcaa aataaaaaac tcaccaaggc
catggcaccg agcgttggtt ttcttgtatt 3900ccccttagta tgcagcgcgc ggcgatgtat
gaggaaggtc ctcctccctc ctacgagagc 3960gtggtgagcg cggcgccagt ggcggcggcg
ctgggttccc ccttcgatgc tcccctggac 4020ccgccgtttg tgcctccgcg gtacctgcgg
cctaccgggg ggagaaacag catccgttac 4080tctgagttgg cacccctatt cgacaccacc
cgtgtgtacc ttgtggacaa caagtcaacg 4140gatgtggcat ccctgaacta ccagaacgac
cacagcaact ttctaaccac ggtcattcaa 4200aacaatgact acagcccggg ggaggcaagc
acacagacca tcaatcttga cgaccgttcg 4260cactggggcg gcgacctgaa aaccatcctg
cataccaaca tgccaaatgt gaacgagttc 4320atgtttacca ataagtttaa ggcgcgggtg
atggtgtcgc gctcgcttac taaggacaaa 4380caggtggagc tgaaatatga gtgggtggag
ttcacgctgc ccgagggcaa ctactccgag 4440accatgacca tagaccttat gaacaacgcg
atcgtggagc actacttgaa agtgggcagg 4500cagaacgggg ttctggaaag cgacatcggg
gtaaagtttg acacccgcaa cttcagactg 4560gggtttgacc cagtcactgg tcttgtcatg
cctggggtat atacaaacga agccttccat 4620ccagacatca ttttgctgcc aggatgcggg
gtggacttca cccacagccg cctgagcaac 4680ttgttgggca tccgcaagcg gcaacccttc
caggagggct ttaggatcac ctacgatgac 4740ctggagggtg gtaacattcc cgcactgttg
gatgtggacg cctaccaggc aagcttaaaa 4800gatgacaccg aacagggcgg ggatggcgca
ggcggcggca acaacagtgg cagcggcgcg 4860gaagagaact ccaacgcggc agccgcggca
atgcagccgg tggaggacat gaacgatcat 4920gccattcgcg gcgacacctt tgccacacgg
gcggaggaga agcgcgctga ggccgaggca 4980gcggcagaag ctgccgcccc cgctgcgcaa
cccgaggtcg agaagcctca gaagaaaccg 5040gtgatcaaac ccctgacaga ggacagcaag
aaacgcagtt acaacctaat aagcaatgac 5100agcaccttca cccagtaccg cagctggtac
cttgcataca actacggcga ccctcagacc 5160gggatccgct catggaccct cctttgcact
cctgacgtaa cctgcggctc ggagcaggtc 5220tactggtcgt tgccagacat gatgcaagac
cccgtgacct tccgctccac gagccagatc 5280agcaactttc cggtggtggg cgccgagctg
ttgcccgtgc actccaagag cttctacaac 5340gaccaggccg tctactccca gctcatccgc
cagtttacct ctctgaccca cgtgttcaat 5400cgctttcccg agaaccagat tttggcgcgc
ccgccagccc ccaccatcac caccgtcagt 5460gaaaacgttc ctgctctcac agatcacggg
acgctaccgc tgcgcaacag catcggagga 5520gtccagcgag tgaccattac tgacgccaga
cgccgcacct gcccctacgt ttacaaggcc 5580ctgggcatag tctcgccgcg cgtcctatcg
agccgcactt tttgagcaaa catgtccatc 5640cttatatcgc ccagcaataa cacaggctgg
ggcctgcgct tcccaagcaa gatgtttggc 5700ggggcaaaga agcgctccga ccaacaccca
gtgcgcgtgc gcgggcacta ccgcgcgccc 5760tggggcgcgc acaaacgcgg ccgcactggg
cgcaccaccg tcgatgacgc cattgacgcg 5820gtggtggagg aggcgcgcaa ctacacgccc
acgccgccac cagtgtccac agtggacgcg 5880gccattcaga ccgtggtgcg cggagcccgg
cgttatgcta aaatgaagag acggcggagg 5940cgcgtagcac gtcgccaccg ccgccgaccc
ggcactgccg cccaacgcgc ggcggcggcc 6000ctgcttaacc gcgcacgtcg caccggccga
cgggcggcca tgcgggccgc tcgaaggctg 6060gccgcgggta ttgtcactgt gccccccagg
tccaggcgac gagcggccgc cgcagcagcc 6120gcggccatta gtgctatgac tcagggtcgc
aggggcaacg tgtactgggt gcgcgactcg 6180gttagcggcc tgcgcgtgcc cgtgcgcacc
cgccccccgc gcaactagat tgcaagaaaa 6240aactacttag actcgtactg ttgtatgtat
ccagcggcgg cggcgcgcaa cgaagctatg 6300tccaagcgca aaatcaaaga agagatgctc
caggtcatcg cgccggagat ctatggcccc 6360ccgaagaagg aagagcagga ttacaagccc
cgaaagctaa agcgggtcaa aaagaaaaag 6420aaagatgatg atgatgatga acttgacgac
gaggtggaac tgctgcacgc aaccgcgccc 6480aggcggcggg tacagtggaa aggtcgacgc
gtaagacgtg ttttgcgacc cggcaccacc 6540gtagttttta cgcccggtga gcgctccacc
cgcacctaca agcgcgtgta tgatgaggtg 6600tacggcgacg aggacctgct tgagcaggcc
aacgagcgcc tcggggagtt tgcctacgga 6660aagcggcata aggacatgtt ggcgttgccg
ctggacgagg gcaacccaac acctagccta 6720aagcccgtga cactgcagca ggtgctgccc
acgcttgcac cgtccgaaga aaagcgcggc 6780ctaaagcgcg agtctggtga cttggcaccc
accgtgcagc tgatggtacc caagcgccag 6840cgactggaag atgtcttgga aaaaatgacc
gtggagcctg ggctggagcc cgaggtccgc 6900gtgcggccaa tcaagcaggt ggcaccggga
ctgggcgtgc agaccgtgga cgttcagata 6960cccaccacca gtagcactag tattgccact
gccacagagg gcatggagac acaaacgtcc 7020ccggttgcct cggcggtggc agatgccgcg
gtgcaggcgg ccgctgcggc cgcgtccaaa 7080acctctacgg aggtgcaaac ggacccgtgg
atgtttcgcg tttcagcccc ccggcgcccg 7140cgccgttcca ggaagtacgg caccgccagc
gcactactgc ccgaatatgc cctacatcct 7200tccatcgcgc ctacccccgg ctatcgtggc
tacacctacc gccccagaag acgagcgact 7260acccgacgcc gaaccaccac tggaacccgc
cgccgccgtc gccgtcgcca gcccgtgctg 7320gccccgattt ccgtgcgcag ggtggctcgc
gaaggaggca ggaccctggt gctgccaaca 7380gcgcgctacc accccagcat cgtttaaaag
ccggtctttg tggttcttgc agatatggcc 7440ctcacctgcc gcctccgttt cccggtgccg
ggattccgag gaagaatgca ccgtaggagg 7500ggcatggccg gccacggcct gacgggcggc
atgcgtcgtg cgcaccaccg gcggcggcgc 7560gcgtcgcacc gtcgcatgcg cggcggtatc
ctgcccctcc ttattccact gatcgccgcg 7620gcgattggcg ccgtgcccgg aattgcatcc
gtggccttgc aggcgcagag acactgatta 7680aaaacaagtt gcatgtggaa aaatcaaaat
aaaaagtctg gagtctcacg ctcgcttggt 7740cctgtaacta ttttgtagaa tggaagacat
caactttgcg tctctggccc cgcgacacgg 7800ctcgcgcccg ttcatgggaa actggcaaga
tatcggcacc agcaatatga gcggtggcgc 7860cttcagctgg ggctcgctgt ggagcggcat
taaaaatttc ggttccacca ttaagaacta 7920tggcagcaag gcctggaaca gcagcacagg
ccagatgctg agggacaagt tgaaagagca 7980aaatttccaa caaaaggtgg tagatggcct
ggcctctggc attagcgggg tggtggacct 8040ggccaaccag gcagtgcaaa ataagattaa
cagtaagctt gatccccgcc ctcccgtaga 8100ggagcctcca ccggccgtgg agacagtgtc
tccagagggg cgtggcgaaa agcgtccgcg 8160gcccgacagg gaagaaactc tggtgacgca
aatagatgag cctccctcgt acgaggaggc 8220actaaagcaa ggcctgccca ccacccgtcc
catcgcgccc atggctaccg gagtgctggg 8280ccagcacaca cctgtaacgc tggacctgcc
tccccccgct gacacccagc agaaacctgt 8340gctgccaggg ccgtccgccg ttgttgtaac
ccgccctagc cgcgcgtccc tgcgccgtgc 8400cgccagcggt ccgcgatcga cctgcaggca
tgcaagcttg gcgtaatcat ggtcatagct 8460gtttcctgtg tgaaattgtt atccgctcac
aattccacac aacatacgag ccggaagcat 8520aaagtgtaaa gcctggggtg cctaatgagt
gagctaactc acattaattg cgttgcgctc 8580actgcccgct ttccagtcgg gaaacctgtc
gtgccagctg cattaatgaa tcggccaacg 8640cgcggggaga ggcggtttgc gtattgggcg
ctcttccgct tcctcgctca ctgactcgct 8700gcgctcggtc gttcggctgc ggcgagcggt
atcagctcac tcaaaggcgg taatacggtt 8760atccacagaa tcaggggata acgcaggaaa
gaacatgtga gcaaaaggcc agcaaaaggc 8820caggaaccgt aaaaaggccg cgttgctggc
gtttttccat aggctccgcc cccctgacga 8880gcatcacaaa aatcgacgct caagtcagag
gtggcgaaac ccgacaggac tataaagata 8940ccaggcgttt ccccctggaa gctccctcgt
