NOVEL DNA CLONING METHOD RELYING ON THE E.COLI recE/recT RECOMBINATION SYSTEM

STEWART; Francis ;   et al.

Patent Application Summary

U.S. patent application number 13/342621 was filed with the patent office on 2012-08-16 for novel dna cloning method relying on the e.coli rece/rect recombination system. This patent application is currently assigned to Europaisches Laboratorium fur Molekularbiologie (EMBL). Invention is credited to Frank BUCHHOLZ, Francis STEWART, Youming ZHANG.

Application Number20120208277 13/342621
Document ID /
Family ID26145961
Filed Date2012-08-16

United States Patent Application 20120208277
Kind Code A1
STEWART; Francis ;   et al. August 16, 2012

NOVEL DNA CLONING METHOD RELYING ON THE E.COLI recE/recT RECOMBINATION SYSTEM

Abstract

The invention relates to methods for cloning DNA molecules using recE/recT-mediated homologous recombination mechanism between at least two DNA molecules where one DNA molecule is a circular or linear DNA molecule and the second DNA molecule is a circular DNA molecule, and the second DNA molecule contains two regions with sequence homology to the first DNA molecule. Competent cells and vectors are also described.


Inventors: STEWART; Francis; (Leimen, DE) ; ZHANG; Youming; (Heidelberg, DE) ; BUCHHOLZ; Frank; (Bremen, DE)
Assignee: Europaisches Laboratorium fur Molekularbiologie (EMBL)
Heidelberg
DE

Family ID: 26145961
Appl. No.: 13/342621
Filed: January 3, 2012

Related U.S. Patent Documents

Application Number Filing Date Patent Number
12757129 Apr 9, 2010
13342621
10842534 May 11, 2004 7736851
12757129
10231013 Aug 30, 2002 6787316
10842534
09555510 Jun 5, 2000 6509156
PCT/EP1998/007945 Dec 7, 1998
10231013

Current U.S. Class: 435/446 ; 435/252.33; 435/471
Current CPC Class: C12N 15/10 20130101; C12N 15/902 20130101
Class at Publication: 435/446 ; 435/471; 435/252.33
International Class: C12N 15/01 20060101 C12N015/01; C12N 1/21 20060101 C12N001/21; C12N 15/70 20060101 C12N015/70

Foreign Application Data

Date Code Application Number
Dec 5, 1997 EP 97 121 462.2
Oct 5, 1998 EP 98 118 756.0

Claims



1. A genetically engineered prokaryotic cell comprising a homologous recombinant DNA molecule made by homologous recombination between a circular first molecule and a linear second DNA molecule, and prepared by a method comprising the following steps: a) providing a prokaryotic host cell capable of performing homologous recombination, wherein the host cell expresses recE and recT genes; b) contacting in said host cell said circular first DNA molecule which is capable of being replicated in said host cell, said first DNA molecule being the host cell chromosome, with said linear second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule and further comprising a DNA fragment to be integrated into the first DNA molecule, under conditions which favor homologous recombination between said first and second DNA molecules; wherein when said homologous recombination occurs, it is mediated by gene products of said recE and recT genes; and c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred, thereby obtaining the genetically engineered prokaryotic cell.

2. A genetically engineered prokaryotic cell comprising a homologous recombinant DNA molecule made by homologous recombination between a circular first molecule and a linear second DNA molecule and free of at least one marker gene, and prepared by a method comprising the following steps: a) providing a prokaryotic host cell capable of performing homologous recombination, wherein the host cell expresses recE and recT genes; b) contacting in said host cell said circular first DNA molecule which is capable of being replicated in said host cell, said first DNA molecule being the host cell chromosome, with said linear second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule and further comprising a DNA fragment to be integrated into the first DNA molecule, wherein the second DNA molecule contains said at least one marker gene placed between the two regions of sequence homology, under conditions which favor homologous recombination between said first and second DNA molecules, wherein when said homologous recombination occurs, it is mediated by gene products of said recE and recT genes; c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred, wherein homologous recombination is detected by expression of said at least one marker gene; and d) removing said at least one marker gene from a DNA molecule generated by the homologous recombination between the first and second DNA molecules in said host cell of step c), thereby obtaining the genetically engineered prokaryotic cell.

3. The prokaryotic cell according to claim 1 or 2, wherein the recE and recT genes are selected from .lamda.red.alpha. and red.beta. genes or from E. coli recE and recT genes.

4. The prokaryotic cell according to claim 1 or 2, wherein the host cell comprises at least one vector capable of expressing recE and/or recT genes.

5. The prokaryotic cell according to claim 1 or 2, wherein the expression of the recE and/or recT genes is under control of a regulatable promoter.

6. The prokaryotic cell according to claim 1 or 2, wherein the recE gene is selected from a nucleic acid molecule comprising (a) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAC) of SEQ ID NO.: 2, (b) the nucleic acid sequence from position 1320 (ATG) to 1998 (CGA) of SEQ ID NO.: 10, (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (a), (b) and/or (c).

7. The prokaryotic cell according to claim 1 or 2, wherein the recT gene is selected from a nucleic acid molecule comprising (a) the nucleic acid sequence from position 2155 (ATG) to 2961 (GAA) of SEQ ID NO.: 4, (b) the nucleic acid sequence from position 2086 (ATG) to 2868 (GCA) of SEQ ID NO.: 10, (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequences from (a), (b) and/or (c).

8. The prokaryotic cell according to claim 1 or 2, wherein the host cell is a gram-negative bacterial cell.

9. The prokaryotic cell according to claim 8 wherein the host cell is an Escherichia coli cell.

10. The prokaryotic cell according to claim 9 wherein the host cell is an Escherichia coli K12 strain.

11. The prokaryotic cell according to claim 1 or 2, wherein the host cell further is capable of expressing a recBC inhibitor gene.

12. The prokaryotic cell according to claim 11 wherein the host cell comprises a vector expressing the recBC inhibitor gene.

13. The prokaryotic cell according to claim 11 wherein the recBC inhibitor gene is selected from a nucleic acid molecule comprising (a) the nucleic acid sequence from position 3588 (ATG) to 4002 (GTA) of SEQ ID NO.: 10, (b) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (c) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (a) and/or (b).

14. The prokaryotic cell according to claim 1 or 2, wherein the regions of sequence homology are at least 15 nucleotides each.

15. The prokaryotic cell according to claim 1 or 2, wherein the second DNA molecule is obtained by an amplification reaction.

16. The prokaryotic cell according to claim 1 or 2, wherein the second DNA molecule is introduced into the host cells by transformation.

17. The prokaryotic cell according to claim 16 wherein the transformation method is electroporation.

18. The prokaryotic cell according to claim 1, wherein the second DNA molecule contains at least one marker gene placed between the two regions of sequence homology and wherein homologous recombination is detected by expression of said marker gene.

19. The prokaryotic cell according to claim 18, wherein said marker gene on the second DNA molecule is selected from antibiotic resistance genes, deficiency complementation genes and reporter genes.

20. The prokaryotic cell according to claim 2, wherein said marker gene on the second DNA molecule is selected from antibiotic resistance genes, deficiency complementation genes and reporter genes.

21. The prokaryotic cell according to claim 1 or 2, wherein the first DNA molecule contains at least one marker gene between the two regions of sequence homology and wherein homologous recombination is detected by lack of expression of said marker gene.

22. The prokaryotic cell according to claim 21 wherein said marker gene on the first DNA molecule is selected from genes which, under selected conditions, convey a toxic or bacteriostatic effect on the cell, and reporter genes.

23. The prokaryotic cell according to claim 1 or 2, wherein the first DNA molecule contains at least one target site for a site specific recombinase between the two regions of sequence homology and wherein homologous recombination is detected by removal of said target site.

24. The prokaryotic cell according to claim 1, wherein the chromosome of the prokaryotic cell obtained in step c) differs from the chromosome of the prokaryotic host cell of step a) by at least one mutation selected from the group consisting of point mutations, insertions, and deletions.

25. The prokaryotic cell according to claim 2, wherein the chromosome of the prokaryotic cell obtained in step d) differs from the chromosome of the prokaryotic host cell of step a) by at least one mutation selected from the group consisting of point mutations, insertions, and deletions.

26. A method for preparing a genetically engineered prokaryotic cell comprising the steps of: a) providing a prokaryotic host cell capable of performing homologous recombination, wherein the host cell expresses recE and recT genes; b) contacting in said host cell a circular first DNA molecule which is capable of being replicated in said host cell, said first DNA molecule being the host cell chromosome, with a linear second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule and further comprising a DNA fragment to be integrated into the first DNA molecule, under conditions which favor homologous recombination between said first and second DNA molecules, wherein when said homologous recombination occurs, it is mediated by gene products of said recE and recT genes; and c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred, thereby obtaining a genetically engineered prokaryotic cell.

27. The method according to claim 26 wherein the recE and recT genes are selected from .lamda.red.alpha. and red.beta. genes or from E. coli recE and recT genes.

28. The method according to claim 26 wherein the host cell is transformed with at least one vector capable of expressing recE and/or recT genes.

29. The method of claim 26 wherein the expression of the recE and/or recT genes is under control of a regulatable promoter.

30. The method according to claim 26 wherein the recE gene is selected from a nucleic acid molecule comprising (a) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAC) of SEQ ID NO.: 2, (b) the nucleic acid sequence from position 1320 (ATG) to 1998 (CGA) of SEQ ID NO.: 10, (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (a), (b) and/or (c).

31. The method according to claim 26 wherein the recT gene is selected from a nucleic acid molecule comprising (a) the nucleic acid sequence from position 2155 (ATG) to 2961 (GAA) of SEQ ID NO.: 4, (b) the nucleic acid sequence from position 2086 (ATG) to 2868 (GCA) of SEQ ID NO.: 10, (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequences from (a), (b) and/or (c).

32. The method according to claim 26 wherein the host cell is a gram-negative bacterial cell.

33. The method according to claim 32 wherein the host cell is an Escherichia coli cell.

34. The method according to claim 33 wherein the host cell is an Escherichia coli K12 strain.

35. The method according to claim 26 wherein the host cell further is capable of expressing a recBC inhibitor gene.

36. The method according to claim 35 wherein the host cell is transformed with a vector expressing the recBC inhibitor gene.

37. The method according to claim 35 wherein the recBC inhibitor gene is selected from a nucleic acid molecule comprising (a) the nucleic acid sequence from position 3588 (ATG) to 4002 (GTA) of SEQ ID NO.: 10, (b) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (c) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (a) and/or (b).

38. The method according to claim 26 wherein the regions of sequence homology are at least 15 nucleotides each.

39. The method according to claim 26 wherein the second DNA molecule is obtained by an amplification reaction.

40. The method according to claim 26 wherein the second DNA molecule is introduced into the host cells by transformation.

41. The method according to claim 40 wherein the transformation method is electroporation.

42. The method according to claim 26 wherein the second DNA molecule contains at least one marker gene placed between the two regions of sequence homology and wherein homologous recombination is detected by expression of said marker gene.

43. The method according to claim 42 wherein said marker gene on the second DNA molecule is selected from antibiotic resistance genes, deficiency complementation genes and reporter genes.

44. The method according to claim 43 wherein the first DNA molecule contains at least one marker gene between the two regions of sequence homology and wherein homologous recombination is detected by lack of expression of said marker gene.

45. The method of claim 44 wherein said marker gene on the first DNA molecule is selected from genes which, under selected conditions, convey a toxic or bacteriostatic effect on the cell, and reporter genes.

46. The method according to claim 26 wherein the first DNA molecule contains at least one target site for a site specific recombinase between the two regions of sequence homology and wherein homologous recombination is detected by removal of said target site.

47. The method according to claim 26 wherein the chromosome of the prokaryotic cell obtained in step c) differs from the chromosome of the prokaryotic host cell of step a) by at least one mutation selected from the group consisting of point mutations, insertions, and deletions.

48. The method of claim 42, further comprising the step of: d) removing said at least one marker gene from a DNA molecule generated by the homologous recombination between the first and second DNA molecules in said host cell of step c), thereby obtaining a genetically engineered prokaryotic cell.

49. The method according to claim 48, wherein the chromosome of the prokaryotic cell obtained in step d) differs from the chromosome of the prokaryotic host cell of step a) by at least one mutation selected from the group consisting of point mutations, insertions, and deletions.
Description



[0001] This application is a divisional of U.S. Ser. No. 12/757,129, filed Apr. 9, 2010 which is a divisional of 10/842,534 filed May 11, 2004, U.S. Pat. No. 7,736,851 issued Jun. 15, 2010 which is a divisional of 10/231,013 Aug. 30, 2002, now U.S. Pat. No. 6,787,316 issued Sep. 7, 2004, which is a divisional of 09/555,510 filed Jun. 5, 2000, now U.S. Pat. No. 6,509,156, issued Jan. 21, 2003, which is a 35 U.S.C. 371 National Phase Entry Application from PCT/EP1998/07945, filed Dec. 7, 1998, which claims the benefit of European Patent Application Nos. 98 118 756.0 filed on Oct. 5, 1998 and 97 121 462.2 filed on Dec. 5, 1997, the disclosures of which are incorporated herein in their entirety by reference.

[0002] The invention refers to a novel method for cloning DNA molecules using a homologous recombination mechanism between at least two DNA molecules. Further, novel reagent kits suitable for DNA cloning are provided.

[0003] Current methods for cloning foreign DNA in bacterial cells usually comprise the steps of providing a suitable bacterial vector, cleaving said vector with a restriction enzyme and in vitro-inserting a foreign DNA fragment in said vector. The resulting recombinant vectors are then used to transform bacteria. Although such cloning methods have been used successfully for about 20 years they suffer from several drawbacks. These drawbacks are, in particular, that the in vitro steps required for inserting foreign DNA in a vector are often very complicated and time-consuming, if no suitable restriction sites are available on the foreign DNA or the vector.

[0004] Furthermore, current methods usually rely on the presence of suitable restriction enzyme cleavage sites in the vector into which the foreign DNA fragment is placed. This imposes two, limitations on the final cloning product. First, the foreign DNA fragment can usually only be inserted into the vector at the position of such a restriction site or sites. Thus, the cloning product is limited by the disposition of suitable restriction sites and cloning into regions of the vector where there is no suitable restriction site, is difficult and often imprecise. Second, since restriction sites are typically 4 to 8 base pairs in length, they occur a multiple number of times as the size of the DNA molecules being used increases. This represents a practical limitation to the size of the DNA molecules that can be manipulated by most current cloning techniques. In particular, the larger sizes of DNA cloned into vectors such as cosmids, BACs, PACs and P1s are such that it is usually impractical to manipulate them directly by restriction enzyme based techniques. Therefore, there is a need for providing a new cloning method, from which the drawbacks of the prior art have at least partly been eliminated.

