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 Number | 20120208277 13/342621 |
Document ID | / |
Family ID | 26145961 |
Filed Date | 2012-08-16 |
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
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Application
Number |
Filing Date |
Patent Number |
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12757129 |
Apr 9, 2010 |
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13342621 |
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10842534 |
May 11, 2004 |
7736851 |
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12757129 |
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10231013 |
Aug 30, 2002 |
6787316 |
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10842534 |
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09555510 |
Jun 5, 2000 |
6509156 |
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PCT/EP1998/007945 |
Dec 7, 1998 |
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10231013 |
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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
* * * * *
References