gcgctctcct gttccgaccc tgccgcttac 9000cggatacctg tccgcctttc tcccttcggg
aagcgtggcg ctttctcaat gctcacgctg 9060taggtatctc agttcggtgt aggtcgttcg
ctccaagctg ggctgtgtgc acgaaccccc 9120cgttcagccc gaccgctgcg ccttatccgg
taactatcgt cttgagtcca acccggtaag 9180acacgactta tcgccactgg cagcagccac
tggtaacagg attagcagag cgaggtatgt 9240aggcggtgct acagagttct tgaagtggtg
gcctaactac ggctacacta gaaggacagt 9300atttggtatc tgcgctctgc tgaagccagt
taccttcgga aaaagagttg gtagctcttg 9360atccggcaaa caaaccaccg ctggtagcgg
tggttttttt gtttgcaagc agcagattac 9420gcgcagaaaa aaaggatctc aagaagatcc
tttgatcttt tctacggggt ctgacgctca 9480gtggaacgaa aactcacgtt aagggatttt
ggtcatgaga ttatcaaaaa ggatcttcac 9540ctagatcctt ttaaattaaa aatgaagttt
taaatcaatc taaagtatat atgagtaaac 9600ttggtctgac agttaccaat gcttaatcag
tgaggcacct atctcagcga tctgtctatt 9660tcgttcatcc atagttgcct gactccccgt
cgtgtagata actacgatac gggagggctt 9720accatctggc cccagtgctg caatgatacc
gcgagaccca cgctcaccgg ctccagattt 9780atcagcaata aaccagccag ccggaagggc
cgagcgcaga agtggtcctg caactttatc 9840cgcctccatc cagtctatta attgttgccg
ggaagctaga gtaagtagtt cgccagttaa 9900tagtttgcgc aacgttgttg ccattgctac
aggcatcgtg gtgtcacgct cgtcgtttgg 9960tatggcttca ttcagctccg gttcccaacg
atcaaggcga gttacatgat cccccatgtt 10020gtgcaaaaaa gcggttagct ccttcggtcc
tccgatcgtt gtcagaagta agttggccgc 10080agtgttatca ctcatggtta tggcagcact
gcataattct cttactgtca tgccatccgt 10140aagatgcttt tctgtgactg gtgagtactc
aaccaagtca ttctgagaat agtgtatgcg 10200gcgaccgagt tgctcttgcc cggcgtcaat
acgggataat accgcgccac atagcagaac 10260tttaaaagtg ctcatcattg gaaaacgttc
ttcggggcga aaactctcaa ggatcttacc 10320gctgttgaga tccagttcga tgtaacccac
tcgtgcaccc aactgatctt cagcatcttt 10380tactttcacc agcgtttctg ggtgagcaaa
aacaggaagg caaaatgccg caaaaaaggg 10440aataagggcg acacggaaat gttgaatact
catactcttc ctttttcaat attattgaag 10500catttatcag ggttattgtc tcatgagcgg
atacatattt gaatgtattt agaaaaataa 10560acaaataggg gttccgcgca catttccccg
aaaagtgcca cctgacgtct aagaaaccat 10620tattatcatg acattaacct ataaaaatag
gcgtatcacg aggccctttc gtc 1067310422563DNAartificialplasmid pXba
104tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat
420ccttctagac cgtgcaaaag gagagcctgt aagcgggcac tcttccgtgg tctggtggat
480aaattcgcaa gggtatcatg gcggacgacc ggggttcgaa ccccggatcc ggccgtccgc
540cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt gtgcgacgtc agacaacggg
600ggagcgctcc ttttggcttc cttccaggcg cggcggctgc tgcgctagct tttttggcca
660ctggccgcgc gcggcgtaag cggttaggct ggaaagcgaa agcattaagt ggctcgctcc
720ctgtagccgg agggttattt tccaagggtt gagtcgcagg acccccggtt cgagtctcgg
780gccggccgga ctgcggcgaa cgggggtttg cctccccgtc atgcaagacc ccgcttgcaa
840attcctccgg aaacagggac gagccccttt tttgcttttc ccagatgcat ccggtgctgc
900ggcagatgcg cccccctcct cagcagcggc aagagcaaga gcagcggcag acatgcaggg
960caccctcccc ttctcctacc gcgtcaggag gggcaacatc cgcggctgac gcggcggcag
1020atggtgatta cgaacccccg cggcgccggg cccggcacta cctggacttg gaggagggcg
1080agggcctggc gcggctagga gcgccctctc ctgagcgaca cccaagggtg cagctgaagc
1140gtgacacgcg cgaggcgtac gtgccgcggc agaacctgtt tcgcgaccgc gagggagagg
1200agcccgagga gatgcgggat cgaaagttcc acgcagggcg cgagttgcgg catggcctga
1260accgcgagcg gttgctgcgc gaggaggact ttgagcccga cgcgcggacc gggattagtc
1320ccgcgcgcgc acacgtggcg gccgccgacc tggtaaccgc gtacgagcag acggtgaacc
1380aggagattaa ctttcaaaaa agctttaaca accacgtgcg cacgcttgtg gcgcgcgagg
1440aggtggctat aggactgatg catctgtggg actttgtaag cgcgctggag caaaacccaa
1500atagcaagcc gctcatggcg cagctgttcc ttatagtgca gcacagcagg gacaacgagg
1560cattcaggga tgcgctgcta aacatagtag agcccgaggg ccgctggctg ctcgatttga
1620taaacattct gcagagcata gtggtgcagg agcgcagctt gagcctggct gacaaggtgg
1680ccgccattaa ctattccatg ctcagtctgg gcaagtttta cgcccgcaag atataccata
1740ccccttacgt tcccatagac aaggaggtaa agatcgaggg gttctacatg cgcatggcgt
1800tgaaggtgct taccttgagc gacgacctgg gcgtttatcg caacgagcgc atccacaagg
1860ccgtgagcgt gagccggcgg cgcgagctca gcgaccgcga gctgatgcac agcctgcaaa
1920gggccctggc tggcacgggc agcggcgata gagaggccga gtcctacttt gacgcgggcg
1980ctgacctgcg ctgggcccca agccgacgcg ccctggaggc agctggggcc ggacctgggc
2040tggcggtggc acccgcgcgc gctggcaacg tcggcggcgt ggaggaatat gacgaggacg
2100atgagtacga gccagaggac ggcgagtact aagcggtgat gtttctgatc agatgatgca
2160agacgcaacg gacccggcgg tgcgggcggc gctgcagagc cagccgtccg gccttaactc
2220cacggacgac tggcgccagg tcatggaccg catcatgtcg ctgactgcgc gtaaccctga
2280cgcgttccgg cagcagccgc aggccaaccg gctctccgca attctggaag cggtggtccc
2340ggcgcgcgca aaccccacgc acgagaaggt gctggcgatc gtaaacgcgc tggccgaaaa
2400cagggccatc cggcccgatg aggccggcct ggtctacgac gcgctgcttc agcgcgtggc
2460tcgttacaac agcggcaacg tgcagaccaa cctggaccgg ctggtggggg atgtgcgcga
2520ggccgtggcg cagcgtgagc gcgcgcagca gcagggcaac ctgggctcca tggttgcact
2580aaacgccttc ctgagtacac agcccgccaa cgtgccgcgg ggacaggagg actacaccaa
2640ctttgtgagc gcactgcggc taatggtgac tgagacaccg caaagtgagg tgtaccagtc
2700cgggccagac tattttttcc agaccagtag acaaggcctg cagaccgtaa acctgagcca
2760ggctttcaag aacttgcagg ggctgtgggg ggtgcgggct cccacaggcg accgcgcgac
2820cgtgtctagc ttgctgacgc ccaactcgcg cctgttgctg ctgctaatag cgcccttcac
2880ggacagtggc agcgtgtccc gggacacata cctaggtcac ttgctgacac tgtaccgcga
2940ggccataggt caggcgcatg tggacgagca tactttccag gagattacaa gtgtcagccg
3000cgcgctgggg caggaggaca cgggcagcct ggaggcaacc ctgaactacc tgctgaccaa
3060ccggcggcag aagatcccct cgttgcacag tttaaacagc gaggaggagc gcatcttgcg
3120ctatgtgcag cagagcgtga gccttaacct gatgcgcgac ggggtaacgc ccagcgtggc
3180gctggacatg accgcgcgca acatggaacc gggcatgtat gcctcaaacc ggccgtttat
3240caatcgccta atggactact tgcatcgcgc ggccgccgtg aaccccgagt atttcaccaa
3300tgccatcttg aacccgcact ggctaccgcc ccctggtttc tacaccgggg gatttgaggt
3360gcccgagggt aacgatggat tcctctggga cgacatagac gacagcgtgt tttccccgca
3420accgcagacc ctgctagagt tgcaacagcg cgagcaggca gaggcggcgc tgcgaaagga
3480aagcttccgc aggccaagca gcttgtccga tctaggcgct gcggccccgc ggtcagatgc
3540gagtagccca tttccaagct tgatagggtc ttttaccagc actcgcacca cccgcccgcg
3600cctgctgggc gaggaggagt acctaaacaa ctcgctgctg cagccgcagc gcgaaaagaa