[0005] According to the present invention it was found that an efficient homologous recombination mechanism between two DNA molecules occurs at usable frequencies in a bacterial host cell which is capable of expressing the products of the recE and recT genes or functionally related genes such as the red.alpha. and red.beta. genes, or the phage P22 recombination system (Kolodner at al., Mol. Microbiol. 11 (1994) 23-30; Fenton, A. C. and Poteete, A. R., Virology 134 (1984) 148-160; Poteete, A. R. and Fenton, A. C., Virology 134 (1984) 161-167). This novel method of cloning DNA fragments is termed "ET cloning".

[0006] The identification and characterization of the E. coli RecE and RecT proteins is described Gillen et al. (J. Bacteriol. 145 (1981), 521-532) and Hall et al. (J. Bacteriol. 175 (1993), 277-287). Hall and Kolodner (Proc. Natl. Acad. Sci. <USA 91 (1994), 3205-3209) disclose in vitro homologous pairing and strand exchange of linear double-stranded DNA and homologous circular single-stranded DNA promoted by the RecT protein. Any references to the use of this method for the cloning of DNA molecules in cells cannot be found therein.

[0007] The recET pathway of genetic recombination in E. coli is known (Hall and Kolodner (1994), supra; Gillen et al. (1981), supra). This pathway requires the expression of two genes, recE and recT. The DNA sequence of these genes has been published (Hall et al., supra). The RecE protein is similar to bacteriophage proteins, such as .lamda. exo or .lamda. Red.alpha. (Gillen at al., J. Mol. Biol. 113 (1977), 27-41; Little, J. Biol. Chem. 242 (1.967), 679-686; Redding and Carter, J. Biol. Chem. 246 (1971), 2513-2518; Joseph and Kolodner, J. Biol. Chem. 258 (1983), 10418-10424). The RecT protein is similar to bacteriophage proteins, such as .lamda. .beta.-protein or .lamda. Red.beta. (Hall at al. (1993), supra; Muniyappa and Redding, J. Biol. Chem. 261 (1986), 7472-7478; Kmiec and Hollomon, J. Biol. Chem. 256 (1981), 12636-12639). The content of the above-cited documents is incorporated herein by reference.

[0008] Oliner et al. (Nucl. Acids Res. 21 (1993), 5192-5197) describe in vivo cloning of PCR products in. E. coli by intermolecular homologous recombination between a linear PCR product and a linearized plasmid vector. Other previous attempts to develop new cloning methods based on homologous recombination in prokaryotes, too, relied on the use of restriction enzymes to linearise the vector (Bubeck et al., Nucleic Acids Res. 21 (1993), 3601-3602; Oliver et al., Nucleic Acids Res. 21 (1993), 5192-5197; Degryse, Gene 170 (1996), 45-50) or on the host-specific recA-dependent recombination system (Hamilton et al., J. Bacteriol. 171 (1989), 4617-4622; Yang et al., Nature Biotech. 15 (1997), 859-865; Dabert and Smith, Genetics 145 (1997), 877-889). These methods are of very limited applicability and are hardly used in practice.

[0009] The novel method of cloning DNA according to the present invention does not require in vitro treatments with restriction enzymes or DNA ligases and is therefore fundamentally distinct from the standard methodologies of DNA cloning. The method relies on a pathway of homologous recombination in E. coli involving the recE and recT gene products, or the red.alpha. and red.beta. gene products, or functionally equivalent gene products. The method covalently combines one preferably linear and preferably extrachromosomal DNA fragment, the DNA fragment to be cloned, with one second preferably circular DNA vector molecule, either an episome or the endogenous host chromosome or chromosomes. It is therefore distinct from previous descriptions of cloning in E. coli by homologous recombination which either rely on the use of two linear DNA fragments or different recombination pathways.

[0010] The present invention provides a flexible way to use homologous recombination to engineer large DNA molecules including an intact >76 kb plasmid and the E. coli chromosome. Thus, there is practically no limitation of target choice either according to size or site. Therefore, any recipient DNA in a host cell, from high copy plasmid to the genome, is amenable to precise alteration. In addition to engineering large DNA molecules, the invention outlines new, restriction enzyme-independent approaches to DNA design. For example, deletions between any two chosen base pairs in a target episome can be made by choice of oligonucleotide homology arms. Similarly, chosen DNA sequences can be inserted at a chosen base pair to create, for example, altered protein reading frames. Concerted combinations of insertions and deletions, as well as point mutations, are also possible. The application of these strategies is particularly relevant to complex or difficult DNA constructions, for example, those intended for homologous recombinations in eukaryotic cells, e.g. mouse embryonic stem cells. Further, the present invention provides a simple way to position site specific recombination target sites exactly where desired. This will simplify applications of site specific recombination in other living systems, such as plants and mice.

[0011] A subject matter of the present invention is a method for cloning DNA molecules in cells comprising the steps: [0012] a) providing a host cell capable of performing homologous recombination, [0013] b) contacting in said host cell a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and [0014] c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred.

[0015] In the method of the present invention the homologous recombination preferably occurs via the recET mechanism, i.e. the homologous recombination is mediated by the gene products of the recE and the recT genes which are preferably selected from the E. coli genes recE and recT or functionally related genes such as the phage .lamda. red.alpha. and red.beta. genes.

[0016] The host cell suitable for the method of the present invention preferably is a bacterial cell, e.g. a gram-negative bacterial cell. More preferably, the host cell is an enterobacterial cell, such as Salmonella, Klebsiella or Escherichia. Most preferably the host cell is an Escherichia coli cell. It should be noted, however, that the cloning method of the present invention is also suitable for eukaryotic cells, such as fungi, plant or animal cells.

[0017] Preferably, the host cell used for homologous recombination and propagation of the cloned. DNA can be any cell, e.g. a bacterial strain in which the products of the recE and recT, or red.alpha. and red.beta., genes are expressed. The host cell may comprise the recE and recT genes located on the host cell chromosome or on non-chromosomal DNA, preferably on a vector, e.g. a plasmid. In a preferred case, the RecE and RecT, or Red.alpha. and Red.beta., gene products are expressed from two different regulatable promoters, such as the arabinose-inducible BAD promoter or the lac promoter or from non-regulatable promoters. Alternatively, the recE and recT, or red.alpha. and red.beta., genes are expressed on a polycistronic mRNA from a single regulatable or non-regulatable promoter. Preferably the expression is controlled by regulatable promoters.

[0018] Especially preferred is also an embodiment, wherein the recE or red.alpha. gene is expressed by a regulatable promoter. Thus, the recombinogenic potential of the system is only elicited when required and, at other times, possible undesired recombination reactions are limited. The recT or red.beta. gene, on the other hand, is preferably overexpressed with respect to recE or red.alpha.. This may be accomplished by using a strong constitutive promoter, e.g. the EM7 promoter and/or by using a higher copy number of recT, or red.beta., versus recE, or red.alpha., genes.

[0019] For the purpose of the present invention any recE and recT genes are suitable insofar as they allow a homologous recombination of first and second DNA molecules with sufficient efficiency to give rise to recombination products in more than 1 in 10.sup.9 cells transfected with DNA. The recE and recT genes may be derived from any bacterial strain or from bacteriophages or may be mutants and variants thereof. Preferred are recE and recT genes which are derived from E. coli or from E. coli bacteriophages, such as the red.alpha. and red.beta. genes from lambdoid phages, e.g. bacteriophage .lamda..

[0020] More preferably, the recE or red.alpha. gene is selected from a nucleic acid molecule comprising

(a) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAO) as depicted in FIG. 7B, (b) the nucleic acid sequence from position 1320 (ATG) to 1998(CGA) as depicted in FIG. 14B, (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (a), (b) and/or (c).

[0021] More preferably, the recT or red.beta. gene is selected from a nucleic acid molecule comprising

(a) the nucleic acid sequence from position 21 55 (ATG) to 2961 (GAA) as depicted in FIG. 7B, (b) the nucleic acid sequence from position 2086 (ATG) to 2868 (GCA) as depicted in FIG. 14B, (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequences from (a), (b) and/or (c).

[0022] It should be noted that the present invention also encompasses mutants and variants of the given sequences, e.g. naturally occurring mutants and variants or mutants and variants obtained by genetic engineering. Further it should be noted that the recE gene depicted in FIG. 7B is an already truncated gene encoding amino acids 588-866 of the native protein. Mutants and variants preferably have a nucleotide sequence identity of at least 60%, preferably of at least 70% and more preferably of at least 80% of the recE and recT sequences depicted in FIGS. 7B and 13B, and of the redo and red.beta. sequences depicted in FIG. 14B.

[0023] According to the present invention hybridization under stringent conditions preferably is defined according to Sambrook et al. (1989), infra, and comprises a detectable hybridization signal after washing for 30 min in 0.1.times.SSC, 0.5% SDS at 55.degree. C., preferably at 62.degree. C. and more preferably at 68.degree. C.

[0024] In a preferred case the recE and recT genes are derived from the corresponding endogenous genes present in the E. coli K12 strain and its derivatives or from bacteriophages. In particular, strains that carry the sbcA mutation are suitable. Examples of such strains are JC8679 and JC 9604 (Gillen et al. (1981), supra). Alternatively, the corresponding genes may also be obtained from other coliphages such as lambdoid phages or phage P22.

[0025] The genotype of JC 8679 and JC 9604 is Sex (Hfr, F+, F-, or F'): F-.JC 8679 comprises the mutations: recBC 21, recC 22, sbcA 23, thr-1, ara-14, leu B 6, DE (gpt-proA) 62, lacY1, tsx-33, gluV44 (AS), galK2 (Oc), LAM-, his-60, relA 1, rps L31 (strR), xyl A5, mtl-1, argE3 (Oc) and thi-1. JC 9604 comprises the same mutations and further the mutation recA 56.

[0026] Further, it should be noted that the recE and recT, or red.alpha. and red.beta., genes can be isolated from a first donor source, e.g. a donor bacterial cell and transformed into a second receptor source, e.g. a receptor bacterial or eukaryotic cell in which they are expressed by recombinant DNA means.

[0027] In one embodiment of the invention, the host cell used is a bacterial strain having an sbcA mutation, e.g. one of E. coli strains JC 8679 and JC 9604 mentioned above. However, the method of the invention is not limited to host cells having an sbcA mutation or analogous cells. Surprisingly, it has been found that the cloning method of the invention also works in cells without sbcA mutation, whether recBC+ or recBC-, e.g. also in prokaryotic recBC+ host cells, e.g. in E. coli recBC+ cells. In that case preferably those host cells are used in which the product of a recBC type exonuclease inhibitor gene is expressed. Preferably, the exonuclease inhibitor is capable of inhibiting the host recBC system or an equivalent thereof. A suitable example of such exonuclease inhibitor gene is the .lamda. red.gamma. gene (Murphy, J. Bacteriol. 173 (1991), 5808-5821) and functional equivalents thereof, respectively, which, for example, can be obtained from other coliphages such as from phage P22 (Murphy, J. Biol. Chem. 269 (1994), 22507-22516).

[0028] More preferably, the exonuclease inhibitor gene is selected from a nucleic acid molecule comprising

(a) the nucleic acid sequence from position 3588 (ATG) to 4002 (GTA) as depicted in FIG. 14A, (b) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (c) a nucleic acid sequence which hybridizes under stringent conditions (as defined above) with the nucleic acid sequence from (a) and/or (b).

[0029] Surprisingly, it has been found that the expression of an exonuclease inhibitor gene in both recBC+ and recBC- strains leads to significant improvement of cloning efficiency.

[0030] The cloning method according to the present invention employs a homologous recombination between a first DNA molecule and a second DNA molecule. The first DNA molecule can be any DNA molecule that carries an origin of replication which is operative in the host cell, e.g. an E. coli replication origin. Further, the first DNA molecule is present in a form which is capable of being replicated in the host cell. The first DNA molecule, i.e. the vector, can be any extrachromosomal DNA molecule containing an origin of replication which is operative in said host cell, e.g. a plasmid including single, low, medium or high copy plasmids or other extrachromosomal circular DNA molecules based on cosmid, P1, BAC or PAC vector technology. Examples of such vectors are described, for example, by Sambrook et al. (Molecular Cloning, Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press) and Ioannou et al. (Nature Genet. 6 (1994), 84-89) or references cited therein. The first DNA molecule can also be a host cell chromosome, particularly the E. coli chromosome. Preferably, the first DNA molecule is a double-stranded DNA molecule.

[0031] The second DNA molecule is preferably a linear DNA molecule and comprises at least two regions of sequence homology, preferably of sequence identity to regions on the first. DNA molecule. These homology or identity regions are preferably at least 15 nucleotides each, more preferably at least 20 nucleotides and, most preferably, at least 30 nucleotides each. Especially good results were obtained when using sequence homology regions having a length of about 40 or more nucleotides, e.g. 60 or more nucleotides. The two sequence homology regions can be located on the linear DNA fragment so that one is at one end and the other is at the other end, however they may also be located internally. Preferably, also the second DNA molecule is a double-stranded DNA molecule.

[0032] The two sequence homology regions are chosen according to the experimental design. There are no limitations on which regions of the first DNA molecule can be chosen for the two sequence homology regions located on the second DNA molecule, except that the homologous recombination event cannot delete the origin of replication of the first DNA molecule. The sequence homology regions can be interrupted by non-identical sequence regions as long as sufficient sequence homology is retained for the homologous recombination reaction. By using sequence homology arms having non-identical sequence regions compared to the target site mutations such as substitutions, e.g. point mutations, insertions and/or deletions may be introduced into the target site by ET cloning.

[0033] The second foreign DNA molecule which is to be cloned in the bacterial cell may be derived from any source. For example, the second DNA molecule may be synthesized by a nucleic acid amplification reaction such as a PCR where both of the DNA oligonucleotides used to prime the amplification contain in addition to sequences at the 3'-ends that serve as a primer for the amplification, one or the other of the two homology regions. Using oligonucleotides of this design, the DNA product of the amplification can be any DNA sequence suitable for amplification and will additionally have a sequence homology region at each end.

