CN114107302A

CN114107302A - CRISPR-Cas9 gene editing vector for bacteria and application thereof

Info

Publication number: CN114107302A
Application number: CN202111484187.3A
Authority: CN
Inventors: 张沨; 黄潮勇
Original assignee: Shandong Qibang Huikang Biotechnology Co ltd
Current assignee: Qingdao Kinstar Biotechnology Co ltd; Shandong Qibang Huikang Biotechnology Co ltd; Beijing Institute of Technology BIT
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-03-01

Abstract

The invention discloses a CRISPR-Cas9 gene editing vector for bacteria and application thereof, and relates to the field of gene editing. The invention provides a CRISPR-Cas9 gene editing vector for bacteria, which comprises a plasmid 1 and a plasmid 2; the plasmid 1 comprises a replication initiation site, a screening marker gene, a Cas9 protein expression element, a recombinant protein expression element and a counter-screening marker gene; the plasmid 2 comprises a replication initiation site, a selection marker gene, a sgRNA expression element and a homologous recombination element. The CRISPR-Cas9 gene editing vector constructed by the invention can edit bacteria such as escherichia coli, specifically comprises operations such as DNA sequence knockout, insertion and replacement, and has the advantages of short test period, high efficiency, low cost and the like.

Description

CRISPR-Cas9 gene editing vector for bacteria and application thereof

Technical Field

The invention relates to the field of gene editing, in particular to a CRISPR-Cas9 gene editing vector for bacteria and application thereof.

Background

Bacteria, as a class of multifunctional living systems, have important applications in many areas of synthetic biology. In bacteria, the genetic information carried on a single copy of the genome determines the characteristics of a strain. In order to better study the characteristics of bacteria and to utilize bacteria to explore the world and serve humans, researchers often need to reprogram the genetic information of bacteria through genomic engineering. By DNA editing, researchers can add specific exogenous genetic information to the genome of a bacterium or delete specific genetic information from the genome of a bacterium. The long fragment editing technology can greatly accelerate the genome engineering of bacteria and obtain strains with stable genetic information. For example, long fragment deletion techniques can be used to streamline the bacterial genome, facilitating the study of the minimal genome of a particular strain; the long fragment insertion technology can be used for expanding the genetic information of bacteria and provides an important tool for the research of storing human information by utilizing the bacteria. In the field of metabolic engineering, the maintenance of plasmid DNA is not independent of the use of antibiotics, which not only raises biosafety issues, but also leads to increased industrial costs. These problems are well solved by the use of long fragment editing techniques to construct plasmid DNA independent high producing strains.

To accelerate the progress of bacterial genome engineering, researchers have developed many gene editing methods for DNA insertion and deletion in bacteria. Recombineering is a classical bacterial gene editing method that is very convenient in the insertion and deletion of short sequences, but is significantly less efficient when editing long fragments. This is because the transformation efficiency of linear DNA templates decreases dramatically with increasing length; on the other hand, because the recombination process relies on the formation of replication forks, the linear DNA template will anneal to the replication forks before recombination can occur, and the range of the replication forks is limited.

Introduction of double-strand breaks into target DNA is an effective strategy to improve gene editing efficiency, because double-strand breaks can maximally activate DNA repair pathways and promote homologous recombination. The enzyme tools for cutting double-stranded DNA are mainly homing endonucleases represented by I-SceI and Cas nucleases represented by Cas 9. When the I-SceI is used for cutting double-stranded DNA, an 18bp recognition site needs to be introduced on a genome firstly, and when the Cas9 is used, the operation is not needed. The Cas9 nuclease can specifically cut the target DNA under the guidance of a single-stranded guide RNA, generating a double-strand break. Meanwhile, a double-strand break introduced by the Cas9 nuclease can kill wild cells which are not subjected to homologous recombination, so that the Cas9 nuclease can be used as a screening tool, and the use of a screening marker gene or a counter-screening system is avoided. In addition, the sequence specificity of reprogramming Cas9 nuclease is simple, only the spacer sequence of single stranded guide RNA needs to be changed.

At present, a plurality of gene editing methods based on the CRISPR-Cas9 technology are reported, the methods are convenient to use and save time, but the problem of low efficiency of large fragment editing is still not fundamentally solved. In many methods, the CRISPR-Cas9 system is only used as a screening tool, and its advantages are not fully utilized. The CRISPR-Cas9 mediated non-homologous end connection method well utilizes the potential of the system, and is very suitable for genome large fragment deletion. However, this approach inevitably introduces random sequence deletions and insertions, and even genomic rearrangements. In addition, this method cannot be used for long segment insertion.

Among all CRISPR-Cas 9-mediated gene editing methods, the CRISPR-Cas 9-mediated recombineering method is more advantageous. The CRISPR-Cas 9-mediated recombination engineering method can utilize circular double-stranded DNA (plasmid DNA) as an editing template, and can also utilize linear DNA (double-stranded DNA obtained by PCR or chemically synthesized single-stranded DNA) as an editing template, and the two strategies have advantages respectively. The circular editing template can not only avoid being degraded by DNA exonuclease in cells, but also increase the copy number of the circular editing template along with the replication of plasmids, thereby greatly improving the recombination efficiency and the editing efficiency. However, during homologous recombination, the circular editing template is easily integrated into the genome, and these recombination events are difficult to distinguish from the recombination events we want by conventional PCR methods, thereby increasing the false positive rate. Linear editing of the template avoids the problem of plasmid integration into the genome and thus increases the positive rate. However, the linear editing template is easily degraded by DNA exonuclease in the cell, resulting in a decrease in homologous recombination efficiency and editing efficiency.