3660cctgcctccg gcatttccca acaacgggat agagagccta gtggacaaga tgagtagatg
3720gaagacgtat gcgcaggagc acagggatgt gcccggcccg cgcccgccca cccgtcgtca
3780aaggcacgac cgtcagcggg gtctggtgtg ggaggacgat gactcggcag acgacagcag
3840cgtcctggat ttgggaggga gtggcaaccc gtttgcgcac cttcgcccca ggctggggag
3900aatgttttaa aaaaaaaaaa aaaaagcatg atgcaaaata aaaaactcac caaggccatg
3960gcaccgagcg ttggttttct tgtattcccc ttagtatgca gcgcgcggcg atgtatgagg
4020aaggtcctcc tccctcctac gagagcgtgg tgagcgcggc gccagtggcg gcggcgctgg
4080gttccccctt cgatgctccc ctggacccgc cgtttgtgcc tccgcggtac ctgcggccta
4140ccggggggag aaacagcatc cgttactctg agttggcacc cctattcgac accacccgtg
4200tgtaccttgt ggacaacaag tcaacggatg tggcatccct gaactaccag aacgaccaca
4260gcaactttct aaccacggtc attcaaaaca atgactacag cccgggggag gcaagcacac
4320agaccatcaa tcttgacgac cgttcgcact ggggcggcga cctgaaaacc atcctgcata
4380ccaacatgcc aaatgtgaac gagttcatgt ttaccaataa gtttaaggcg cgggtgatgg
4440tgtcgcgctc gcttactaag gacaaacagg tggagctgaa atatgagtgg gtggagttca
4500cgctgcccga gggcaactac tccgagacca tgaccataga ccttatgaac aacgcgatcg
4560tggagcacta cttgaaagtg ggcaggcaga acggggttct ggaaagcgac atcggggtaa
4620agtttgacac ccgcaacttc agactggggt ttgacccagt cactggtctt gtcatgcctg
4680gggtatatac aaacgaagcc ttccatccag acatcatttt gctgccagga tgcggggtgg
4740acttcaccca cagccgcctg agcaacttgt tgggcatccg caagcggcaa cccttccagg
4800agggctttag gatcacctac gatgacctgg agggtggtaa cattcccgca ctgttggatg
4860tggacgccta ccaggcaagc ttaaaagatg acaccgaaca gggcggggat ggcgcaggcg
4920gcggcaacaa cagtggcagc ggcgcggaag agaactccaa cgcggcagcc gcggcaatgc
4980agccggtgga ggacatgaac gatcatgcca ttcgcggcga cacctttgcc acacgggcgg
5040aggagaagcg cgctgaggcc gaggcagcgg cagaagctgc cgcccccgct gcgcaacccg
5100aggtcgagaa gcctcagaag aaaccggtga tcaaacccct gacagaggac agcaagaaac
5160gcagttacaa cctaataagc aatgacagca ccttcaccca gtaccgcagc tggtaccttg
5220catacaacta cggcgaccct cagaccggga tccgctcatg gaccctcctt tgcactcctg
5280acgtaacctg cggctcggag caggtctact ggtcgttgcc agacatgatg caagaccccg
5340tgaccttccg ctccacgagc cagatcagca actttccggt ggtgggcgcc gagctgttgc
5400ccgtgcactc caagagcttc tacaacgacc aggccgtcta ctcccagctc atccgccagt
5460ttacctctct gacccacgtg ttcaatcgct ttcccgagaa ccagattttg gcgcgcccgc
5520cagcccccac catcaccacc gtcagtgaaa acgttcctgc tctcacagat cacgggacgc
5580taccgctgcg caacagcatc ggaggagtcc agcgagtgac cattactgac gccagacgcc
5640gcacctgccc ctacgtttac aaggccctgg gcatagtctc gccgcgcgtc ctatcgagcc
5700gcactttttg agcaaacatg tccatcctta tatcgcccag caataacaca ggctggggcc
5760tgcgcttccc aagcaagatg tttggcgggg caaagaagcg ctccgaccaa cacccagtgc
5820gcgtgcgcgg gcactaccgc gcgccctggg gcgcgcacaa acgcggccgc actgggcgca
5880ccaccgtcga tgacgccatt gacgcggtgg tggaggaggc gcgcaactac acgcccacgc
5940cgccaccagt gtccacagtg gacgcggcca ttcagaccgt ggtgcgcgga gcccggcgtt
6000atgctaaaat gaagagacgg cggaggcgcg tagcacgtcg ccaccgccgc cgacccggca
6060ctgccgccca acgcgcggcg gcggccctgc ttaaccgcgc acgtcgcacc ggccgacggg
6120cggccatgcg ggccgctcga aggctggccg cgggtattgt cactgtgccc cccaggtcca
6180ggcgacgagc ggccgccgca gcagccgcgg ccattagtgc tatgactcag ggtcgcaggg
6240gcaacgtgta ctgggtgcgc gactcggtta gcggcctgcg cgtgcccgtg cgcacccgcc
6300ccccgcgcaa ctagattgca agaaaaaact acttagactc gtactgttgt atgtatccag
6360cggcggcggc gcgcaacgaa gctatgtcca agcgcaaaat caaagaagag atgctccagg
6420tcatcgcgcc ggagatctat ggccccccga agaaggaaga gcaggattac aagccccgaa
6480agctaaagcg ggtcaaaaag aaaaagaaag atgatgatga tgatgaactt gacgacgagg
6540tggaactgct gcacgcaacc gcgcccaggc ggcgggtaca gtggaaaggt cgacgcgtaa
6600gacgtgtttt gcgacccggc accaccgtag tttttacgcc cggtgagcgc tccacccgca
6660cctacaagcg cgtgtatgat gaggtgtacg gcgacgagga cctgcttgag caggccaacg
6720agcgcctcgg ggagtttgcc tacggaaagc ggcataagga catgttggcg ttgccgctgg
6780acgagggcaa cccaacacct agcctaaagc ccgtgacact gcagcaggtg ctgcccacgc
6840ttgcaccgtc cgaagaaaag cgcggcctaa agcgcgagtc tggtgacttg gcacccaccg
6900tgcagctgat ggtacccaag cgccagcgac tggaagatgt cttggaaaaa atgaccgtgg
6960agcctgggct ggagcccgag gtccgcgtgc ggccaatcaa gcaggtggca ccgggactgg
7020gcgtgcagac cgtggacgtt cagataccca ccaccagtag cactagtatt gccactgcca
7080cagagggcat ggagacacaa acgtccccgg ttgcctcggc ggtggcagat gccgcggtgc
7140aggcggccgc tgcggccgcg tccaaaacct ctacggaggt gcaaacggac ccgtggatgt
7200ttcgcgtttc agccccccgg cgcccgcgcc gttccaggaa gtacggcacc gccagcgcac
7260tactgcccga atatgcccta catccttcca tcgcgcctac ccccggctat cgtggctaca
7320cctaccgccc cagaagacga gcgactaccc gacgccgaac caccactgga acccgccgcc
7380gccgtcgccg tcgccagccc gtgctggccc cgatttccgt gcgcagggtg gctcgcgaag
7440gaggcaggac cctggtgctg ccaacagcgc gctaccaccc cagcatcgtt taaaagccgg
7500tctttgtggt tcttgcagat atggccctca cctgccgcct ccgtttcccg gtgccgggat
7560tccgaggaag aatgcaccgt aggaggggca tggccggcca cggcctgacg ggcggcatgc
7620gtcgtgcgca ccaccggcgg cggcgcgcgt cgcaccgtcg catgcgcggc ggtatcctgc
7680ccctccttat tccactgatc gccgcggcga ttggcgccgt gcccggaatt gcatccgtgg
7740ccttgcaggc gcagagacac tgattaaaaa caagttgcat gtggaaaaat caaaataaaa
7800agtctggagt ctcacgctcg cttggtcctg taactatttt gtagaatgga agacatcaac
7860tttgcgtctc tggccccgcg acacggctcg cgcccgttca tgggaaactg gcaagatatc
7920ggcaccagca atatgagcgg tggcgccttc agctggggct cgctgtggag cggcattaaa
7980aatttcggtt ccaccattaa gaactatggc agcaaggcct ggaacagcag cacaggccag
8040atgctgaggg acaagttgaa agagcaaaat ttccaacaaa aggtggtaga tggcctggcc
8100tctggcatta gcggggtggt ggacctggcc aaccaggcag tgcaaaataa gattaacagt
8160aagcttgatc cccgccctcc cgtagaggag cctccaccgg ccgtggagac agtgtctcca
8220gaggggcgtg gcgaaaagcg tccgcggccc gacagggaag aaactctggt gacgcaaata
8280gatgagcctc cctcgtacga ggaggcacta aagcaaggcc tgcccaccac ccgtcccatc
8340gcgcccatgg ctaccggagt gctgggccag cacacacctg taacgctgga cctgcctccc
8400cccgctgaca cccagcagaa acctgtgctg ccagggccgt ccgccgttgt tgtaacccgc
8460cctagccgcg cgtccctgcg ccgtgccgcc agcggtccgc gatcgatgcg gcccgtagcc
8520agtggcaact ggcaaagcac actgaacagc atcgtgggtc tgggggtgca atccctgaag
8580cgccgacgat gcttctaaat agctaacgtg tcgtatgtgt catgtatgcg tccatgtcgc
8640cgccagagga gctgctgagc cgccgtgcgc ccgctttcca agatggctac cccttcgatg
8700atgccgcagt ggtcttacat gcacatctcg ggccaggacg cctcggagta cctgagcccc