[0034] A specific example of the generation of the second DNA molecule is the amplification of a gene that serves to convey a phenotypic difference to the bacterial host cells, in particular, antibiotic resistance. A simple variation of this procedure involves the use of oligonucleotides that include other sequences in addition to the PCR primer sequence and the sequence homology region. A further simple variation is the use of more than two amplification primers to generate the amplification product. A further simple variation is the use of more than one amplification reaction to generate the amplification product. A further variation is the use of DNA fragments obtained by methods other than PCR, for example, by endonuclease or restriction enzyme cleavage to linearize fragments from any source of DNA.

[0035] It should be noted that the second DNA molecule is not necessarily a single species of DNA molecule. It is of course possible to use a heterogenous population of second DNA molecules, e.g. to generate a DNA library, such as a genomic or cDNA library.

[0036] The method of the present invention may comprise the contacting of the first and second DNA molecules in vivo. In one embodiment of the present invention the second DNA fragment is transformed into a bacterial strain that already harbors the first vector DNA molecule. In a different embodiment the second DNA molecule and the first DNA molecule are mixed together in vitro before co-transformation in the bacterial host cell. These two embodiments of the present invention are schematically depicted in FIG. 1. The method of transformation can be any method known in the art (e.g. Sambrook et al. supra). The preferred method of transformation or co-transformation, however, is electroporation.

[0037] After contacting the first and second DNA molecules under conditions which favour homologous recombination between first and second DNA molecules via the ET cloning mechanism a host cell is selected, in which homologous recombination between said first and second. DNA molecules has occurred. This selection procedure can be carried out by several different methods. In the following three preferred selection methods are depicted in FIG. 2 and described in detail below.

[0038] In a first selection method a second DNA fragment is employed which carries a gene for a marker placed between the two regions of sequence homology wherein homologous recombination is detectable by expression of the marker gene. The marker gene may be a gene for a phenotypic marker which is not expressed in the host or from the first DNA molecule. Upon recombination by ET cloning, the change in phenotype of the host strain conveyed by the stable acquisition of the second DNA fragment identifies the ET cloning product.

[0039] In a preferred case, the phenotypic marker is a gene that conveys resistance to an antibiotic, in particular, genes that convey resistance to kanamycin, ampillicin, chloramphenicol, tetracyclin or any other substance that shows bacteriocidal or bacteriostatic effects on the bacterial strain employed.

[0040] A simple variation is the use of a gene that complements a deficiency present within the bacterial host strain employed. For example, the host strain may be mutated so that it is incapable of growth without a metabolic supplement. In the absence of this supplement, a gene on the second DNA fragment can complement the mutational defect thus permitting growth. Only those cells which contain the episome carrying the intended DNA rearrangement caused by the ET cloning step will grow.

[0041] In another example, the host strain carries a phenotypic marker gene which is mutated so that one of its codons is a stop codon that truncates the open reading frame. Expression of the full length protein from this phenotypic marker gene requires the introduction of a suppressor tRNA gene which, once expressed, recognizes the stop codon and permits translation of the full open reading frame. The suppressor tRNA gene is introduced by the ET cloning step and successful recombinants identified by selection for, or identification of, the expression of the phenotypic marker gene. In these cases, only those cells which contain the intended DNA rearrangement caused by the ET cloning step will grow.

[0042] A further simple variation is the use of a reporter gene that conveys a readily detectable change in colony colour or morphology. In a preferred case, the green fluorescence protein (GFP) can be used and colonies carrying the ET cloning product identified by the fluorescence emissions of GFP. In another preferred case, the lacZ gene can be used and colonies carrying the ET cloning product identified by a blue colony colour when X-gal is added to the culture medium.

[0043] In a second selection method the insertion of the second DNA fragment into the first DNA molecule by ET cloning alters the expression of a marker present on the first DNA molecule. In this embodiment the first DNA molecule contains at least one marker gene between the two regions of sequence homology and homologous recombination may be detected by an altered expression, e.g. lack of expression of the marker gene.

[0044] In a preferred application, the marker present on the first DNA molecule is a counter-selectable gene product, such as the sacB, ccdB or tetracycline-resistance genes. In these cases, bacterial cells that carry the first DNA molecule unmodified by the ET cloning step after transformation with the second DNA fragment, or co-transformation with the second DNA fragment and the first DNA molecule, are plated onto a medium so the expression of the counter-selectable marker conveys a toxic or bacteriostatic effect on the host. Only those bacterial cells which contain the first DNA molecule carrying the intended DNA rearrangement caused by the ET cloning step will grow.

[0045] In another preferred application, the first DNA molecule carries a reporter gene that conveys a readily detectable change in colony colour or morphology. In a preferred case, the green fluorescence protein (GFP) can be present on the first DNA molecule and colonies carrying the first DNA molecule with or without the ET cloning product can be distinguished by differences in the fluorescence emissions of GFP. In another preferred case, the lacZ gene can be present on the first DNA molecule and colonies carrying the first DNA molecule with or without the ET cloning product identified by a blue or white colony colour when X-gal is added to the culture medium.

[0046] In a third selection method the integration of the second DNA fragment into the first DNA molecule by ET cloning removes a target site for a site specific recombinase, termed here an RT (for recombinase target) present on the first DNA molecule between the two regions of sequence-homology. A homologous recombination event may be detected by removal of the target site.

[0047] In the absence of the ET cloning product, the RT is available for use by the corresponding site specific recombinase. The difference between the presence or not of this. RT is the basis for selection of the ET cloning product. In the presence of this RT and the corresponding site specific recombinase, the site specific recombinase mediates recombination at this RT and changes the phenotype of the host so that it is either not able to grow or presents a readily observable phenotype. In the absence of this RT, the corresponding site specific recombinase is not able to mediate recombination.

[0048] In a preferred case, the first DNA molecule to which the second DNA fragment is directed, contains two RTs, one of which is adjacent to, but not part of, an antibiotic resistance gene. The second DNA fragment is directed, by design, to remove this RT. Upon exposure to the corresponding site specific recombinase, those, first DNA molecules that do not carry the ET cloning product will be subject to a site specific recombination reaction between the RTs that remove the antibiotic resistance gene and therefore the first DNA molecule fails to convey resistance to the corresponding antibiotic. Only those first DNA molecules that contain the ET cloning product, or have failed to be site specifically recombined for some other reason, will convey resistance to the antibiotic.

[0049] In another preferred case, the RT to be removed by ET cloning of the second DNA fragment is adjacent to a gene that complements a deficiency present within the host strain employed. In another preferred case, the RT to be removed by ET cloning of the second DNA fragment is adjacent to a reporter gene that conveys a readily detectable change in colony colour or morphology.

[0050] In another preferred case, the RT to be removed by ET cloning of the second DNA fragment is anywhere on a first episomal DNA molecule and the episome carries an origin of replication incompatible with survival of the bacterial host cell if it is integrated into the host genome. In this case the host genome carries a second RT, which may or may not be a mutated RT so that the corresponding site specific recombinase can integrate the episome, via its RT, into the RT sited in the host genome. Other preferred. RTs include RTs for site specific recombinases of the resolvase/transposase class. RTs include those described from existing examples of site specific recombination as well as natural or mutated variations thereof.

[0051] The preferred site specific recombinases include Cre, FLP, Kw or any site specific recombinase of the integrase class. Other preferred site specific recombinases include site specific recombinases of the resolvase/transposase class.

[0052] There are no limitations on the method of expression of the site specific recombinase in the host cell. In a preferred method, the expression of the site specific recombinase is regulated so that expression can be induced and quenched according to the optimisation of the ET cloning efficiency. In this case, the site specific recombinase gene can be either integrated into the host genome or carried on an episome. In another preferred case, the site specific recombinase is expressed from an episome that carries a conditional origin of replication so that it can be eliminated from the host cell.

[0053] In another preferred case, at least two of the above three selection methods are combined. A particularly preferred case involves a two-step use of the first selection method above, followed by use of the second selection method. This combined use requires, most simply, that the DNA fragment to be cloned includes a gene, or genes that permits the identification, in the first step, of correct ET cloning products by the acquisition of a phenotypic change. In a second step, expression of the gene or genes introduced in the first step is altered so that a second round of ET cloning products can be identified. In a preferred example, the gene employed is the tetracycline resistance gene and the first step ET cloning products are identified by the acquisition of tetracycline resistance. In the second step, loss of expression of the tetracycline gene is identified by loss of sensitivity to nickel chloride, fusaric acid or any other agent that is toxic to the host cell when the tetracycline gene is expressed. This two-step procedure permits the identification of ET cloning products by first the integration of a gene that conveys a phenotypic change on the host, and second by the loss of a related phenotypic change, most simply by removal of some of the DNA sequences integrated in the first step. Thereby the genes used to identify ET cloning products can be inserted and then removed to leave ET cloning products that are free of these genes.

[0054] In a further embodiment of the present invention the ET cloning may also be used for a recombination method comprising the steps of

a) providing a source of RecE and RecT, or Red.alpha. and Red.beta., proteins, b) contacting a first DNA molecule which is capable of being replicated in a suitable host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and c) selecting DNA molecules in which a homologous recombination between said first and second DNA molecules has occurred.

[0055] The source of RecE and RecT, or Red.alpha. and Red.beta., proteins may be either purified or partially purified RecE and RecT, or Red.alpha. and Red.beta., proteins or cell extracts comprising RecE and RecT, or Red.alpha. and Red.beta., proteins.

[0056] The homologous recombination event in this embodiment may occur in vitro, e.g. when providing a cell extract containing further components required for homologous recombination. The homologous recombination event, however, may also occur in vivo, e.g. by introducing RecE and RecT, or Red.alpha. and Red.beta., proteins or the extract in a host cell (which may be recET positive or not, or red.alpha..beta. positive or not) and contacting the DNA molecules in the host cell. When the recombination occurs in vitro the selection of DNA molecules may be accomplished by transforming the recombination mixture in a suitable host cell and selecting for positive clones as described above. When the recombination occurs in vivo the selection methods as described above may directly be applied.

[0057] A further subject matter of the invention is the use of cells, preferably bacterial cells, most preferably, E. coli cells capable of expressing the recE and recT, or red.alpha. and red.beta., genes as a host cell for a cloning method involving homologous recombination.

[0058] Still a further subject matter of the invention is a vector system capable of expressing recE and recT, or red.alpha. and red.beta., genes in a host cell and its use for a cloning method involving homologous recombination. Preferably, the vector system is also capable of expressing an exonuclease inhibitor gene as defined above, e.g. the .lamda. red.gamma. gene. The vector system may comprise at least one vector. The recE and recT, or red.alpha. and red.beta., genes are preferably located on a single vector and more preferably under control of a regulatable promoter which may be the same for both genes or a single promoter for each gene. Especially preferred is a vector system which is capable of overexpressing the recT, or red.beta., gene versus the recE, or red.alpha., gene.

[0059] Still a further subject matter of the invention is the use of a source of RecE and RecT, or Red.alpha. and Red.beta., proteins for a cloning method involving homologous recombination.

[0060] A still further subject matter of the invention is a reagent kit for cloning comprising [0061] (a) a host cell, preferably a bacterial host cell, [0062] (b) means of expressing recE and recT, or red.alpha. and red.beta., genes in said host cell, e.g. comprising a vector system, and [0063] (c) a recipient cloning vehicle, e.g. a vector, capable of being replicated in said cell.

[0064] On the one hand, the recipient cloning vehicle which corresponds to the first DNA molecule of the process of the invention can already be present in the bacterial cell. On the other hand, it can be present separated from the bacterial cell.

[0065] In a further embodiment the reagent kit comprises

(a) a source for RecE and RecT, or Red.alpha. and Red.beta., proteins and (b) a recipient cloning vehicle capable of being propagated in a host cell and (c) optionally a host cell suitable for propagating said recipient cloning vehicle.

[0066] The reagent kit furthermore contains, preferably, means for expressing a site specific recombinase in said host cell, in particular, when the recipient ET cloning product contains at least one site specific recombinase target site. Moreover, the reagent kit can also contain DNA molecules suitable for use as a source of linear DNA fragments used for ET cloning, preferably by serving as templates for PCR generation of the linear fragment, also as specifically designed DNA vectors from which the linear DNA fragment is released by restriction enzyme cleavage, or as prepared linear fragments included in the kit for use as positive controls or other tasks. Moreover, the reagent kit can also contain nucleic acid amplification primers comprising a region of homology to said vector. Preferably, this region of homology is located at the 5'-end of the nucleic acid amplification primer.

[0067] The invention is further illustrated by the following Sequence listings, Figures and Examples. [0068] SEQ ID NO. 1: shows the nucleic acid sequence of the Plasmid pBAD24-rec ET (FIG. 7). [0069] SEQ ID NOs 2/3: show the nucleic acid and amino acid sequences of the truncated recE gene (t-recE) present on pBAD24-recET at positions 1320-2162. [0070] SEQ ID NOs 4/5: show the nucleic acid and amino acid sequences of the recT gene present on pBAD24-recET at position 2155-2972. [0071] SEQ ID NOs 6/7: show the nucleic acid and amino acid sequences of the araC gene present on the complementary stand to the one shown of pBAD24-recET at positions 974-996. [0072] SEQ ID NOs 8/9: show the nucleic acid an amino acid sequences of the bla gene present on pBAD24-recET at positions 3493-4353. [0073] SEQ ID NO 10: shows the nucleic acid sequence of the plasmid pBAD-ET.gamma. (FIG. 13): [0074] SEQ ID No 11: shows the nucleic acid sequence of the plasmid pBAD-.alpha..beta..gamma. (FIG. 14) as well as the coding regions for the genes red.alpha. (1320-200), red.beta. (2086-2871) and red.gamma. (3403-3819). [0075] SEQ ID NOs 12-14: show the amino acid sequences of the Red.alpha., Red.beta. and Red.gamma. proteins, respectively. The red.gamma. sequence is present on each of pBAD-ET.gamma. (FIG. 13) and pBAD-.alpha..beta..gamma. (FIG. 14).

FIG. 1

[0076] A preferred method for ET cloning is shown by diagram. The linear DNA fragment to be cloned is synthesized by PCR using oligonucleotide primers that contain a left homology arm chosen to match sequences in the recipient episome and a sequence for priming in the PCR reaction, and a right homology arm chosen to match another sequence in the recipient episome and a sequence for priming in the PCR reaction. The product of the PCR reaction, here a selectable marker gene (sm1), is consequently flanked by the left and right homology arms and can be mixed together in vitro with the episome before co-transformation, or transformed into a host cell harboring the target episome. The host cell contains the products of the recE and recT genes. ET cloning products are identified by the combination of two selectable markers, sm1 and sm2 on the recipient episome.