8760gggctggtgc agtttgcccg cgccaccgag acgtacttca gcctgaataa caagtttaga
8820aaccccacgg tggcacctac gcacgacgta accacagacc ggtcccagcg tttgacgctg
8880cggttcatcc ctgtggaccg cgaggatacc gcgtactcgt acaaagcgcg gttcaccctg
8940gctgtgggtg acaaccgtgt gcttgatatg gcttccacgt actttgacat ccgcggcgtg
9000ctggacaggg ggcctacttt taagccctac tccggcactg cctacaacgc tctagctccc
9060aagggcgctc ctaactcctg tgagtgggaa caaaccgaag atagcggccg ggcagttgcc
9120gaggatgaag aagaggaaga tgaagatgaa gaagaggaag aagaagagca aaacgctcga
9180gatcaggcta ctaagaaaac acatgtctat gcccaggctc ctttgtctgg agaaacaatt
9240acaaaaagcg ggctacaaat aggatcagac aatgcagaaa cacaagctaa acctgtatac
9300gcagatcctt cctatcaacc agaacctcaa attggcgaat ctcagtggaa cgaagctgat
9360gctaatgcgg caggagggag agtgcttaaa aaaacaactc ccatgaaacc atgctatgga
9420tcttatgcca ggcctacaaa tccttttggt ggtcaatccg ttctggttcc ggatgaaaaa
9480ggggtgcctc ttccaaaggt tgacttgcaa ttcttctcaa atactacctc tttgaacgac
9540cggcaaggca atgctactaa accaaaagtg gttttgtaca gtgaagatgt aaatatggaa
9600accccagaca cacatctgtc ttacaaacct ggaaaaggtg atgaaaattc taaagctatg
9660ttgggtcaac aatctatgcc aaacagaccc aattacattg ctttcaggga caattttatt
9720ggcctaatgt attataacag cactggcaac atgggtgttc ttgctggtca ggcatcgcag
9780ctaaatgccg tggtagattt gcaagacaga aacacagagc tgtcctatca actcttgctt
9840gattccatag gtgatagaac cagatatttt tctatgtgga atcaggctgt agacagctat
9900gatccagatg ttagaatcat tgaaaaccat ggaactgagg atgaattgcc aaattattgt
9960tttcctcttg ggggtattgg ggtaactgac acctatcaag ctattaaggc taatggcaat
10020ggctcaggcg ataatggaga tactacatgg acaaaagatg aaacttttgc aacacgtaat
10080gaaataggag tgggtaacaa ctttgccatg gaaattaacc taaatgccaa cctatggaga
10140aatttccttt actccaatat tgcgctgtac ctgccagaca agctaaaata caaccccacc
10200aatgtggaaa tatctgacaa ccccaacacc tacgactaca tgaacaagcg agtggtggct
10260cccgggcttg tagactgcta cattaacctt ggggcgcgct ggtctctgga ctacatggac
10320aacgttaatc cctttaacca ccaccgcaat gcgggcctcc gttatcgctc catgttgttg
10380ggaaacggcc gctacgtgcc ctttcacatt caggtgcccc aaaagttttt tgccattaaa
10440aacctcctcc tcctgccagg ctcatataca tatgaatgga acttcaggaa ggatgttaac
10500atggttctgc agagctctct gggaaacgat cttagagttg acggggctag cattaagttt
10560gacagcattt gtctttacgc caccttcttc cccatggccc acaacacggc ctccacgctg
10620gaagccatgc tcagaaatga caccaacgac cagtccttta atgactacct ttccgccgcc
10680aacatgctat accccatacc cgccaacgcc accaacgtgc ccatctccat cccatcgcgc
10740aactgggcag catttcgcgg ttgggccttc acacgcttga agacaaagga aaccccttcc
10800ctgggatcag gctacgaccc ttactacacc tactctggct ccataccata ccttgacgga
10860accttctatc ttaatcacac ctttaagaag gtggccatta cctttgactc ttctgttagc
10920tggccgggca acgaccgcct gcttactccc aatgagtttg agattaaacg ctcagttgac
10980ggggagggct acaacgtagc tcagtgcaac atgaccaagg actggttcct ggtgcagatg
11040ttggccaact acaatattgg ctaccagggc ttctacattc cagaaagcta caaggaccgc
11100atgtactcgt tcttcagaaa cttccagccc atgagccggc aagtggttga cgatactaaa
11160tacaaggagt atcagcaggt tggaattctt caccagcata acaactcagg attcgtaggc
11220tacctcgctc ccaccatgcg cgagggacag gcttaccccg ccaacgtgcc ctacccacta
11280ataggcaaaa ccgcggttga cagtattacc cagaaaaagt ttctttgcga tcgcaccctt
11340tggcgcatcc cattctccag taactttatg tccatgggcg cactcacaga cctgggccaa
11400aaccttctct acgccaactc cgcccacgcg ctagacatga cttttgaggt ggatcccatg
11460gacgagccca cccttcttta tgttttgttt gaagtctttg acgtggtccg tgtgcaccag
11520ccgcaccgcg gcgtcatcga gaccgtgtac ctgcgcacgc ccttctcggc cggcaacgcc
11580acaacataaa agaagcaagc aacatcaaca acagctgccg ccatgggctc cagtgagcag
11640gaactgaaag ccattgtcaa agatcttggt tgtgggccat attttttggg cacctatgac
11700aagcgctttc caggctttgt ttctccacac aagctcgcct gcgccatagt caatacggcc
11760ggtcgcgaga ctgggggcgt acactggatg gcctttgcct ggaacccgcg ctcaaaaaca
11820tgctacctct ttgagccctt tggcttttct gaccaacgac tcaagcaggt ttaccagttt
11880gagtacgagt cactcctgcg ccgtagcgcc attgcttctt cccccgaccg ctgtataacg
11940ctggaaaagt ccacccaaag cgtgcagggg cccaactcgg ccgcctgtgg actattctgc
12000tgcatgtttc tccacgcctt tgccaactgg ccccaaactc ccatggatca caaccccacc
12060atgaacctta ttaccggggt acccaactcc atgcttaaca gtccccaggt acagcccacc
12120ctgcgtcgca accaggaaca gctctacagc ttcctggagc gccactcgcc ctacttccgc
12180agccacagtg cgcagattag gagcgccact tctttttgtc acttgaaaaa catgtaaaaa
12240taatgtacta ggagacactt tcaataaagg caaatgtttt tatttgtaca ctctcgggtg
12300attatttacc ccccaccctt gccgtctgcg ccgtttaaaa atcaaagggg ttctgccgcg
12360catcgctatg cgccactggc agggacacgt tgcgatactg gtgtttagtg ctccacttaa
12420actcaggcac aaccatccgc ggcagctcgg tgaagttttc actccacagg ctgcgcacca
12480tcaccaacgc gtttagcagg tcgggcgccg atatcttgaa gtcgcagttg gggcctccgc
12540cctgcgcgcg cgagttgcga tacacagggt tgcagcactg gaacactatc agcgccgggt
12600ggtgcacgct ggccagcacg ctcttgtcgg agatcagatc cgcgtccagg tcctccgcgt
12660tgctcagggc gaacggagtc aactttggta gctgccttcc caaaaagggt gcatgcccag
12720gctttgagtt gcactcgcac cgtagtggca tcagaaggtg accgtgcccg gtctgggcgt
12780taggatacag cgcctgcatg aaagccttga tctgcttaaa agccacctga gcctttgcgc
12840cttcagagaa gaacatgccg caagacttgc cggaaaactg attggccgga caggccgcgt
12900catgcacgca gcaccttgcg tcggtgttgg agatctgcac cacatttcgg ccccaccggt
12960tcttcacgat cttggccttg ctagactgct ccttcagcgc gcgctgcccg ttttcgctcg
13020tcacatccat ttcaatcacg tgctccttat ttatcataat gctcccgtgt agacacttaa
13080gctcgccttc gatctcagcg cagcggtgca gccacaacgc gcagcccgtg ggctcgtggt
13140gcttgtaggt tacctctgca aacgactgca ggtacgcctg caggaatcgc cccatcatcg
13200tcacaaaggt cttgttgctg gtgaaggtca gctgcaaccc gcggtgctcc tcgtttagcc
13260aggtcttgca tacggccgcc agagcttcca cttggtcagg cagtagcttg aagtttgcct
13320ttagatcgtt atccacgtgg tacttgtcca tcaacgcgcg cgcagcctcc atgcccttct
13380cccacgcaga cacgatcggc aggctcagcg ggtttatcac cgtgctttca ctttccgctt
13440cactggactc ttccttttcc tcttgcgtcc gcataccccg cgccactggg tcgtcttcat
13500tcagccgccg caccgtgcgc ttacctccct tgccgtgctt gattagcacc ggtgggttgc
13560tgaaacccac catttgtagc gccacatctt ctctttcttc ctcgctgtcc acgatcacct
13620ctggggatgg cgggcgctcg ggcttgggag aggggcgctt ctttttcttt ttggacgcaa
13680tggccaaatc cgccgtcgag gtcgatggcc gcgggctggg tgtgcgcggc accagcgcat
13740cttgtgacga gtcttcttcg tcctcggact cgagacgccg cctcagccgc ttttttgggg