FIG. 2

[0077] Three ways to identify ET cloning products are depicted. The first, (on the left of the figure), shows the acquisition, by ET cloning, of a gene that conveys a phenotypic difference to the host, here a selectable marker gene (sm). The second (in the centre of the figure) shows the loss, by ET cloning, of a gene that conveys a phenotypic difference to the host, here a counter selectable marker gene (counter-sm). The third shows the loss of a target site (RT, shown as triangles on the circular episome) for a site specific recombinase (SSR), by ET cloning. In this case, the correct ET cloning product deletes one of the target sites required by the SSR to delete a selectable marker gene (sm). The failure of the SSR to delete the sm gene identifies the correct ET cloning product.

FIG. 3.

[0078] A simple example of ET cloning is presented.

(a) Top panel--PCR products (left lane) synthesized from oligonucleotides designed as described in FIG. 1 to amplify by PCR a kanamycin resistance gene and to be flanked by homology arms present in the recipient vector, were mixed in vitro with the recipient vector (2nd lane) and cotransformed into a recET+E. coli host. The recipient vector carried an ampillicin resistance gene. (b) Transformation of the sbcA E. coli strain JC9604 with either the PCR product alone (0.2 .mu.g) or the vector alone (0.3 .mu.g) did not convey resistance to double selection with ampicillin and kanamycin (amp+kan), however cotransformation of both the FOR product and the vector produced double resistant colonies. More than 95% of these colonies contained the correct ET cloning product where the kanamycin gene had precisely integrated into the recipient vector according to the choice of homology arms. The two lanes on the right of (a) show Pvu II restriction enzyme digestion of the recipient vector before and after ET cloning. (c) As for b, except that six PCR products (0.2 .mu.g each) were cotransformed with pSVpaZ11 (0.3 .mu.g each) into JC9604 and plated onto Amp+Kan plates or Amp plates. Results are plotted as Amp+Kan-resistant colonies, representing recombination products, divided by Amp-resistant colonies, representing the plasmid transformation efficiency of the competent cell preparation, .times.10.sup.6. The PCR products were equivalent to the a-b PCR product except that homology arm lengths were varied. Results are from five experiments that used the same batches of competent cells and DNAs. Error bars represent standard deviation. (d) Eight products flanked by 50 by homology arms were cotransformed with pSVpaZ11 into JC9604. All eight PCR products contained the same left homology arm and amplified neo gene. The right homology arms were chosen from the pSVpaZ11 sequence to be adjacent to (O), or at increasing distances (7-3100 bp), from the left. Results are from four experiments.

FIG. 4

[0079] ET cloning in an approximately 100 kb P1 vector to exchange the selectable marker.

[0080] A P1 clone which uses a kanamycin resistance gene as selectable marker and which contains at least 70 kb of the mouse Hox a gene cluster was used. Before ET cloning, this episome conveys kanamycin resistance (top panel, upper left) to its host E. coli which are ampillicin sensitive (top panel, upper right). A linear DNA fragment designed to replace the kanamycin resistance gene with an ampillicin resistance gene was made by PCR as outlined in FIG. 1 and transformed into E. coli host cells in which the recipient Hox a/P1 vector was resident. ET cloning resulted in the deletion of the kanamycin resistance gene, and restoration of kanamycin sensitivity (top panel, lower left) and the acquisition of ampillicin resistance (top panel, lower right). Precise DNA recombination was verified by restriction digestion and Southern blotting analyses of isolated DNA before and after ET cloning (lower panel).

FIG. 5

ET Cloning to Remove a Counter Selectable Marker

[0081] A PCR fragment (upper panel, left, third lane) made as outlined in FIGS. 1 and 2 to contain the kanamycin resistance gene was directed by its chosen homology arms to delete the counter selectable ccdB gene present in the vector, pZero-2.1. The PCR product and the pZero vector were mixed in vitro (upper panel, left, 1st lane) before cotransformation into a recE/recT+E. coli host. Transformation of pZero-2.1 alone and plating onto kanamycin selection medium resulted in little colony growth (lower panel, left). Cotransformation of pZero-2.1 and the PCR product presented ET cloning products (lower panel, right) which showed the intended molecular event as visualized by Pvu II digestion (upper panel, right).

FIG. 6

[0082] ET cloning mediated by inducible expression of recE and recT from an episome.

[0083] RecE/RecT mediate homologous recombination between linear and circular DNA molecules. (a) The plasmid pBAD24-recET was transformed into E. coli JC5547, and then batches of competent cells were prepared after induction of RecE/RecT expression by addition of L-arabinose for the times indicated before harvesting. A PCR product, made using oligonucleotides e and f to contain the chloramphenicol resistance gene (cm) of pMAK705 and 50 bp homology arms chosen to flank the ampicillin resistance gene (bla) of pBAD24-recET, was then transformed and recombinants identified on chloramphenicol plates. (b) Arabinose was added to cultures of pBAD24-recETtransformed JC5547 for different times immediately before harvesting for competent cell preparation. Total protein expression was analyzed by SOS-PAGE and Coomassie blue staining. (c) The number of chloramphenicol resistant colonies per .mu.g of PCR product was normalized against a control for transformation efficiency, determined by including 5 .mu.g pZero2.1, conveying kanamycin resistance, in the transformation and plating an aliquot onto Kan plates.

FIG. 7A

[0084] The plasmid pBAD24-recET is shown by diagram. The plasmid contains the genes recE (in a truncated form) and recT under control of the inducible BAD promoter (P.sub.BAD). The plasmid further contains an ampillicin resistance gene (Amp.sup.r) and an araC gene.

FIG. 7B

[0085] The nucleic acid sequence and the protein coding portions of pBAD24-recET are depicted.

FIG. 8

[0086] Manipulation of a large E. coli episome by multiple recombination steps a Scheme of the recombination reactions. A P1 clone of the Mouse Hoxa complex, resident in JC9604, was modified by recombination with PCR products that contained the neo gene and two Hp recombination targets (FRTs). The two PCR products were identical except that one was flanked by g and h homology arms (insertion), and the other was flanked by i and h homology arms (deletion). In a second step, the neo gene was removed by Flp recombination between the FRTs by transient transformation of a Flp expression plasmid based on the pSC101 temperature-sensitive origin (ts ori). b Upper panel; ethidium bromide stained agarose gel showing EcoR1 digestions of P1 DNA preparations from three independent colonies for each step. Middle panel; a Southern blot of the upper panel hybridized with a neo gene probe. Lower panel; a Southern blot of the upper panel hybridized with a Hoxa3 probe to visualize the site of recombination. Lanes 1, the original Hoxa3 P1 clone grown in E. coli strain NS3145. Lanes 2, replacement of the Tn903 kanamycin resistance gene resident in the P1 vector with an ampicillin resistance gene increased the 8.1 kb band (lanes 1), to 9.0 kb. Lanes 3, insertion of the Tn5-neo gene with g-h homology arms upstream of Hoxa3, increased the 6.7 kb band (lanes 1,2) to 9.0 kb. Lanes 4, Flp recombinase deleted the g-h neo gene reducing the 9.0 kb band (lanes 3) back to 6.7 kb. Lanes 5, deletion of 6 kb of Hoxa3-4 intergenic DNA by replacement with the 1-h neo gene, decreased the 6.7 kb band (lanes 2) to 4.5 kb. Lanes 6, Flp recombinase deleted the i-h neo gene reducing the 4.5 kb band to 2.3 kb.

FIG. 9

[0087] Manipulation of the E. coli chromosome. A Scheme of the recombination reactions. The endogenous lacZ gene of JC9604 at 7.8.degree. of the E. coli chromosome, shown in expanded form with relevant Ava I sites and coordinates, was targeted by a PCR fragment that contained the neo gene flanked by homology arms j and k, and loxP sites, as depicted. Integration of the neo gene removed most of the lacZ gene including an Ava I site to alter the 1443 and 3027 by bands into a 3277 by band. In a second step, the neo gene was removed by Cre recombination between the loxPs by transient transformation of a Cre expression plasmid based on the pSC101 temperature-sensitive origin (ts ori). Removal of the neo gene by Cre recombinase reduces the 3277 band to 2111 bp. b .beta.-galactosidase expression evaluated by streaking colonies on X-Gal plates'. The top row of three streaks show .beta.-galactosidase expression in the host JC9604 strain (w.t.), the lower three rows (Km) show 24 independent primary colonies, 20 of which display a loss of .beta.-galactosidase expression indicative of the intended recombination event. c Southern analysis of E. coli chromosomal DNA digested with Ava I using a random primed probe made from the entire lacZ coding region; lanes 1,2, w.t.; lanes 3-6, four independent white colonies after integration of the j-k neo gene; lanes 7-10; the same four colonies after transient transformation with the Cre expression plasmid.

FIG. 10

[0088] Two rounds of ET cloning to introduce a point mutation. a Scheme of the recombination reactions. The lacZ gene of pSVpaX1 was disrupted in JC9604lacZ, a strain made by the experiment of FIG. 9 to ablate endogenous lacZ expression and remove competitive sequences, by a sacB-neo gene cassette, synthesized by PCR to pIB279 and flanked by I and m homology arms. The recombinants, termed pSV-sacB-neo, were selected on Amp+Kan plates. The lacZ gene of pSV-sacB-neo was then repaired by a PCR fragment made from the intact lacZ gene using l* and m* homology arms. The m* homology arm included a silent C to G change that created a BamH1 site. The recombinants, termed pSVpaX1', were identified by counter selection against the sacB gene using 7% sucrose. b .beta.-galactosidase expression from pSVpaX1 was disrupted in pSV-sacB-neo and restored in pSVpaX1 Expression was analyzed on X-gal plates. Three independent colonies of each pSV-sacB-neo and pSVpaX1* are shown. c Ethidium bromide stained agarose gels of BamH1 digested DNA prepared from independent colonies taken after counter selection with sucrose. All .beta.-galactosidase expressing colonies (blue) contained the introduced BamH1 restriction site (upper panel). All white colonies displayed large rearrangements and no product carried the diagnostic 1.5 kb BamH1 restriction fragment (lower panel).

FIG. 11

[0089] Transference of ET cloning into a recBC+host to modify a large episome. a Scheme of the plasmid, pBAD-ET.gamma., which carries the mobile ET system, and the strategy employed to target the Hoxa P1 episome. pBAD-ET.gamma. is based on pBAD24 and includes (i) the truncated recE gene (t-recE) under the arabinose-inducible P.sub.BAD promoter; (ii) the recT gene under the EM7 promoter; and (iii) the red.gamma. gene under the Tn5 promoter. It was transformed into NS3145, a recA E. coli strain which contained the Hoxa P1 episome. After arabinose induction, competent cells were prepared and transformed with a PCR product carrying the chloramphenicol resistance gene (cm) flanked by n and p homology arms. n and p were chosen to recombine with a segment of the P1 vector. b Southern blots of Pvu II digested DNAs hybridized with a probe made from the P1 vector to visualize the recombination target site (upper panel) and a probe made from the chloramphenicol resistance gene (lower panel). Lane 1, DNA prepared from cells harboring the Hoxa P1 episome before ET cloning. Lanes 2-17, DNA prepared from 16 independent chloramphenicol resistant colonies.

FIG. 12

[0090] Comparison of ET cloning using the recE/recT genes in pBAD-ET.gamma. with red.alpha./red.beta. genes in pBAD-.alpha..beta..gamma..

[0091] The plasmids pBAD-ET.gamma. or pBAD-.alpha..beta..gamma., depicted, were transformed into the E. coli recA-, recBC+ strain, DK1 and targeted by a chloramphenicol gene as described in FIG. 6 to evaluate ET cloning efficiencies. Arabinose induction of protein expression was for 1 hour.

FIG. 13A

[0092] The plasmid pBAD-ET.gamma. is shown by diagram.

FIG. 13B

[0093] The nucleic acid sequence and the protein coding portions of pBAD-ET.gamma. are depicted.

FIG. 14A

[0094] The plasmid pBAD-.alpha..beta..gamma. is shown by diagram. This plasmid substantially corresponds to the plasmid shown in FIG. 13 except that the recE and recT genes are substituted by the red.alpha. and red.beta. genes.

FIG. 14B

[0095] The nucleic acid sequence and the protein coding portions of pBAD-.alpha..beta..gamma. are depicted.

1. Methods

1.1. Preparation of Linear Fragments

[0096] Standard PCR reaction conditions were used to amplify linear DNA fragments. The sequences of the primers used are depicted in Table 1.

Table 1

[0097] The Tn5-neo gene from pJP5603 (Penfold and Pemberton, Gene 118 (1992), 145-146) was amplified by using oligo pairs a/b and c/d. The chloramphenicol (cm) resistant gene from pMAK705 (Hashimoto-Gotoh and Sekiguchi, J. Bacteriol. 131 (1977), 405-412) was amplified by using primer pairs elf and n/p. The Tn5-neo gene flanked by FRT or loxP sites was amplified from pKaZ or pKaX (http://www.embl-heidelberg.de/ExternalInfo/stewart) using oligo pairs i/h, g/h and j/k. The sacB-neo cassette from pIB279 (Blomfield et al., Mol. Microbiol. 5 (1991), 1447-1457) was amplified by using oligo pair Urn. The lacZ gene fragment from pSVpaZ11 (Buchholz et al., Nucleic Acids Res. 24 (1996), 4256-4262) was amplified using oligo pair l*/m*. FOR products were purified using the QIAGEN FOR Purification Kit and eluted with H.sub.2O.sub.2, followed by digestion of any residual template DNA with Dpn I. After digestion, PCR products were extracted once with Phenol:CHCl.sub.3, ethanol precipitated and resuspended in H.sub.2O at approximately 0.5 .mu.g/.mu.l.

1.2 Preparation of Competent Cells and Electroporation

[0098] Saturated overnight cultures were diluted 50 fold into LB medium, grown to an OD600 of 0.5, following by chilling on ice for 15 min. Bacterial cells were centrifuged at 7,000 rpm for 10 min at 0.degree. C. The pellet was resuspended in ice-cold 10% glycerol and centrifuged again (7,000 rpm, -5.degree. C., 10 min). This was repeated twice more and the cell pellet was suspended in an equal volume of ice-cold 10% glycerol. Aliquots of 50 .mu.l were frozen in liquid nitrogen and stored at -80.degree. C. Cells were thawed on ice and 1 .mu.l DNA solution (containing, for co-transformation, 0.3 .mu.g plasmid and 0.2 .mu.g PCR products; or, for transformation, 0.2 .mu.g PCR products) was added. Electroporation was performed using ice-cold cuvettes and a Bio-Rad Gene Pulser set to 25 .mu.FD, 2.3 kV. with Pulse Controller set at 200 ohms. LB medium (1 ml) was added after electroporation. The cells were incubated at 37.degree. C. for 1 hour with shaking and then spread on antibiotic plates.