13800gcgcgcgggg aggcggcggc gacggcgacg gggacgacac gtcctccatg gttggtggac
13860gtcgcgccgc accgcgtccg cgctcggggg tggtttcgcg ctgctcctct tcccgactgg
13920ccatttcctt ctcctatagg cagaaaaaga tcatggagtc agtcgagaag gaggacagcc
13980taaccgcccc ctttgagttc gccaccaccg cctccaccga tgccgccaac gcgcctacca
14040ccttccccgt cgaggcaccc ccgcttgagg aggaggaagt gattatcgag caggacccag
14100gttttgtaag cgaagacgac gaggatcgct cagtaccaac agaggataaa aagcaagacc
14160aggacgacgc agaggcaaac gaggaacaag tcgggcgggg ggaccaaagg catggcgact
14220acctagatgt gggagacgac gtgctgttga agcatctgca gcgccagtgc gccattatct
14280gcgacgcgtt gcaagagcgc agcgatgtgc ccctcgccat agcggatgtc agccttgcct
14340acgaacgcca cctgttctca ccgcgcgtac cccccaaacg ccaagaaaac ggcacatgcg
14400agcccaaccc gcgcctcaac ttctaccccg tatttgccgt gccagaggtg cttgccacct
14460atcacatctt tttccaaaac tgcaagatac ccctatcctg ccgtgccaac cgcagccgag
14520cggacaagca gctggccttg cggcagggcg ctgtcatacc tgatatcgcc tcgctcgacg
14580aagtgccaaa aatctttgag ggtcttggac gcgacgagaa acgcgcggca aacgctctgc
14640aacaagaaaa cagcgaaaat gaaagtcact gtggagtgct ggtggaactt gagggtgaca
14700acgcgcgcct agccgtgctg aaacgcagca tcgaggtcac ccactttgcc tacccggcac
14760ttaacctacc ccccaaggtt atgagcacag tcatgagcga gctgatcgtg cgccgtgcac
14820gacccctgga gagggatgca aacttgcaag aacaaaccga ggagggccta cccgcagttg
14880gcgatgagca gctggcgcgc tggcttgaga cgcgcgagcc tgccgacttg gaggagcgac
14940gcaagctaat gatggccgca gtgcttgtta ccgtggagct tgagtgcatg cagcggttct
15000ttgctgaccc ggagatgcag cgcaagctag aggaaacgtt gcactacacc tttcgccagg
15060gctacgtgcg ccaggcctgc aaaatttcca acgtggagct ctgcaacctg gtctcctacc
15120ttggaatttt gcacgaaaac cgcctcgggc aaaacgtgct tcattccacg ctcaagggcg
15180aggcgcgccg cgactacgtc cgcgactgcg tttacttatt tctgtgctac acctggcaaa
15240cggccatggg cgtgtggcag caatgcctgg aggagcgcaa cctaaaggag ctgcagaagc
15300tgctaaagca aaacttgaag gacctatgga cggccttcaa cgagcgctcc gtggccgcgc
15360acctggcgga cattatcttc cccgaacgcc tgcttaaaac cctgcaacag ggtctgccag
15420acttcaccag tcaaagcatg ttgcaaaact ttaggaactt tatcctagag cgttcaggaa
15480ttctgcccgc cacctgctgt gcgcttccta gcgactttgt gcccattaag taccgtgaat
15540gccctccgcc gctttggggt cactgctacc ttctgcagct agccaactac cttgcctacc
15600actccgacat catggaagac gtgagcggtg acggcctact ggagtgtcac tgtcgctgca
15660acctatgcac cccgcaccgc tccctggtct gcaattcgca actgcttagc gaaagtcaaa
15720ttatcggtac ctttgagctg cagggtccct cgcctgacga aaagtccgcg gctccggggt
15780tgaaactcac tccggggctg tggacgtcgg cttaccttcg caaatttgta cctgaggact
15840accacgccca cgagattagg ttctacgaag accaatcccg cccgccaaat gcggagctta
15900ccgcctgcgt cattacccag ggccacatcc ttggccaatt gcaagccatc aacaaagccc
15960gccaagagtt tctgctacga aagggacggg gggtttacct ggacccccag tccggcgagg
16020agctcaaccc aatccccccg ccgccgcagc cctatcagca gccgcgggcc cttgcttccc
16080aggatggcac ccaaaaagaa gctgcagctg ccgccgccgc cacccacgga cgaggaggaa
16140tactgggaca gtcaggcaga ggaggttttg gacgaggagg aggagatgat ggaagactgg
16200gacagcctag acgaagcttc cgaggccgaa gaggtgtcag acgaaacacc gtcaccctcg
16260gtcgcattcc cctcgccggc gccccagaaa ttggcaaccg ttcccagcat cgctacaacc
16320tccgctcctc aggcgccgcc ggcactgcct gttcgccgac ccaaccgtag atgggacacc
16380actggaacca gggccggtaa gtctaagcag ccgccgccgt tagcccaaga gcaacaacag
16440cgccaaggct accgctcgtg gcgcgggcac aagaacgcca tagttgcttg cttgcaagac
16500tgtgggggca acatctcctt cgcccgccgc tttcttctct accatcacgg cgtggccttc
16560ccccgtaaca tcctgcatta ctaccgtcat ctctacagcc cctactgcac cggcggcagc
16620ggcagcggca gcaacagcag cggtcacaca gaagcaaagg cgaccggata gcaagactct
16680gacaaagccc aagaaatcca cagcggcggc agcagcagga ggaggagcgc tgcgtctggc
16740gcccaacgaa cccgtatcga cccgcgagct tagaaatagg atttttccca ctctgtatgc
16800tatatttcaa caaagcaggg gccaagaaca agagctgaaa ataaaaaaca ggtctctgcg
16860ctccctcacc cgcagctgcc tgtatcacaa aagcgaagat cagcttcggc gcacgctgga
16920agacgcggag gctctcttca gcaaatactg cgcgctgact cttaaggact agtttcgcgc
16980cctttctcaa atttaagcgc gaaaactacg tcatctccag cggccacacc cggcgccagc
17040acctgtcgtc agcgccatta tgagcaagga aattcccacg ccctacatgt ggagttacca
17100gccacaaatg ggacttgcgg ctggagctgc ccaagactac tcaacccgaa taaactacat
17160gagcgcggga ccccacatga tatcccgggt caacggaatc cgcgcccacc gaaaccgaat
17220tctcctcgaa caggcggcta ttaccaccac acctcgtaat aaccttaatc cccgtagttg
17280gcccgctgcc ctggtgtacc aggaaagtcc cgctcccacc actgtggtac ttcccagaga
17340cgcccaggcc gaagttcaga tgactaactc aggggcgcag cttgcgggcg gctttcgtca
17400cagggtgcgg tcgcccgggc agggtataac tcacctgaaa atcagagggc gaggtattca
17460gctcaacgac gagtcggtga gctcctctct tggtctccgt ccggacggga catttcagat
17520cggcggcgct ggccgctctt catttacgcc ccgtcaggcg atcctaactc tgcagacctc
17580gtcctcggag ccgcgctccg gaggcattgg aactctacaa tttattgagg agttcgtgcc
17640ttcggtttac ttcaacccct tttctggacc tcccggccac tacccggacc agtttattcc
17700caactttgac gcggtgaaag actcggcgga cggctacgac tgaatgacca gtggagaggc
17760agagcgactg cgcctgacac acctcgacca ctgccgccgc cacaagtgct ttgcccgcgg
17820ctccggtgag ttttgttact ttgaattgcc cgaagagcat atcgagggcc cggcgcacgg
17880cgtccggctc accacccagg tagagcttac acgtagcctg attcgggagt ttaccaagcg
17940ccccctgcta gtggagcggg agcggggtcc ctgtgttctg accgtggttt gcaactgtcc
18000taaccctgga ttacatcaag atctttgttg tcatctctgt gctgagtata ataaatacag
18060aaattagaat ctactggggc tcctgtcgcc atcctgtgaa cgccaccgtt tttacccacc
18120caaagcagac caaagcaaac ctcacctccg gtttgcacaa gcgggccaat aagtacctta
18180cctggtactt taacggctct tcatttgtaa tttacaacag tttccagcga gacgaagtaa
18240gtttgccaca caaccttctc ggcttcaact acaccgtcaa gaaaaacacc accaccacca
18300ccctcctcac ctgccgggaa cgtacgagtg cgtcaccggt tgctgcgccc acacctacag
18360cctgagcgta accagacatt actcccattt ttccaaaaca ggaggtgagc tcaactcccg
18420gaactcaggt caaaaaagca ttttgcgggg tgctgggatt ttttaattaa gtatatgagc
18480aattcaagta actctacaag cttgtctaat ttttctggaa ttggggtcgg ggttatcctt
18540actcttgtaa ttctgtttat tcttatacta gcacttctgt gccttagggt tgccgcctgc
18600tgcacgcacg tttgtaccta ttgtcagctt tttaaacgct gggggcaaca tccaagatga
18660ggtacatgat tttaggcttg ctcgcccttg cggcagtctg cagcgctgcc aaaaaggttg
18720agtttaagga accagcttgc aatgttacat ttaaatcaga agctaatgaa tgcactactc
18780ttataaaatg caccacagaa catgaaaagc ttattattcg ccacaaagac aaaattggca