1.3 Induction of RecE and RecT Expression

[0099] E. coli JC5547 carrying pBAD24-recET was cultured, overnight in LB medium plus 0.2% glucose, 100 .mu.g/ml ampicillin, Five parallel LB cultures, one of which (O) included 0.2% glucose, were started by a 1/100 inoculation. The cultures were incubated at 37.degree. C. with shaking for 4 hours and 0.1% L-arabinose was added 3, 2, 1 or 1/2 hour before harvesting and processing as above. Immediately before harvesting, 100 .mu.l was removed for analysis on a 10% SDS-polyacrylamide gel. E. coli NS3145 carrying Hoxa-P1 and pBAD-ET.gamma. was induced by 0.1% L-arabinose for 90 min before harvesting.

1.4 Transient Transformation of FLP and Cre Expression Plasmids

[0100] The FLP and Cre expression plasmids, 705-Cre and 705-FLP (Buchholz et al, Nucleic Acids Res. 24 (1996), 3118-3119), based on the pSC101 temperature sensitive origin, were transformed into rubidium chloride competent bacterial cells. Cells were spread on 25 .mu.g/ml chloramphenicol plates, and grown for 2 days at 30.degree. C., whereupon colonies were picked, replated on L-agar plates without any antibiotics and incubated at 40.degree. C. overnight. Single colonies were analyzed on various antibiotic plates and all showed the expected loss of chloramphenicol and kanamycin resistance.

1.5 Sucrose Counter Selection of sacB Expression

[0101] The E. coli JC9604lacZ strain, generated as described in FIG. 11, was cotransformed with a sacB-neo PCR fragment and pSVpaX1 (Buchholz et al, Nucleic Acids Res. 24 (1996), 4256-4262). After selection on 100 .mu.g/ml ampicillin, 50 .mu.g/ml kanamycin plates, pSVpaX-sacB-neo plasmids were isolated and cotransformed into fresh JC9604lacZ cells with a PCR fragment amplified from pSVpaX1 using primers l*/m*. Oligo m* carried a silent point mutation which generated a BamHI site. Cells were plated on 7% sucrose, 100 .mu.g/ml ampicillin, 40 .mu.g/ml X-gal plates and incubated at 28.degree. C. for 2 days. The blue and white colonies grown on sucrose plates were counted and further checked by restriction analysis.

1.6 Other Methods

[0102] DNA preparation and Southern analysis were performed according to standard procedures. Hybridization probes were generated by random priming of fragments isolated from the Tn5 neo gene (PvuII), Hoxa3 gene (both HindIII fragments), lacZ genes (EcoRI and BamH1 fragments from pSVpaX1), cm gene (BstB1 fragments from pMAK705) and P1 vector fragments (2.2 kb EcoR1 fragments from P1 vector).

2. Results

[0103] 2.1 Identification of Recombination Events in E. coli

[0104] To identify a flexible homologous recombination reaction in E. coli, an assay based on recombination between linear and circular DNAs was designed (FIG. 1, FIG. 3). Linear DNA carrying the Tn5 kanamycin resistance gene (neo) was made by PCR (FIG. 3a). Initially, the oligonucleotides used for PCR amplification of neo were 60mers consisting of 42 nucleotides at their 5' ends identical to chosen regions in the plasmid and, at the 3' ends, 18 nucleotides to serve as PCR primers. Linear and circular DNAs were mixed in equimolar proportions and co-transformed into a variety of E. coli hosts. Homologous recombination was only detected in sbcA E. coli hosts. More than 95% of double ampicillin/kanamycin resistant colonies (FIG. 3b) contained the expected homologously recombined plasmid as determined by restriction digestion and sequencing. Only a low background of kanamycin resistance, due to genomic integration of the neo gene, was apparent (not shown).

[0105] The linear plus circular recombination reaction was characterized in two ways. The relationship between homology arm length and recombination efficiency was simple, with longer arms recombining more efficiently (FIG. 3c). Efficiency increased within the range tested, up to 60 bp. The effect of distance between the two chosen homology sites in the recipient plasmid was examined (FIG. 3d). A set of eight FOR fragments was generated by use of a constant left homology arm with differing right homology arms. The right homology arms were chosen from the plasmid sequence to be 0-3100 by from the left. Correct products were readily obtained from all, with less than 4 fold difference between them, although the insertional product (O) was least efficient. Correct products also depended on the presence of both homology arms, since PCR fragments containing only one arm failed to work.

2.2 Involvement of RecE and RecT

[0106] The relationship between host genotype and this homologous recombination reaction was more systematically examined using a panel of E. coli strains deficient in various recombination components (Table 2).

Table 2

[0107] Only the two sbcA strains, JC8679 and JC9604 presented the intended recombination products and RecA was not required. In sbcA strains, expression of RecE and RecT is activated. Dependence on recE can be inferred from comparison of JC8679 with JC8691. Notably no recombination products were observed in JC9387 suggesting that the sbcBC background is not capable of supporting homologous recombination based on 50 nucleotide homology arms.

[0108] To demonstrate that RecE and RecT are involved, part of the recET operon was cloned into an inducible expression vector to create pBAD24-recET (FIG. 6a). the recE gene was truncated at its N-terminal end, as the first 588 a.a.s of RecE are dispensable. The recBC strain, JC5547, was transformed with pBAD24-recET and a time course of RecE/RecT induction performed by adding arabinose to the culture media at various times before harvesting for competent cells. The batches of harvested competent cells were evaluated for protein expression by gel electrophoresis (FIG. 6b) and for recombination between a linear DNA fragment and the endogenous pBAD24-recET plasmid (FIG. 6c). Without induction of RecE/RecT, no recombinant products were found, whereas recombination increased in approximate concordance with increased RecE/RecT expression. This experiment also shows that co-transformation of linear and circular DNAs is not essential and the circular recipient can be endogenous in the host. From the results shown in FIGS. 3, 6 and Table 2, we conclude that RecE and RecT mediate a very useful homologous recombination reaction in recBC E. coli at workable frequencies. Since RecE and RecT are involved, we refer to this way of recombining linear and circular DNA fragments as "ET cloning".

2.3 Application of ET Cloning to Large Target DNAs

[0109] To show that large. DNA episomes could be manipulated in E. coli, a >76 kb P1 clone that contains at least 59 kb of the intact mouse. Hoxa complex, (confirmed by DNA sequencing and Southern blotting), was transferred to an E. coli strain having an sbcA background (JC9604) and subjected to two rounds of ET cloning. In the first round, the Tn903 kanamycin resistance gene resident in the P1 vector was replaced by an ampicillin resistance gene (FIG. 4). In the second round, the interval between the Hoxa3 and a4 genes was targeted either by inserting the neb gene between two base pairs upstream of the Hoxa3 proximal promoter, or by deleting 6203 bp between the Hoxa3 and a4 genes' (FIG. 8a). Both insertional and deletional ET cloning products were readily obtained (FIG. 8b, lanes 2, 3 and 5) showing that the two rounds of ET cloning took place in this large E. coli episome with precision and no apparent unintended recombination.

[0110] The general applicability of ET cloning was further examined by targeting a gene in the E. coli chromosome (FIG. 9a). The .beta.-galactosidase (lacZ) gene of JC9604 was chosen so that the ratio between correct and incorrect recombinants could be determined by evaluating .beta.-galactosidase expression. Standard conditions (0.2 .mu.g PCR fragment; 50 .mu.l competent cells), produced 24 primary colonies, 20 of which were correct as determined by .beta.-galactosidase expression (FIG. 9b), and DNA analysis (FIG. 9c, lanes 3-6).

2.4 Secondary Recombination Reactions to Remove Operational Sequences

[0111] The products of ET cloning as described above are limited by the necessary inclusion of selectable marker genes. Two different ways to use a further recombination step to remove this limitation were developed. In the first way, site specific recombination mediated by either Flp or Cre recombinase was employed. In the experiments of FIGS. 8 and 9, either Flp recombination target sites. (FRTs) or Cre recombination target sites (loxPs) were included to flank the neo gene in the linear substrates. Recombination between the FRTs or loxPs was accomplished by Flp or Cre, respectively, expressed from plasmids with the pSC101 temperature sensitive replication origin (Hashimoto-Gotoh and Sekiguchj, J. Bacteriol. 131 (1977), 405-412) to permit simple elimination of these plasmids after site specific recombination by temperature shift. The precisely recombined Hoxa P1 vector was recovered after both ET and Hp recombination with no other recombination products apparent (FIG. 8, lanes 4 and 6). Similarly, Cre recombinase precisely recombined the targeted lacZ allele (FIG. 9, lanes 7-10). Thus site specific recombination can be readily coupled with ET cloning to remove operational sequences and leave a 34 by site specific recombination target site at the point of DNA manipulation.

[0112] In the second way to remove the selectable marker gene, two rounds of ET cloning, combining positive and counter selection steps, were used to leave the DNA product free of any operational sequences (FIG. 10a).

[0113] Additionally this experiment was designed to evaluate, by a functional test based on .beta.-galactosidase activity, whether ET cloning promoted small mutations such as frame shift or point mutations within the region being manipulated. In the first round, the lacZ gene of pSVpaX1 was disrupted with a 3.3 kb PCR fragment carrying the neo and B. subtilis sacB (Blomfield et al., Mol. Microbiol. 5 (1991), 1447-1457) genes, by selection for kanamycin resistance (FIG. 10a). As shown above for other positively selected recombination products, virtually all selected colonies were white (FIG. 10b), indicative of successful lacZ disruption, and 17 of 17 were confirmed as correct recombinants by DNA analysis. In the second round, a 1.5 kb PCR fragment designed to repair lacZ was introduced by counter selection against the sacB gene. Repair of lacZ included a silent point mutation to create a BamH1 restriction site. Approximately one quarter of sucrose resistant colonies expressed .beta.-galactosidase, and all analyzed (17 of 17; FIG. 10c) carried the repaired lacZ gene with the BamH1 point mutation. The remaining three quarters of sucrose resistant colonies did not express .beta.-galactosidase, and all analyzed (17 of 17; FIG. 10c) had undergone a variety of large mutational events, none of which resembled the ET cloning product. Thus, in two rounds of ET cloning directed at the lacZ gene, no disturbances of .beta.-galactosidase activity by small mutations were observed, indicating the RecE/RecT recombination works with high fidelity. The significant presence of incorrect products observed in the counter selection step is an inherent limitation of the use of counter selection, since any mutation that ablates expression of the counter selection gene will be selected. Notably, all incorrect products were large mutations and therefore easily distinguished from the correct ET product by DNA analysis. In a different experiment (FIG. 5), we observed that ET cloning into pZerb2.1 (InVitroGen) by counter selection against the ccdB gene gave a lower background of incorrect products (8%), indicating that the counter selection background is variable according to parameters that differ from those that influence ET cloning efficiencies.

2.5 Transference of ET Cloning Between E. coli Hosts

[0114] The experiments shown above were performed in recBC-E. coli hosts since the sbcA mutation had been identified as a suppressor of recBC (Barbour et al., Proc. Natl. Acad. Sci. USA 67 (1970), 128-135; Clark, Genetics 78 (1974), 259-271). However, many useful E. coli strains are recBC+, including strains commonly used for propagation of P1, BAC or PAC episomes. To transfer ET cloning into recBC+ strains, we developed pBAD-ET.gamma. and pBAD-.alpha..beta..gamma. (FIGS. 13 and 14). These plasmids incorporate three features important to the mobility of ET cloning. First, RecBC is the major E. coli exonuclease and degrades introduced linear fragments. Therefore the RecBC inhibitor, Red.gamma. (Murphy, J. Bacteriol. 173 (1991), 5808-5821), was included. Second, the recombinogenic potential of RecE/RecT, or Red.alpha./Red.beta., was regulated by placing recE or red.alpha. under an inducible promoter. Consequently ET cloning can be induced when required and undesired recombination events which are restricted at other times. Third, we observed that ET cloning efficiencies are enhanced when RecT, or Red.beta., but not RecE, or Red.alpha., is overexpressed. Therefore we placed recT, or red.beta., under the strong, constitutive, EM7 promoter.

[0115] pBAD-ET.gamma. was transformed into NS3145 E. coli harboring the original Hoxa P1 episome (FIG. 11a). A region in the P1 vector backbone was targeted by FOR amplification of the chloramphenicol resistance gene (cm) flanked by n and p homology arms. As described above for positively selected ET cloning reactions, most (>9.0%) chloramphenicol resistant colonies were correct. Notably, the overall efficiency of ET cloning, in terms of linear DNA transformed, was nearly three times better using pBAD-ET.gamma. than with similar experiments based on targeting the same episome in the sbcA host, JC9604. This is consistent with our observation that overexpression of RecT improves ET cloning efficiencies.

[0116] A comparison between ET cloning efficiencies mediated by RecE/RecT, expressed from pBAD-ET.gamma., and Red.alpha./Red.beta., expressed from pBAD-.alpha..beta..gamma. was made in the recA-, recBC+ E. coli strain, DK1 (FIG. 12). After transformation of E. coli DK1 with either pBAD-ET.gamma. or pBAD-.alpha..beta..gamma., the same experiment as described in FIG. 6a,c, to replace the bla gene of the pBAD vector with a chloramphenicol gene was performed. Both pBAD-ET.gamma. or pBAD-.alpha..beta..gamma. presented similar ET cloning efficiencies in terms of responsiveness to arabinose induction of RecE and Red.alpha., and number of targeted events.

Sequence CWU 1

1

1416150DNAArtificial Sequencemisc_feature(1)..(6150)plasmid pBAD24-rec ET 1atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga 720tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacca 1320tggatcccgt aatcgtagaa gacatagagc caggtattta ttacggaatt tcgaatgaga 1380attaccacgc gggtcccggt atcagtaagt ctcagctcga tgacattgct gatactccgg 1440cactatattt gtggcgtaaa aatgcccccg tggacaccac aaagacaaaa acgctcgatt 1500taggaactgc tttccactgc cgggtacttg aaccggaaga attcagtaac cgctttatcg 1560tagcacctga atttaaccgc cgtacaaacg ccggaaaaga agaagagaaa gcgtttctga 1620tggaatgcgc aagcacagga aaaacggtta tcactgcgga agaaggccgg aaaattgaac 1680tcatgtatca aagcgttatg gctttgccgc tggggcaatg gcttgttgaa agcgccggac 1740acgctgaatc atcaatttac tgggaagatc ctgaaacagg aattttgtgt cggtgccgtc 1800cggacaaaat tatccctgaa tttcactgga tcatggacgt gaaaactacg gcggatattc 1860aacgattcaa aaccgcttat tacgactacc gctatcacgt tcaggatgca ttctacagtg 1920acggttatga agcacagttt ggagtgcagc caactttcgt ttttctggtt gccagcacaa 1980ctattgaatg cggacgttat ccggttgaaa ttttcatgat gggcgaagaa gcaaaactgg 2040caggtcaaca ggaatatcac cgcaatctgc gaaccctgtc tgactgcctg aataccgatg 2100aatggccagc tattaagaca ttatcactgc cccgctgggc taaggaatat gcaaatgact 2160aagcaaccac caatcgcaaa agccgatctg caaaaaactc agggaaaccg tgcaccagca 2220gcagttaaaa atagcgacgt gattagtttt attaaccagc catcaatgaa agagcaactg 2280gcagcagctc ttccacgcca tatgacggct gaacgtatga tccgtatcgc caccacagaa 2340attcgtaaag ttccggcgtt aggaaactgt gacactatga gttttgtcag tgcgatcgta 2400cagtgttcac agctcggact tgagccaggt agcgccctcg gtcatgcata tttactgcct 2460tttggtaata aaaacgaaaa gagcggtaaa aagaacgttc agctaatcat tggctatcgc 2520ggcatgattg atctggctcg ccgttctggt caaatcgcca gcctgtcagc ccgtgttgtc 2580cgtgaaggtg acgagtttag cttcgaattt ggccttgatg aaaagttaat acaccgcccg 2640ggagaaaacg aagatgcccc ggttacccac gtctatgctg tcgcaagact gaaagacgga 2700ggtactcagt ttgaagttat gacgcgcaaa cagattgagc tggtgcgcag cctgagtaaa 2760gctggtaata acgggccgtg ggtaactcac tgggaagaaa tggcaaagaa aacggctatt 2820cgtcgcctgt tcaaatattt gcccgtatca attgagatcc agcgtgcagt atcaatggat 2880gaaaaggaac cactgacaat cgatcctgca gattcctctg tattaaccgg ggaatacagt 2940gtaatcgata attcagagga atagatctaa gcttggctgt tttggcggat gagagaagat 3000tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 3060tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 3120tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 3180taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 3240acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 3300ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 3360ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 3420cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 3480aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 3540ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 3600cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 3660agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 3720gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 3780cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 3840gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 3900ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 3960gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 4020gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 4080cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 4140ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 4200gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 4260gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 4320gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 4380ctttagattg atttacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 4440gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 4500ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 4560agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg 4620ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 4680gttctttaat agtggactct tgttccaaac ttgaacaaca ctcaacccta tctcgggcta 4740ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 4800ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt aaaaggatct 4860aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 4920actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 4980gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 5040atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 5100atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 5160ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 5220gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 5280cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 5340tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 5400cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 5460ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 5520gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 5580tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 5640ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 5700gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc 5760atctgtgcgg tatttcacac cgcatagggt catggctgcg ccccgacacc cgccaacacc 5820cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 5880cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca 5940gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga 6000aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga 6060tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt 6120agaggatctg ctcatgtttg acagcttatc 61502843DNAArtificial Sequencemisc_feature(1)..(843)t-recE on plasmid pBAD24-recET at 1320-2162 2atg gat ccc gta atc gta gaa gac ata gag cca ggt att tat tac gga 48Met Asp Pro Val Ile Val Glu Asp Ile Glu Pro Gly Ile Tyr Tyr Gly1 5 10 15att tcg aat gag aat tac cac gcg ggt ccc ggt atc agt aag tct cag 96Ile Ser Asn Glu Asn Tyr His Ala Gly Pro Gly Ile Ser Lys Ser Gln 20 25 30ctc gat gac att gct gat act ccg gca cta tat ttg tgg cgt aaa aat 144Leu Asp Asp Ile Ala Asp Thr Pro Ala Leu Tyr Leu Trp Arg Lys Asn 35 40 45gcc ccc gtg gac acc aca aag aca aaa acg ctc gat tta gga act gct 192Ala Pro Val Asp Thr Thr Lys Thr Lys Thr Leu Asp Leu Gly Thr Ala 50 55 60ttc cac tgc cgg gta ctt gaa ccg gaa gaa ttc agt aac cgc ttt atc 240Phe His Cys Arg Val Leu Glu Pro Glu Glu Phe Ser Asn Arg Phe Ile65 70 75 80gta gca cct gaa ttt aac cgc cgt aca aac gcc gga aaa gaa gaa gag 288Val Ala Pro Glu Phe Asn Arg Arg Thr Asn Ala Gly Lys Glu Glu Glu 85 90 95aaa gcg ttt ctg atg gaa tgc gca agc aca gga aaa acg gtt atc act 336Lys Ala Phe Leu Met Glu Cys Ala Ser Thr Gly Lys Thr Val Ile Thr 100 105 110gcg gaa gaa ggc cgg aaa att gaa ctc atg tat caa agc gtt atg gct 384Ala Glu Glu Gly Arg Lys Ile Glu Leu Met Tyr Gln Ser Val Met Ala 115 120 125ttg ccg ctg ggg caa tgg ctt gtt gaa agc gcc gga cac gct gaa tca 432Leu Pro Leu Gly Gln Trp Leu Val Glu Ser Ala Gly His Ala Glu Ser 130 135 140tca att tac tgg gaa gat cct gaa aca gga att ttg tgt cgg tgc cgt 480Ser Ile Tyr Trp Glu Asp Pro Glu Thr Gly Ile Leu Cys Arg Cys Arg145 150 155 160ccg gac aaa att atc cct gaa ttt cac tgg atc atg gac gtg aaa act 528Pro Asp Lys Ile Ile Pro Glu Phe His Trp Ile Met Asp Val Lys Thr 165 170 175acg gcg gat att caa cga ttc aaa acc gct tat tac gac tac cgc tat 576Thr Ala Asp Ile Gln Arg Phe Lys Thr Ala Tyr Tyr Asp Tyr Arg Tyr 180 185 190cac gtt cag gat gca ttc tac agt gac ggt tat gaa gca cag ttt gga 624His Val Gln Asp Ala Phe Tyr Ser Asp Gly Tyr Glu Ala Gln Phe Gly 195 200 205gtg cag cca act ttc gtt ttt ctg gtt gcc agc aca act att gaa tgc 672Val Gln Pro Thr Phe Val Phe Leu Val Ala Ser Thr Thr Ile Glu Cys 210 215 220gga cgt tat ccg gtt gaa att ttc atg atg ggc gaa gaa gca aaa ctg 720Gly Arg Tyr Pro Val Glu Ile Phe Met Met Gly Glu Glu Ala Lys Leu225 230 235 240gca ggt caa cag gaa tat cac cgc aat ctg cga acc ctg tct gac tgc 768Ala Gly Gln Gln Glu Tyr His Arg Asn Leu Arg Thr Leu Ser Asp Cys 245 250 255ctg aat acc gat gaa tgg cca gct att aag aca tta tca ctg ccc cgc 816Leu Asn Thr Asp Glu Trp Pro Ala Ile Lys Thr Leu Ser Leu Pro Arg 260 265 270tgg gct aag gaa tat gca aat gac taa 843Trp Ala Lys Glu Tyr Ala Asn Asp * 275 2803280PRTArtificial Sequencemisc_feature(1)..(280)t-recE on plasmid pBAD24-recET at 1320-2162 3Met Asp Pro Val Ile Val Glu Asp Ile Glu Pro Gly Ile Tyr Tyr Gly1 5 10 15Ile Ser Asn Glu Asn Tyr His Ala Gly Pro Gly Ile Ser Lys Ser Gln 20 25 30Leu Asp Asp Ile Ala Asp Thr Pro Ala Leu Tyr Leu Trp Arg Lys Asn 35 40 45Ala Pro Val Asp Thr Thr Lys Thr Lys Thr Leu Asp Leu Gly Thr Ala 50 55 60Phe His Cys Arg Val Leu Glu Pro Glu Glu Phe Ser Asn Arg Phe Ile65 70 75 80Val Ala Pro Glu Phe Asn Arg Arg Thr Asn Ala Gly Lys Glu Glu Glu 85 90 95Lys Ala Phe Leu Met Glu Cys Ala Ser Thr Gly Lys Thr Val Ile Thr 100 105 110Ala Glu Glu Gly Arg Lys Ile Glu Leu Met Tyr Gln Ser Val Met Ala 115 120 125Leu Pro Leu Gly Gln Trp Leu Val Glu Ser Ala Gly His Ala Glu Ser 130 135 140Ser Ile Tyr Trp Glu Asp Pro Glu Thr Gly Ile Leu Cys Arg Cys Arg145 150 155 160Pro Asp Lys Ile Ile Pro Glu Phe His Trp Ile Met Asp Val Lys Thr 165 170 175Thr Ala Asp Ile Gln Arg Phe Lys Thr Ala Tyr Tyr Asp Tyr Arg Tyr 180 185 190His Val Gln Asp Ala Phe Tyr Ser Asp Gly Tyr Glu Ala Gln Phe Gly 195 200 205Val Gln Pro Thr Phe Val Phe Leu Val Ala Ser Thr Thr Ile Glu Cys 210 215 220Gly Arg Tyr Pro Val Glu Ile Phe Met Met Gly Glu Glu Ala Lys Leu225 230 235 240Ala Gly Gln Gln Glu Tyr His Arg Asn Leu Arg Thr Leu Ser Asp Cys 245 250 255Leu Asn Thr Asp Glu Trp Pro Ala Ile Lys Thr Leu Ser Leu Pro Arg 260 265 270Trp Ala Lys Glu Tyr Ala Asn Asp 275 2804810DNAArtificial Sequencemisc_feature(1)..(810)recT on plasmid pBAD24-recET at 2155-2972 4atg act aag caa cca cca atc gca aaa gcc gat ctg caa aaa act cag 48Met Thr Lys Gln Pro Pro Ile Ala Lys Ala Asp Leu Gln Lys Thr Gln 285 290 295gga aac cgt gca cca gca gca gtt aaa aat agc gac gtg att agt ttt 96Gly Asn Arg Ala Pro Ala Ala Val Lys Asn Ser Asp Val Ile Ser Phe 300 305 310att aac cag cca tca atg aaa gag caa ctg gca gca gct ctt cca cgc 144Ile Asn Gln Pro Ser Met Lys Glu Gln Leu Ala Ala Ala Leu Pro Arg 315 320 325cat atg acg gct gaa cgt atg atc cgt atc gcc acc aca gaa att cgt 192His Met Thr Ala Glu Arg Met Ile Arg Ile Ala Thr Thr Glu Ile Arg330 335 340 345aaa gtt ccg gcg tta gga aac tgt gac act atg agt ttt gtc agt gcg 240Lys Val Pro Ala Leu Gly Asn Cys Asp Thr Met Ser Phe Val Ser Ala 350 355 360atc gta cag tgt tca cag ctc gga ctt gag cca ggt agc gcc ctc ggt 288Ile Val Gln Cys Ser Gln Leu Gly Leu Glu Pro Gly Ser Ala Leu Gly 365 370 375cat gca tat tta ctg cct ttt ggt aat aaa aac gaa aag agc ggt aaa 336His Ala Tyr Leu Leu Pro Phe Gly Asn Lys Asn Glu Lys Ser Gly Lys 380 385 390aag aac gtt cag cta atc att ggc tat cgc ggc atg att gat ctg gct 384Lys Asn Val Gln Leu Ile Ile Gly Tyr Arg Gly Met Ile Asp Leu Ala 395 400 405cgc cgt tct ggt caa atc gcc agc ctg tca gcc cgt gtt gtc cgt gaa 432Arg Arg Ser Gly Gln Ile Ala Ser Leu Ser Ala Arg Val Val Arg Glu410 415 420 425ggt gac gag ttt agc ttc gaa ttt ggc ctt gat gaa aag tta ata cac 480Gly Asp Glu Phe Ser Phe Glu Phe Gly Leu Asp Glu Lys Leu Ile His 430 435 440cgc ccg gga gaa aac gaa gat gcc ccg gtt acc cac gtc tat gct gtc 528Arg Pro Gly Glu Asn Glu Asp Ala Pro Val Thr His Val Tyr Ala Val 445 450 455gca aga ctg aaa gac gga ggt act cag ttt gaa gtt atg acg cgc aaa 576Ala Arg Leu Lys Asp Gly Gly Thr Gln Phe Glu Val Met Thr Arg Lys 460 465 470cag att gag ctg gtg cgc agc ctg agt aaa gct ggt aat aac ggg ccg 624Gln Ile Glu Leu Val Arg Ser Leu Ser Lys Ala Gly Asn Asn Gly Pro 475 480 485tgg gta act cac tgg gaa gaa atg gca aag aaa acg gct att cgt cgc 672Trp Val Thr His Trp Glu Glu Met Ala Lys Lys Thr Ala Ile Arg Arg490 495 500 505ctg ttc aaa tat ttg ccc gta tca att gag atc cag cgt gca gta tca 720Leu Phe Lys Tyr Leu Pro Val Ser Ile Glu Ile Gln Arg Ala Val Ser 510 515 520atg gat gaa aag gaa cca ctg aca atc gat cct gca gat tcc tct gta 768Met Asp Glu Lys Glu Pro Leu Thr Ile Asp Pro Ala Asp Ser Ser Val 525 530 535tta acc ggg gaa tac agt gta atc gat aat tca gag gaa tag 810Leu Thr Gly Glu Tyr Ser Val Ile Asp Asn Ser Glu Glu * 540 545 5505269PRTArtificial Sequencemisc_feature(1)..(269)recT on plasmid pBAD24-recET at 2155-2972 5Met Thr Lys Gln Pro Pro Ile Ala Lys Ala Asp Leu Gln Lys Thr Gln1 5 10 15Gly Asn Arg Ala Pro Ala Ala Val Lys Asn Ser Asp Val Ile Ser Phe 20 25 30Ile Asn Gln Pro Ser Met Lys Glu Gln Leu Ala Ala Ala Leu Pro Arg 35 40 45His Met Thr Ala Glu Arg Met Ile Arg Ile Ala Thr Thr Glu Ile Arg 50 55 60Lys Val Pro Ala Leu Gly Asn Cys Asp Thr Met Ser Phe Val Ser Ala65 70 75 80Ile Val Gln Cys Ser Gln Leu Gly Leu Glu Pro Gly Ser Ala Leu Gly 85 90 95His Ala Tyr Leu Leu Pro Phe Gly Asn Lys Asn Glu Lys Ser Gly Lys 100 105 110Lys Asn Val Gln Leu Ile Ile Gly Tyr Arg Gly Met Ile Asp Leu Ala 115 120 125Arg Arg Ser Gly Gln Ile Ala Ser Leu Ser Ala Arg Val Val Arg Glu 130 135 140Gly Asp Glu Phe Ser Phe Glu Phe Gly Leu Asp Glu Lys Leu Ile His145 150 155 160Arg Pro Gly Glu Asn Glu Asp Ala Pro Val Thr His Val Tyr Ala Val 165 170 175Ala Arg Leu Lys Asp Gly Gly Thr Gln Phe Glu Val Met Thr Arg Lys 180 185 190Gln Ile Glu Leu Val Arg Ser Leu Ser Lys Ala Gly Asn Asn Gly Pro 195 200 205Trp Val Thr His Trp Glu Glu Met Ala Lys Lys Thr Ala Ile Arg Arg 210 215 220Leu Phe Lys