18840agtatgctgt atatgctatt tggcagccag gtgacactaa cgactataat gtcacagtct
18900tccaaggtga aaatcgtaaa acttttatgt ataaatttcc attttatgaa atgtgcgata
18960ttaccatgta catgagcaaa cagtacaagt tgtggccccc acaaaagtgt ttagagaaca
19020ctggcacctt ttgttccacc gctctgctta ttacagcgct tgctttggta tgtaccttac
19080tttatctcaa atacaaaagc agacgcagtt ttattgatga aaagaaaatg ccttgatttt
19140ccgcttgctt gtattcccct ggacaattta ctctatgtgg gatatgctcc aggcgggcaa
19200gattataccc acaaccttca aatcaaactt tcctggacgt tagcgcctga tttctgccag
19260cgcctgcact gcaaatttga tcaaacccag cttcagcttg cctgctccag agatgaccgg
19320ctcaaccatc gcgcccacaa cggactatcg caacaccact gctaccggac taacatctgc
19380cctaaattta ccccaagttc atgcctttgt caatgactgg gcgagcttgg acatgtggtg
19440gttttccata gcgcttatgt ttgtttgcct tattattatg tggcttattt gttgcctaaa
19500gcgcagacgc gccagacccc ccatctatag gcctatcatt gtgctcaacc cacacaatga
19560aaaaattcat agattggacg gtctgaaacc atgttctctt cttttacagt atgattaaat
19620gagacatgat tcctcgagtt cttatattat tgacccttgt tgcgcttttc tgtgcgtgct
19680ctacattggc cgcggtcgct cacatcgaag tagattgcat cccacctttc acagtttacc
19740tgctttacgg atttgtcacc cttatcctca tctgcagcct cgtcactgta gtcatcgcct
19800tcattcagtt cattgactgg gtttgtgtgc gcattgcgta cctcaggcac catccgcaat
19860acagagacag gactatagct gatcttctca gaattcttta attatgaaac ggagtgtcat
19920ttttgttttg ctgatttttt gcgccctacc tgtgctttgc tcccaaacct cagcgcctcc
19980caaaagacat atttcctgca gattcactca aatatggaac attcccagct gctacaacaa
20040acagagcgat ttgtcagaag cctggttata cgccatcatc tctgtcatgg ttttttgcag
20100taccattttt gccctagcca tatatccata ccttgacatt ggctggaatg ccatagatgc
20160catgaaccac cctactttcc cagtgcccgc tgtcatacca ctgcaacagg ttattgcccc
20220aatcaatcag cctcgccccc cttctcccac ccccactgag attagctact ttaatttgac
20280aggtggagat gactgaatct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat
20340ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
20400ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
20460cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
20520tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
20580ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
20640taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
20700agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
20760cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
20820tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
20880tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat
20940gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
21000acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
21060acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
21120cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
21180gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
21240gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
21300agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
21360ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
21420ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
21480atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
21540tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
21600gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
21660ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
21720caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
21780cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
21840cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
21900cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
21960agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
22020tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
22080agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
22140atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
22200ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
22260cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
22320caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
22380attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
22440agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
22500aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
22560gtc
225631058350DNAartificialplasmid psyx20-lacIq 105cagctggcgt aatagcgaag
aggcccgcac cgatcgccct tcccaacagt tgcgcagcct 60gaatggcgaa tgggacgcgc
cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 120gcgcagcgtg accgctacac
ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 180ttcctttctc gccacgttcg
ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 240agggttccga tttagtgctt
tacggcacct cgaccccaaa aaacttgatt agggtgatgg 300ttcacgtagt gggccatcgc
cctgatagac ggtttttcgc cctttgacgt tggagtccac 360gttctttaat agtggactct
tgttccaaac tggaacaaca ctcaacccta tctcggtcta 420ttcttttgat ttataaggga
ttttgccgat ttcggcctat tggttaaaaa atgagctgat 480ttaacaaaaa tttaacgcga
attttaacaa aattcgaccg atgcccttga gagccttcaa 540cccagtcagc tccttccggt
gggcgcgggg catgactatc gtcgccgcac ttatgactgt 600cttctttatc atgcaactcg
taggacaggt gccggcagcg ctctgggtca ttttcggcga 660ggaccgcttt cgctggagcg
cgacgatgat cggcctgtcg cttgcggtat tcggaatctt 720gcacgccctc gctcaagcct
tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca 780ggccattatc gccggcatgg
cggccgacgc gctgggctac gtcttgctgg cgttcgcgac 840gcgaggctgg atggccttcc
ccattatgat tcttctcgct tccggcggca tcgggatgcc 900cgcgttgcag gccatgctgt
ccaggcaggt agatgacgac catcagggac agcttcaagg 960atcgctcgcg gctcttacca
gcctaacttc gatcattgga ccgctgatcg tcacggcgat 1020ttatgccgcc tcggcgagca
catggaacgg gttggcatgg attgtaggcg ccgccctata 1080ccttgtctgc ctccccgcgt
tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat 1140ggaagccggc ggcacctcgc
taacggattc accactccgc agacccgcca taaaacgccc 1200tgagaagccc gtgacgggct
tttcttgtat tatgggtagt ttccttgcat gaatccataa 1260aaggcgcctg tagtgccatt
tacccccatt cactgccaga gccgtgagcg cagcgaactg 1320aatgtcacga aaaagacagc
gactcaggtg cctgatggtc ggagacaaaa ggaatattca 1380gcgatttgcc cgagcttgcg
agggtgctac ttaagccttt agggttttaa ggtctgtttt 1440gtagaggagc aaacagcgtt
tgcgacatcc ttttgtaata ctgcggaact gactaaagta 1500gtgagttata cacagggctg
ggatctattc tttttatctt tttttattct ttctttattc 1560tataaattat aaccacttga