Tyr Leu Pro Val Ser Ile Glu Ile Gln Arg Ala Val Ser225 230 235 240Met Asp Glu Lys Glu Pro Leu Thr Ile Asp Pro Ala Asp Ser Ser Val 245 250 255Leu Thr Gly Glu Tyr Ser Val Ile Asp Asn Ser Glu Glu 260 2656876DNAArtificial Sequencemisc_feature(1)..(876)araC on plasmid pBAD24-recET at 974-996 6tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg 60gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac 120attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct 180gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg 240tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct 300gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg 360atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat 420cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag 480gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat 540tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg 600ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag 660caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc 720gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc 780gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta 840tcccggcagc aggggatcat tttgcgcttc agccat 8767292PRTArtificial Sequencemisc_feature(1)..(292)araC on plasmid pBAD24-recET at 974-996 7Met Ala Glu Ala Gln Asn Asp Pro Leu Leu Pro Gly Tyr Ser Phe Asn1 5 10 15Ala His Leu Val Ala Gly Leu Thr Pro Ile Glu Ala Asn Gly Tyr Leu 20 25 30Asp Phe Phe Ile Asp Arg Pro Leu Gly Met Lys Gly Tyr Ile Leu Asn 35 40 45Leu Thr Ile Arg Gly Gln Gly Val Val Lys Asn Gln Gly Arg Glu Phe 50 55 60Val Cys Arg Pro Gly Asp Ile Leu Leu Phe Pro Pro Gly Glu Ile His65 70 75 80His Tyr Gly Arg His Pro Glu Ala Arg Glu Trp Tyr His Gln Trp Val 85 90 95Tyr Phe Arg Pro Arg Ala Tyr Trp His Glu Trp Leu Asn Trp Pro Ser 100 105 110Ile Phe Ala Asn Thr Gly Phe Phe Arg Pro Asp Glu Ala His Gln Pro 115 120 125His Phe Ser Asp Leu Phe Gly Gln Ile Ile Asn Ala Gly Gln Gly Glu 130 135 140Gly Arg Tyr Ser Glu Leu Leu Ala Ile Asn Leu Leu Glu Gln Leu Leu145 150 155 160Leu Arg Arg Met Glu Ala Ile Asn Glu Ser Leu His Pro Pro Met Asp 165 170 175Asn Arg Val Arg Glu Ala Cys Gln Tyr Ile Ser Asp His Leu Ala Asp 180 185 190Ser Asn Phe Asp Ile Ala Ser Val Ala Gln His Val Cys Leu Ser Pro 195 200 205Ser Arg Leu Ser His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val Leu 210 215 220Ser Trp Arg Glu Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu Ser225 230 235 240Thr Thr Arg Met Pro Ile Ala Thr Val Gly Arg Asn Val Gly Phe Asp 245 250 255Asp Gln Leu Tyr Phe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala Ser 260 265 270Pro Ser Glu Phe Arg Ala Gly Cys Glu Glu Lys Val Asn Asp Val Ala 275 280 285Val Lys Leu Ser 2908861DNAArtificial Sequencemisc_feature(1)..(861)bla gene on plasmid pBAD24-recET at 3493-4353 8atg agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca 48Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 295 300 305ttt tgc ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa gta aaa 96Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 310 315 320gat gct gaa gat cag ttg ggt gca cga gtg ggt tac atc gaa ctg gat 144Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp325 330 335 340ctc aac agc ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt 192Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 345 350 355cca atg atg agc act ttt aaa gtt ctg cta tgt ggc gcg gta tta tcc 240Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 360 365 370cgt gtt gac gcc ggg caa gag caa ctc ggt cgc cgc ata cac tat tct 288Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 375 380 385cag aat gac ttg gtt gag tac tca cca gtc aca gaa aag cat ctt acg 336Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 390 395 400gat ggc atg aca gta aga gaa tta tgc agt gct gcc ata acc atg agt 384Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser405 410 415 420gat aac act gcg gcc aac tta ctt ctg aca acg atc gga gga ccg aag 432Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys 425 430 435gag cta acc gct ttt ttg cac aac atg ggg gat cat gta act cgc ctt 480Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu 440 445 450gat cgt tgg gaa ccg gag ctg aat gaa gcc ata cca aac gac gag cgt 528Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg 455 460 465gac acc acg atg cct gta gca atg gca aca acg ttg cgc aaa cta tta 576Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 470 475 480act ggc gaa cta ctt act cta gct tcc cgg caa caa tta ata gac tgg 624Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp485 490 495 500atg gag gcg gat aaa gtt gca gga cca ctt ctg cgc tcg gcc ctt ccg 672Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 505 510 515gct ggc tgg ttt att gct gat aaa tct gga gcc ggt gag cgt ggg tct 720Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser 520 525 530cgc ggt atc att gca gca ctg ggg cca gat ggt aag ccc tcc cgt atc 768Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile 535 540 545gta gtt atc tac acg acg ggg agt cag gca act atg gat gaa cga aat 816Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn 550 555 560aga cag atc gct gag ata ggt gcc tca ctg att aag cat tgg taa 861Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp *565 570 5759286PRTArtificial SequenceDOMAIN(1)..(286)bla gene on plasmid pBAD24-recET at 3493-4353 9Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala1 5 10 15Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp 35 40 45Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 50 55 60Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser65 70 75 80Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 85 90 95Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 100 105 110Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser 115 120 125Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys 130 135 140Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu145 150 155 160Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg 165 170 175Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 180 185 190Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp 195 200 205Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 210 215 220Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser225 230 235 240Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile 245 250 255Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn 260 265 270Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp 275 280 285107195DNAArtificial Sequencemisc_feature(1)..(7195)plasmid pBAD-ET-gamma 10atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga 720tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacca 1320tggatcccgt aatcgtagaa gacatagagc caggtattta ttacggaatt tcgaatgaga 1380attaccacgc gggtcccggt atcagtaagt ctcagctcga tgacattgct gatactccgg 1440cactatattt gtggcgtaaa aatgcccccg tggacaccac aaagacaaaa acgctcgatt 1500taggaactgc tttccactgc cgggtacttg aaccggaaga attcagtaac cgctttatcg 1560tagcacctga atttaaccgc cgtacaaacg ccggaaaaga agaagagaaa gcgtttctga 1620tggaatgcgc aagcacagga aaaacggtta tcactgcgga agaaggccgg aaaattgaac 1680tcatgtatca aagcgttatg gctttgccgc tggggcaatg gcttgttgaa agcgccggac 1740acgctgaatc atcaatttac tgggaagatc ctgaaacagg aattttgtgt cggtgccgtc 1800cggacaaaat tatccctgaa tttcactgga tcatggacgt gaaaactacg gcggatattc 1860aacgattcaa aaccgcttat tacgactacc gctatcacgt tcaggatgca ttctacagtg 1920acggttatga agcacagttt ggagtgcagc caactttcgt ttttctggtt gccagcacaa 1980ctattgaatg cggacgttat ccggttgaaa ttttcatgat gggcgaagaa gcaaaactgg 2040caggtcaaca ggaatatcac cgcaatctgc gaaccctgtc tgactgcctg aataccgatg 2100aatggccagc tattaagaca ttatcactgc cccgctgggc taaggaatat gcaaatgact 2160agatctcgag gtacccgagc acgtgttgac aattaatcat cggcatagta tatcggcata 2220gtataatacg acaaggtgag gaactaaacc atggctaagc aaccaccaat cgcaaaagcc 2280gatctgcaaa aaactcaggg aaaccgtgca ccagcagcag ttaaaaatag cgacgtgatt 2340agttttatta accagccatc aatgaaagag caactggcag cagctcttcc acgccatatg 2400acggctgaac gtatgatccg tatcgccacc acagaaattc gtaaagttcc ggcgttagga 2460aactgtgaca ctatgagttt tgtcagtgcg atcgtacagt gttcacagct cggacttgag 2520ccaggtagcg ccctcggtca tgcatattta ctgccttttg gtaataaaaa cgaaaagagc 2580ggtaaaaaga acgttcagct aatcattggc tatcgcggca tgattgatct ggctcgccgt 2640tctggtcaaa tcgccagcct gtcagcccgt gttgtccgtg aaggtgacga gtttagcttc 2700gaatttggcc ttgatgaaaa gttaatacac cgcccgggag aaaacgaaga tgccccggtt 2760acccacgtct atgctgtcgc aagactgaaa gacggaggta ctcagtttga agttatgacg 2820cgcaaacaga ttgagctggt gcgcagcctg agtaaagctg gtaataacgg gccgtgggta 2880actcactggg aagaaatggc aaagaaaacg gctattcgtc gcctgttcaa atatttgccc 2940gtatcaattg agatccagcg tgcagtatca atggatgaaa aggaaccact gacaatcgat 3000cctgcagatt cctctgtatt aaccggggaa tacagtgtaa tcgataattc agaggaatag 3060atctaagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa gacagcatcc 3120ttgaacaagg acaattaaca gttaacaaat aaaaacgcaa aagaaaatgc cgatatccta 3180ttggcatttt cttttatttc ttatcaacat aaaggtgaat cccatacctc gagcttcacg 3240ctgccgcaag cactcagggc gcaagggctg ctaaaaggaa gcggaacacg tagaaagcca 3300gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg 3360aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag 3420actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta 3480aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc 3540gcaggggatc aagatctgat caagagacag gatgaggatc gtttcgcatg gatattaata 3600ctgaaactga gatcaagcaa aagcattcac taaccccctt tcctgttttc ctaatcagcc 3660cggcatttcg cgggcgatat tttcacagct atttcaggag ttcagccatg aacgcttatt 3720acattcagga tcgtcttgag gctcagagct gggcgcgtca ctaccagcag ctcgcccgtg 3780aagagaaaga ggcagaactg gcagacgaca tggaaaaagg cctgccccag cacctgtttg 3840aatcgctatg catcgatcat ttgcaacgcc acggggccag caaaaaatcc attacccgtg 3900cgtttgatga cgatgttgag tttcaggagc gcatggcaga acacatccgg tacatggttg 3960aaaccattgc tcaccaccag gttgatattg attcagaggt ataaaacgag tagaagcttg 4020gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa 4080gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca 4140tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga 4200gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt 4260cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg 4320gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact 4380gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa 4440actcttttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 4500ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 4560cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 4620ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 4680tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 4740cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca 4800actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 4860aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 4920tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 4980ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 5040tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 5100gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 5160gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 5220tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg 5280gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 5340ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 5400gtcagaccaa gtttactcat atatacttta gattgattta cgcgccctgt agcggcgcat 5460taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 5520cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 5580aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 5640ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 5700ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaacttgaa 5760caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 5820cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 5880taacgtttac aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 5940atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6000tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6060ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6120ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 6180cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6240gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6300gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6360acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6420gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6480agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6540tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6600agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 6660cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 6720gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 6780ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat agggtcatgg 6840ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 6900catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 6960cgtcatcacc gaaacgcgcg aggcagcaag gagatggcgc ccaacagtcc cccggccacg 7020gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga 7080tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg 7140atgccggcca

cgatgcgtcc ggcgtagagg atctgctcat gtttgacagc ttatc 7195117010DNAArtificial Sequencemisc_feature(1)..(7010)plasmid pBAD-alpha-beta-gamma 11atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga 720tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacc 1319atg aca ccg gac att atc ctg cag cgt acc ggg atc gat gtg aga gct 1367Met Thr Pro Asp Ile Ile Leu Gln Arg Thr Gly Ile Asp Val Arg Ala 290 295 300gtc gaa cag ggg gat gat gcg tgg cac aaa tta cgg ctc ggc gtc atc 1415Val Glu Gln Gly Asp Asp Ala Trp His Lys Leu Arg Leu Gly Val Ile 305 310 315acc gct tca gaa gtt cac aac gtg ata gca aaa ccc cgc tcc gga aag 1463Thr Ala Ser Glu Val His Asn Val Ile Ala Lys Pro Arg Ser Gly Lys320 325 330 335aag tgg cct gac atg aaa atg tcc tac ttc cac acc ctg ctt gct gag 1511Lys Trp Pro Asp Met Lys Met Ser Tyr Phe His Thr Leu Leu Ala Glu 340 345 350gtt tgc acc ggt gtg gct ccg gaa gtt aac gct aaa gca ctg gcc tgg 1559Val Cys Thr Gly Val Ala Pro Glu Val Asn Ala Lys Ala Leu Ala Trp 355 360 365gga aaa cag tac gag aac gac gcc aga acc ctg ttt gaa ttc act tcc 1607Gly Lys Gln Tyr Glu Asn Asp Ala Arg Thr Leu Phe Glu Phe Thr Ser 370 375 380ggc gtg aat gtt act gaa tcc ccg atc atc tat cgc gac gaa agt atg 1655Gly Val Asn Val Thr Glu Ser Pro Ile Ile Tyr Arg Asp Glu Ser Met 385 390 395cgt acc gcc tgc tct ccc gat ggt tta tgc agt gac ggc aac ggc ctt 1703Arg Thr Ala Cys Ser Pro Asp Gly Leu Cys Ser Asp Gly Asn Gly Leu400 405 410 415gaa ctg aaa tgc ccg ttt acc tcc cgg gat ttc atg aag ttc cgg ctc 1751Glu Leu Lys Cys Pro Phe Thr Ser Arg Asp Phe Met Lys Phe Arg Leu 420 425 430ggt ggt ttc gag gcc ata aag tca gct tac atg gcc cag gtg cag tac 1799Gly Gly Phe Glu Ala Ile Lys Ser Ala Tyr Met Ala Gln Val Gln Tyr 435 440 445agc atg tgg gtg acg cga aaa aat gcc tgg tac ttt gcc aac tat gac 1847Ser Met Trp Val Thr Arg Lys Asn Ala Trp Tyr Phe Ala Asn Tyr Asp 450 455 460ccg cgt atg aag cgt gaa ggc ctg cat tat gtc gtg att gag cgg gat 1895Pro Arg Met Lys Arg Glu Gly Leu His Tyr Val Val Ile Glu Arg Asp 465 470 475gaa aag tac atg gcg agt ttt gac gag atc gtg ccg gag ttc atc gaa 1943Glu Lys Tyr Met Ala Ser Phe Asp Glu Ile Val Pro Glu Phe Ile Glu480 485 490 495aaa atg gac gag gca ctg gct gaa att ggt ttt gta ttt ggg gag caa 1991Lys Met Asp Glu Ala Leu Ala Glu Ile Gly Phe Val Phe Gly Glu Gln 500 505 510tgg cga tag atccggtacc cgagcacgtg ttgacaatta atcatcggca 2040Trp Arg *tagtatatcg gcatagtata atacgacaag gtgaggaact aaacc atg agt act 2094 Met Ser Thr 1gca ctc gca acg ctg gct ggg aag ctg gct gaa cgt gtc ggc atg gat 2142Ala Leu Ala Thr Leu Ala Gly Lys Leu Ala Glu Arg Val Gly Met Asp 5 10 15tct gtc gac cca cag gaa ctg atc acc act ctt cgc cag acg gca ttt 2190Ser Val Asp Pro Gln Glu Leu Ile Thr Thr Leu Arg Gln Thr Ala Phe20 25 30 35aaa ggt gat gcc agc gat gcg cag ttc atc gca tta ctg atc gtt gcc 2238Lys Gly Asp Ala Ser Asp Ala Gln Phe Ile Ala Leu Leu Ile Val Ala 40 45 50aac cag tac ggc ctt aat ccg tgg acg aaa gaa att tac gcc ttt cct 2286Asn Gln Tyr Gly Leu Asn Pro Trp Thr Lys Glu Ile Tyr Ala Phe Pro 55 60 65gat aag cag aat ggc atc gtt ccg gtg gtg ggc gtt gat ggc tgg tcc 2334Asp Lys Gln Asn Gly Ile Val Pro Val Val Gly Val Asp Gly Trp Ser 70 75 80cgc atc atc aat gaa aac cag cag ttt gat ggc atg gac ttt gag cag 2382Arg Ile Ile Asn Glu Asn Gln Gln Phe Asp Gly Met Asp Phe Glu Gln 85 90 95gac aat gaa tcc tgt aca tgc cgg att tac cgc aag gac cgt aat cat 2430Asp Asn Glu Ser Cys Thr Cys Arg Ile Tyr Arg Lys Asp Arg Asn His100 105 110 115ccg atc tgc gtt acc gaa tgg atg gat gaa tgc cgc cgc gaa cca ttc 2478Pro Ile Cys Val Thr Glu Trp Met Asp Glu Cys Arg Arg Glu Pro Phe 120 125 130aaa act cgc gaa ggc aga gaa atc acg ggg ccg tgg cag tcg cat ccc 2526Lys Thr Arg Glu Gly Arg Glu Ile Thr Gly Pro Trp Gln Ser His Pro 135 140 145aaa cgg atg tta cgt cat aaa gcc atg att cag tgt gcc cgt ctg gcc 2574Lys Arg Met Leu Arg His Lys Ala Met Ile Gln Cys Ala Arg Leu Ala 150 155 160ttc gga ttt gct ggt atc tat gac aag gat gaa gcc gag cgc att gtc 2622Phe Gly Phe Ala Gly Ile Tyr Asp Lys Asp Glu Ala Glu Arg Ile Val 165 170 175gaa aat act gca tac act gca gaa cgt cag ccg gaa cgc gac atc act 2670Glu Asn Thr Ala Tyr Thr Ala Glu Arg Gln Pro Glu Arg Asp Ile Thr180 185 190 195ccg gtt aac gat gaa acc atg cag gag att aac act ctg ctg atc gcc 2718Pro Val Asn Asp Glu Thr Met Gln Glu Ile Asn Thr Leu Leu Ile Ala 200 205 210ctg gat aaa aca tgg gat gac gac tta ttg ccg ctc tgt tcc cag ata 2766Leu Asp Lys Thr Trp Asp Asp Asp Leu Leu Pro Leu Cys Ser Gln Ile 215 220 225ttt cgc cgc gac att cgt gca tcg tca gaa ctg aca cag gcc gaa gca 2814Phe Arg Arg Asp Ile Arg Ala Ser Ser Glu Leu Thr Gln Ala Glu Ala 230 235 240gta aaa gct ctt gga ttc ctg aaa cag aaa gcc gca gag cag aag gtg 2862Val Lys Ala Leu Gly Phe Leu Lys Gln Lys Ala Ala Glu Gln Lys Val 245 250 255gca gca tag atctcgagaa gcttcctgct gaacatcaaa ggcaagaaaa 2911Ala Ala *260catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac aaataaaaac 2971gcaaaagaaa atgccgatat cctattggca ttttctttta tttcttatca acataaaggt 3031gaatcccata cctcgagctt cacgctgccg caagcactca gggcgcaagg gctgctaaaa 3091ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca 3151gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca 3211gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat 3271tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt 3331tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag 3391gatcgtttcg c atg gat att aat act gaa act gag atc aag caa aag cat 3441 Met Asp Ile Asn Thr Glu Thr Glu Ile Lys Gln Lys His 1 5 10tca cta acc ccc ttt cct gtt ttc cta atc agc ccg gca ttt cgc ggg 3489Ser Leu Thr Pro Phe Pro Val Phe Leu Ile Ser Pro Ala Phe Arg Gly 15 20 25cga tat ttt cac agc tat ttc agg agt tca gcc atg aac gct tat tac 3537Arg Tyr Phe His Ser Tyr Phe Arg Ser Ser Ala Met Asn Ala Tyr Tyr30 35 40 45att cag gat cgt ctt gag gct cag agc tgg gcg cgt cac tac cag cag 3585Ile Gln Asp Arg Leu Glu Ala Gln Ser Trp Ala Arg His Tyr Gln Gln 50 55 60ctc gcc cgt gaa gag aaa gag gca gaa ctg gca gac gac atg gaa aaa 3633Leu Ala Arg Glu Glu Lys Glu Ala Glu Leu Ala Asp Asp Met Glu Lys 65 70 75ggc ctg ccc cag cac ctg ttt gaa tcg cta tgc atc gat cat ttg caa 3681Gly Leu Pro Gln His Leu Phe Glu Ser Leu Cys Ile Asp His Leu Gln 80 85 90cgc cac ggg gcc agc aaa aaa tcc att acc cgt gcg ttt gat gac gat 3729Arg His Gly Ala Ser Lys Lys Ser Ile Thr Arg Ala Phe Asp Asp Asp 95 100 105gtt gag ttt cag gag cgc atg gca gaa cac atc cgg tac atg gtt gaa 3777Val Glu Phe Gln Glu Arg Met Ala Glu His Ile Arg Tyr Met Val Glu110 115 120 125acc att gct cac cac cag gtt gat att gat tca gag gta taa 3819Thr Ile Ala His His Gln Val Asp Ile Asp Ser Glu Val * 130 135aacgagtaga agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt 3879aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg 3939gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg 3999gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc 4059gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac 4119aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg 4179acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct 4239ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc 4299tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 4359ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 4419ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 4479gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 4539gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg 4599acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 4659actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 4719ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 4779cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 4839gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 4899caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 4959aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 5019ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 5079tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 5139ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 5199ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttacgcg 5259ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 5319cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 5379gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 5439ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg 5499ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 5559ttgttccaaa cttgaacaac actcaaccct atctcgggct attcttttga tttataaggg 5619attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 5679aattttaaca aaatattaac gtttacaatt taaaaggatc taggtgaaga tcctttttga 5739taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 5799agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 5859aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 5919ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 5979gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 6039aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 6099aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 6159gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 6219aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 6279aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 6339cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 6399cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 6459tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 6519tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 6579ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 6639ccgcataggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6699gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6759tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcaaggaga tggcgcccaa 6819cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc 6879gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc 6939acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatct gctcatgttt 6999gacagcttat c 701012226PRTArtificial SequenceDOMAIN(1)..(226)Red-alpha from plasmid pBAD-alpha-beta-gamma 12Met Thr Pro Asp Ile Ile Leu Gln Arg Thr Gly Ile Asp Val Arg Ala1 5 10 15Val Glu Gln Gly Asp Asp Ala Trp His Lys Leu Arg Leu Gly Val Ile 20 25 30Thr Ala Ser Glu Val His Asn Val Ile Ala Lys Pro Arg Ser Gly Lys 35 40 45Lys Trp Pro Asp Met Lys Met Ser Tyr Phe His Thr Leu Leu Ala Glu 50 55 60Val Cys Thr Gly Val Ala Pro Glu Val Asn Ala Lys Ala Leu Ala Trp65 70 75 80Gly Lys Gln Tyr Glu Asn Asp Ala Arg Thr Leu Phe Glu Phe Thr Ser 85 90 95Gly Val Asn Val Thr Glu Ser Pro Ile Ile Tyr Arg Asp Glu Ser Met 100 105 110Arg Thr Ala Cys Ser Pro Asp Gly Leu Cys Ser Asp Gly Asn Gly Leu 115 120 125Glu Leu Lys Cys Pro Phe Thr Ser Arg Asp Phe Met Lys Phe Arg Leu 130 135 140Gly Gly Phe Glu Ala Ile Lys Ser Ala Tyr Met Ala Gln Val Gln Tyr145 150 155 160Ser Met Trp Val Thr Arg Lys Asn Ala Trp Tyr Phe Ala Asn Tyr Asp 165 170 175Pro Arg Met Lys Arg Glu Gly Leu His Tyr Val Val Ile Glu Arg Asp 180 185 190Glu Lys Tyr Met Ala Ser Phe Asp Glu Ile Val Pro Glu Phe Ile Glu 195 200 205Lys Met Asp Glu Ala Leu Ala Glu Ile Gly Phe Val Phe Gly Glu Gln 210 215 220Trp Arg22513261PRTArtificial SequenceDOMAIN(1)..(261)Red-beta from plasmid pBAD-alpha-beta-gamma 13Met Ser Thr Ala Leu Ala Thr Leu Ala Gly Lys Leu Ala Glu Arg Val1 5 10 15Gly Met Asp Ser Val Asp Pro Gln Glu Leu Ile Thr Thr Leu Arg Gln 20 25 30Thr Ala Phe Lys Gly Asp Ala Ser Asp Ala Gln Phe Ile Ala Leu Leu 35 40 45Ile Val Ala Asn Gln Tyr Gly Leu Asn Pro Trp Thr Lys Glu Ile Tyr 50 55 60Ala Phe Pro Asp Lys Gln Asn Gly Ile Val Pro Val Val Gly Val Asp65 70 75 80Gly Trp Ser Arg Ile Ile Asn Glu Asn Gln Gln Phe Asp Gly Met Asp 85 90 95Phe Glu Gln Asp Asn Glu Ser Cys Thr Cys Arg Ile Tyr Arg Lys Asp 100 105 110Arg Asn His Pro Ile Cys Val Thr Glu Trp Met Asp Glu Cys Arg Arg 115 120 125Glu Pro Phe Lys Thr Arg Glu Gly Arg Glu Ile Thr Gly Pro Trp Gln 130 135 140Ser His Pro Lys Arg Met Leu Arg His Lys Ala Met Ile Gln Cys Ala145 150 155 160Arg Leu Ala Phe Gly Phe Ala Gly Ile Tyr Asp Lys Asp Glu Ala Glu 165 170 175Arg Ile Val Glu Asn Thr Ala Tyr Thr Ala Glu Arg Gln Pro Glu Arg 180 185 190Asp Ile Thr Pro Val Asn Asp Glu Thr Met Gln Glu Ile Asn Thr Leu 195 200 205Leu Ile Ala Leu Asp Lys Thr Trp Asp Asp Asp Leu Leu Pro Leu Cys 210 215 220Ser Gln Ile Phe Arg Arg Asp Ile Arg Ala Ser Ser Glu Leu Thr Gln225 230 235 240Ala Glu Ala Val Lys Ala Leu Gly Phe Leu Lys Gln Lys Ala Ala Glu 245 250 255Gln Lys Val Ala Ala 26014138PRTArtificial SequenceDOMAIN(1)..(138)Red-gamma from plasmid pBAD-alpha-beta-gamma and plasmid pBAD-ET-gamma 14Met Asp Ile Asn Thr Glu Thr Glu Ile Lys Gln Lys His Ser Leu Thr1 5 10 15Pro Phe Pro Val Phe Leu Ile Ser Pro Ala Phe Arg Gly Arg Tyr Phe 20 25 30His Ser Tyr Phe Arg Ser Ser Ala Met Asn Ala Tyr Tyr Ile Gln Asp 35 40

45Arg Leu Glu Ala Gln Ser Trp Ala Arg His Tyr Gln Gln Leu Ala Arg 50 55 60Glu Glu Lys Glu Ala Glu Leu Ala Asp Asp Met Glu Lys Gly Leu Pro65 70 75 80Gln His Leu Phe Glu Ser Leu Cys Ile Asp His Leu Gln Arg His Gly 85 90 95Ala Ser Lys Lys Ser Ile Thr Arg Ala Phe Asp Asp Asp Val Glu Phe 100 105 110Gln Glu Arg Met Ala Glu His Ile Arg Tyr Met Val Glu Thr Ile Ala 115 120 125His His Gln Val Asp Ile Asp Ser Glu Val 130 135

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References


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