atataaacaa aaaaaacaca caaaggtcta gcggaattta 1620cagagggtct agcagaattt
acaagttttc cagcaaaggt ctagcagaat ttacagatac 1680ccacaactca aaggaaaagg
actagtaatt atcattgact agcccatctc aattggtata 1740gtgattaaaa tcacctagac
caattgagat gtatgtctga attagttgtt ttcaaagcaa 1800atgaactagc gattagtcgc
tatgacttaa cggagcatga aaccaagcta attttatgct 1860gtgtggcact actcaacccc
acgattgaaa accctacaag gaaagaacgg acggtatcgt 1920tcacttataa ccaatacgct
cagatgatga acatcagtag ggaaaatgct tatggtgtat 1980tagctaaagc aaccagagag
ctgatgacga gaactgtgga aatcaggaat cctttggtta 2040aaggctttga gattttccag
tggacaaact atgccaagtt ctcaagcgaa aaattagaat 2100tagtttttag tgaagagata
ttgccttatc ttttccagtt aaaaaaattc ataaaatata 2160atctggaaca tgttaagtct
tttgaaaaca aatactctat gaggatttat gagtggttat 2220taaaagaact aacacaaaag
aaaactcaca aggcaaatat agagattagc cttgatgaat 2280ttaagttcat gttaatgctt
gaaaataact accatgagtt taaaaggctt aaccaatggg 2340ttttgaaacc aataagtaaa
gatttaaaca cttacagcaa tatgaaattg gtggttgata 2400agcgaggccg cccgactgat
acgttgattt tccaagttga actagataga caaatggatc 2460tcgtaaccga acttgagaac
aaccagataa aaatgaatgg tgacaaaata ccaacaacca 2520ttacatcaga ttcctaccta
cataacggac taagaaaaac actacacgat gctttaactg 2580caaaaattca gctcaccagt
tttgaggcaa aatttttgag tgacatgcaa agtaagtatg 2640atctcaatgg ttcgttctca
tggctcacgc aaaaacaacg aaccacacta gagaacatac 2700tggctaaata cggaaggatc
tgaggttctt atggctcttg tatctatcag tgaagcatca 2760agactaacaa acaaaagtag
aacaactgtt caccgttaca tatcaaaggg aaaactgtcc 2820atatgcacag atgaaaacgg
tgtaaaaaag atagatacat cagagctttt acgagttttt 2880ggtgcattca aagctgttca
ccatgaacag atcgacaatg taacagatga acagcatgta 2940acacctaata gaacaggtga
aaccagtaaa acaaagcaac tagaacatga aattgaacac 3000ctgagacaac ttgttacagc
tcaacagtca cacatagaca gcctgaaaca ggcgatgctg 3060cttatcgaat caaagctgcc
gacaacacgg gagccagtga cgcctcccgt ggggaaaaaa 3120tcatggcaat tctggaagaa
atagcgcttt cagccggcaa accggctgaa gccggatctg 3180cgattctgat aacaaactag
caacaccaga acagcccgtt tgcgggcagc aaaacccgta 3240cttttggacg ttccggcggt
tttttgtggc gagtggtgtt cgggcggtgc gcgattattg 3300aagcatttat cagggttatt
gtctcatgag cggatacata tttgaatgta tttagaaaaa 3360taaacaaata ggggttccgc
gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac 3420cattattatc atgacattaa
cctataaaaa taggcgtatc acgaggccct ttcgtcttca 3480agaattcgcg cgcgaaggcc
aagcggcatg catttacgtt gacaccatcg aatggcgcaa 3540aacctttcgc ggtatggcat
gatagcgccc ggaagagagt caattcaggg tggtgaatgt 3600gaaaccagta acgttatacg
atgtcgcaga gtatgccggt gtctcttatc agaccgtttc 3660ccgcgtggtg aaccaggcca
gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc 3720gatggcggag ctgaattaca
ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc 3780gttgctgatt ggcgttgcca
cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc 3840ggcgattaaa tctcgcgccg
atcaactggg tgccagcgtg gtggtgtcga tggtagaacg 3900aagcggcgtc gaagcctgta
aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg 3960gctgatcatt aactatccgc
tggatgacca ggatgccatt gctgtggaag ctgcctgcac 4020taatgttccg gcgttatttc
ttgatgtctc tgaccagaca cccatcaaca gtattatttt 4080ctcccatgaa gacggtacgc
gactgggcgt ggagcatctg gtcgcattgg gtcaccagca 4140aatcgcgctg ttagcgggcc
cattaagttc tgtctcggcg cgtctgcgtc tggctggctg 4200gcataaatat ctcactcgca
atcaaattca gccgatagcg gaacgggaag gcgactggag 4260tgccatgtcc ggttttcaac
aaaccatgca aatgctgaat gagggcatcg ttcccactgc 4320gatgctggtt gccaacgatc
agatggcgct gggcgcaatg cgcgccatta ccgagtccgg 4380gctgcgcgtt ggtgcggata
tctcggtagt gggatacgac gataccgaag acagctcatg 4440ttatatcccg ccgtcaacca
ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt 4500ggaccgcttg ctgcaactct
ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt 4560ctcactggtg aaaagaaaaa
ccaccctggc gcccaatacg caaaccgcct ctccccgcgc 4620gttggccgat tcattaatgc
agctggcacg acaggtttcc cgactggaaa gcgggcagtg 4680agcgcaacgc aattaatgtg
agttagctca ctcattaggc gaattctcat gtttgacagc 4740ttatcatcga taagctttaa
tgcggtagtt tatcacagtt aaattgctaa cgcagtcagg 4800caccgtgtat gaaatctaac
aatgcgctca tcgtcatcct cggcaccgtc accctggatg 4860ctgtaggcat aggcttggtt
atgccggtac tgccgggcct cttgcgggat atcgtccatt 4920ccgacagcat cgccagtcac
tatggcgtgc tgctagcgct atatgcgttg atgcaatttc 4980tatgcgcacc cgttctcgga
gcactgtccg accgctttgg ccgccgccca gtcctgctcg 5040cttcgctact tggagccact
atcgactacg cgatcatggc gaccacaccc gtcctgtgga 5100tcctctacgc cggacgcatc
gtggccggca tcaccggcgc cacaggtgcg gttgctggcg 5160cctatatcgc cgacatcacc
gatggggaag atcgggctcg ccacttcggg ctcatgagcg 5220cttgtttcgg cgtgggtatg
gtggcaggcc ccgtggccgg gggactgttg ggcgccatct 5280ccttgcatgc accattcctt
gcggcggcgg tgctcaacgg cctcaaccta ctactgggct 5340gcttcctaat gcaggaatcg
cataagggag agcgtcgacc gatgcccttg agagccttca 5400acccagtcag ctccttccgg
tgggcgcggg gcatgactat cgtcgccgca cttatgactg 5460tcttctttat catgcaactc
gtaggacagg tgccggcagc gctctgggtc attttcggcg 5520aggaccgctt tcgctggagc
gcgacgatga tcggcctgtc gcttgcggta ttcggaatct 5580tgcacgccct cgctcaagcc
ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc 5640aggccattat cgccggcatg
gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga 5700cgcgaggctg gatggccttc
cccattatga ttcttctcgc ttccggcggc atcgggatgc 5760ccgcgttgca ggccatgctg
tccaggcagg tagatgacga ccatcaggga cagcttcaag 5820gatcgctcgc ggctcttacc
agcctaactt cgatcattgg accgctgatc gtcacggcga 5880tttatgccgc ctcggcgagc
acatggaacg ggttggcatg gattgtaggc gccgccctat 5940accttgtctg cctccccgcg
ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa 6000tggaagccgg cggcacctcg
ctaacggatt caccactcca agaattggag ccaatcaatt 6060cttgcggaga actgtgaatg
cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc 6120catctccagc agccgcacgc
ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg 6180catgatcgtg ctcctgtcgt
tgaggacccg gctaggctgg cggggttgcc ttactggtta 6240gcagaatgaa tcaccgatac
gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc 6300gacctgagca acaacatgaa
tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg 6360gaagtcagcg ccctgcacca
ttatgttccg gatctgcatc gcaggatgct gctggctacc 6420ctgtggaaca cctacatctg
tattaacgaa gcgctggcat tgaccctgag tgatttttct 6480ctggtcccgc cgcatccata
ccgccagttg tttaccctca caacgttcca gtaaccgggc 6540atgttcatca tcagtaaccc
gtatcgtgag catcctctct cgtttcatcg gtatcattac 6600ccccatgaac agaaatcccc
cttacacgga ggcatcagtg accaaacagg aaaaaaccgc 6660ccttaacatg gcccgcttta
tcagaagcca gacattaacg cttctggaga aactcaacga 6720gctggacgcg gatgaacagg
cagacatctg tgaatcgctt cacgaccacg ctgatgagct 6780ttaccgcagc gcgcagggtc
agcctgaata cgcgtttaat gaccagcaca gtcgtgatgg 6840caaggtcaga atagcgctga
ggtctgcctc gtgaagaagg tgttgctgac tcataccagg 6900cctgaatcgc cccatcatcc
agccagaaag tgagggagcc acggttgatg agagctttgt 6960tgtaggtgga ccagttggtg
attttgaact tttgctttgc cacggaacgg tctgcgttgt 7020cgggaagatg cgtgatctga
tccttcaact cagcaaaagt tcgatttatt caacaaagcc 7080acgttgtgtc tcaaaatctc
tgatgttaca ttgcacaaga taaaaatata tcatcatgaa 7140caataaaact gtctgcttac
ataaacagta atacaagggg tgttatgagc catattcaac 7200gggaaacgtc ttgctcgagg
ccgcgattaa attccaacat ggatgctgat ttatatgggt 7260ataaatgggc tcgcgataat
gtcgggcaat caggtgcgac aatctatcga ttgtatggga 7320agcccgatgc gccagagttg
tttctgaaac atggcaaagg tagcgttgcc aatgatgtta 7380cagatgagat ggtcagacta
aactggctga cggaatttat gcctcttccg accatcaagc 7440attttatccg tactcctgat
gatgcatggt tactcaccac tgcgatcccc gggaaaacag 7500cattccaggt attagaagaa
tatcctgatt caggtgaaaa tattgttgat gcgctggcag 7560tgttcctgcg ccggttgcat
tcgattcctg tttgtaattg tccttttaac agcgatcgcg 7620tatttcgtct cgctcaggcg
caatcacgaa tgaataacgg tttggttgat gcgagtgatt 7680ttgatgacga gcgtaatggc
tggcctgttg aacaagtctg gaaagaaatg cataagcttt 7740tgccattctc accggattca
gtcgtcactc atggtgattt ctcacttgat aaccttattt 7800ttgacgaggg gaaattaata
ggttgtattg atgttggacg agtcggaatc gcagaccgat 7860accaggatct tgccatccta
tggaactgcc tcggtgagtt ttctccttca ttacagaaac 7920ggctttttca aaaatatggt
attgataatc ctgatatgaa taaattgcag tttcatttga 7980tgctcgatga gtttttctaa
tcagaattgg ttaattggtt gtaacactgg cagagcatta 8040cgctgacttg acgggacggc
ggctttgttg aataaatcga acttttgctg agttgaagga 8100tcagatcacg catcttcccg
acaacgcaga ccgttccgtg gcaaagcaaa agttcaaaat 8160caccaactgg tccacctaca
acaaagctct catcaaccgt ggctccctca ctttctggct 8220ggatgatggg gcgattcagg
cctggtatga gtcagcaaca ccttcttcac gaggcagacc 8280tcagcgctat tctgaccttg
ccatcacgac tgtgctggtc attaaacgcg tattcaggct 8340gaccctgcgc
83501063190DNAartificialplasmid pAGR3 106aagtgaccaa acaggaaaaa accgccctta
acatggcccg ctttatcaga agccagacat 60taacgcttct ggagaaactc aacgagctgg
acgcggatga acaggcagac atctgtgaat 120cgcttcacga ccacgctgat gagctttacc
gcagctgcct cgcgcgtttc ggtgatgacg 180gtgaaaacct ctgacacatg cagctcccgg
agacggtcac agcttgtctg taagcggatg 240ccgggagcag acaagcccgt cagggcgcgt
cagcgggtgt tggcgggtgt cggggcgcag 300ccatgaccca gtcacgtagc gatagcggag
tgtatactgg cttaactatg cggcatcaga 360gcagattgta ctgagagtgc accatatgcg
gtgtgaaata ccgcacagat gcgtaaggag 420aaaataccgc atcaggcgct cttccgcttc
ctcgctcact gactcgctgc gctcggtcgt 480tcggctgcgg cgagcggtat cagctcactc
aaaggcggta atacggttat ccacagaatc 540aggggataac gcaggaaaga acatgtgagc
aaaaggccag caaaaggcca ggaaccgtaa 600aaaggccgcg ttgctggcgt ttttccatag
gctccgcccc cctgacgagc atcacaaaaa 660tcgacgctca agtcagaggt ggcgaaaccc
gacaggacta taaagatacc aggcgtttcc 720ccctggaagc tccctcgtgc gctctcctgt
tccgaccctg ccgcttaccg gatacctgtc 780cgcctttctc ccttcgggaa gcgtggcgct
ttctcatagc tcacgctgta ggtatctcag 840ttcggtgtag gtcgttcgct ccaagctggg
ctgtgtgcac gaaccccccg ttcagcccga 900ccgctgcgcc ttatccggta actatcgtct
tgagtccaac ccggtaagac acgacttatc 960gccactggca gcagccactg gtaacaggat
tagcagagcg aggtatgtag gcggtgctac 1020agagttcttg aagtggtggc ctaactacgg
ctacactaga aggacagtat ttggtatctg 1080cgctctgctg aagccagtta ccttcggaaa
aagagttggt agctcttgat ccggcaaaca 1140aaccaccgct ggtagcggtg gtttttttgt
ttgcaagcag cagattacgc gcagaaaaaa 1200aggatctcaa gaagatcctt tgatcttttc
tacggggtct gacgctcagt ggaacgaaaa 1260ctcacgttaa gggattttgg tcatgagatt
atcaaaaagg atcttcacct agatcctttt 1320aaattaaaaa tgaagtttta aatcaatcta
aagtatatat gagtaaactt ggtctgacag 1380ttaccaatgc ttaatcagtg aggcacctat
ctcagcgatc tgtctatttc gttcatccat 1440agttgcctga ctccccgtcg tgtagataac
tacgatacgg gagggcttac catctggccc 1500cagtgctgca atgataccgc gagacccacg
ctcaccggct ccagatttat cagcaataaa 1560ccagccagcc ggaagggccg agcgcagaag
tggtcctgca actttatccg cctccatcca 1620gtctattaat tgttgccggg aagctagagt
aagtagttcg ccagttaata gtttgcgcaa 1680cgttgttgcc attgctgcag gcatcgtggt
gtcacgctcg tcgtttggta tggcttcatt 1740cagctccggt tcccaacgat caaggcgagt
tacatgatcc cccatgttgt gcaaaaaagc 1800ggttagctcc ttcggtcctc cgatcgttgt
cagaagtaag ttggccgcag tgttatcact 1860catggttatg gcagcactgc ataattctct
tactgtcatg ccatccgtaa gatgcttttc 1920tgtgactggt gagtactcaa ccaagtcatt
ctgagaatag tgtatgcggc gaccgagttg 1980ctcttgcccg gcgtcaacac gggataatac
cgcgccacat agcagaactt taaaagtgct 2040catcattgga aaacgttctt cggggcgaaa
actctcaagg atcttaccgc tgttgagatc 2100cagttcgatg taacccactc gtgcacccaa
ctgatcttca gcatctttta ctttcaccag 2160cgtttctggg tgagcaaaaa caggaaggca
aaatgccgca aaaaagggaa taagggcgac 2220acggaaatgt tgaatactca tactcttcct
ttttcaatat tattgaagca tttatcaggg 2280ttattgtctc atgagcggat acatatttga
atgtatttag aaaaataaac aaataggggt 2340tccgcgcaca tttccccgaa aagtgccacc
tgacgtctaa gaaaccatta ttatcatgac 2400attaacctat aaaaataggc gtatcacgag
gccctttcgt cttcaagaat taattcccaa 2460ttccaggcat caaataaaac gaaaggctca
gtcgaaagac tgggcctttc gttttatctg 2520ttgtttgtcg gtgaacgctc tcctgagtag
gacaaatccg ccgggagcgg atttgaacgt 2580tgcgaagcaa cggcccggag ggtggcgggc
aggacgcccg ccataaactg ccaggaatta 2640attccaggca tcaaataaaa cgaaaggctc
agtcgaaaga ctgggccttt cgttttatct 2700gttgtttgtc ggtgaacgct ctcctgagta
ggacaaatcc gccgggagcg gatttgaacg 2760ttgcgaagca acggcccgga gggtggcggg
caggacgccc gccataaact gccaggaatt 2820aattccaggc atcaaataaa acgaaaggct
cagtcgaaag actgggcctt tcgttttatc 2880tgttgtttgt cggtgaacgc tctcctgagt
aggacaaatc cgccgggagc ggatttgaac 2940gttgcgaagc aacggcccgg agggtggcgg
gcaggacgcc cgccataaac tgccaggaat 3000taattccagg catcaaataa aacgaaaggc
tcagtcgaaa gactgggcct ttcgttttat 3060ctgttgtttg tcggtgaacg ctctcctgag
taggacaaat ccgccgggag cggatttgaa 3120cgttgcgaag caacggcccg gagggtggcg
ggcaggacgc ccgccataaa ctgccaggaa 3180ttggggatcg
3190
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