Therefore, how to solve the problem that high recombination efficiency and high positive rate cannot be achieved simultaneously is a technical problem to be solved in the technical field of bacterial gene editing at present.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a CRISPR-Cas9 gene editing vector for bacteria and application thereof.

The concept principle of the invention is as follows: integrating a linear editing template on a CRISPR-Cas9 gene editing vector, wherein a target sequence of a single-stranded guide RNA (sgRNA) is added at one end or two ends of the editing template; before induction of Cas9 protein and sgRNA expression, the editing template replicates as the gene editing vector replicates; after induction, the Cas9-sgRNA complex cleaves a target sequence on a host genome to generate a DNA double strand break, and meanwhile, the Cas9-sgRNA complex cleaves the target sequences at two ends of an editing template to release a linear editing template; and then, under the action of a recombinase, homologous recombination occurs between the linear editing template and the broken genome, so that genome editing is realized.

It should be noted that, this strategy integrates the advantages of the circular editing template and the linear editing template, and at the same time, it avoids the disadvantages of the circular editing template and the linear editing template. The linear editing template is integrated on the gene editing carrier to be changed into a circular editing template, so that the editing template can be copied along with the copying of the gene editing carrier, and the editing template can be prevented from being degraded by exonuclease of a host. Because the target sequences of the sgrnas are added at one end or both ends of the editing template, the circular editing template is cut by Cas9 to become a linear editing template during gene editing, and the linear editing template and the broken genome undergo homologous recombination, thereby avoiding the occurrence of an event that plasmid DNA is integrated into the genome.

Specifically, the technical scheme adopted by the invention is as follows:

a CRISPR-Cas9 gene editing vector for bacteria, comprising plasmid 1 and plasmid 2;

the plasmid 1 comprises a replication initiation site, a screening marker gene, a Cas9 protein expression element, a recombinant protein expression element and a counter-screening marker gene;

the plasmid 2 comprises a replication initiation site, a selection marker gene, a sgRNA expression element and a homologous recombination element.

Preferably, the replication initiation site of the plasmid 1 is a medium copy replication initiation site, and the replication initiation site of the plasmid 2 is a temperature-sensitive replication initiation site.

The copy replication starting site selected in the plasmid 1 not only can ensure that the expression quantity of the Cas9 is most suitable, but also is convenient for eliminating the plasmid 1 after gene editing. The temperature sensitive replication initiation site is selected for the plasmid 2, so that the plasmid 2 is eliminated after gene editing.

Specifically, in this example, the replication start site of the plasmid 1 is the replication start site of p15A, and the replication start site of the plasmid 2 is the replication start site of pSC 101.

Preferably, the selection marker genes of the plasmid 1 and the plasmid 2 are different antibiotic resistance genes;

specifically, the selectable marker gene of the plasmid 1 and the plasmid 2 may be any one of ampicillin resistance gene, chloramphenicol resistance gene, kanamycin resistance gene, tetracycline resistance gene, spectinomycin resistance gene, and apramycin resistance gene, and the selectable marker gene of the plasmid 1 and the plasmid 2 is different antibiotic resistance genes.

In this example, the selectable marker gene of plasmid 1 is a kanamycin resistance gene, and the selectable marker gene of plasmid 2 is an ampicillin resistance gene.

Preferably, the Cas9 protein expression element consists of a highly stringent inducible promoter, a Cas9 encoding gene, a terminator and a regulatory protein gene; the inducible promoter with strong stringency is P_BADPromoter, rhaP_BADOne of a promoter and a tetA promoter.

Specifically, in this embodiment, the inducible promoter with high stringency is P_BADA promoter.

Preferably, the recombinant protein expression element consists of an inducible promoter, a recombinant protein expression gene, a terminator and a regulatory protein; the inducible promoter may be a promoter with high stringency or a promoter with low stringency.

Specifically, in this embodiment, the inducible promoter in the recombinant protein expression element is the T5 promoter.

Preferably, the sgRNA expression element consists of a highly stringent inducible promoter, a sgRNA coding sequence, and a terminator; the sgRNA expression element comprises the same inducible promoter as the Cas9 protein expression element.

Preferably, the sgRNA recognizes a target DNA of a recipient bacterium, and the target DNA of the recipient bacterium contains 5' - (N)_X-NGG-3' structure, (N)_XDenotes X N, N is A, G, C or T, X is a natural number greater than or equal to 12.

Preferably, the homologous recombination element consists of two sequences homologous to the genome or two sequences homologous to the genome and an insertion sequence located between the two homologous sequences, and one or both ends of the homologous recombination element contain 5' - (N) which can be recognized by the sgRNA_X-NGG-3' structure.

Specifically, in this embodiment, the nucleotide sequence of the plasmid 1 is SEQ ID No. 1; the nucleotide sequence of the plasmid 2 is SEQ ID NO.2 or SEQ ID NO.10 or SEQ ID NO. 21.

The invention provides a gene editing kit for bacteria, which comprises the CRISPR-Cas9 gene editing vector for bacteria.

The invention provides application of a CRISPR-Cas9 gene editing vector for bacteria, which is characterized in that the CRISPR-Cas9 gene editing vector for bacteria is used for gene editing in bacteria.

The invention provides a CRISPR-Cas 9-based gene editing method, which comprises the following brief steps: transferring the plasmid 1 and the plasmid 2 into a bacterial strain to be subjected to gene editing, then selecting a transformant for inoculation and culture, then sequentially inducing a recombinase system and a CRISPR-Cas9 system to express, finally coating the culture on a flat plate and selecting a single colony to verify an editing result.

The invention has the beneficial effects that:

1. the editing efficiency is high: the invention can realize gene editing of any type in bacteria, and can simultaneously edit two genome targets;

2. the positive rate is high: the positive rate of the gene editing can be close to 100 percent by applying the invention;

3. and (4) traceless editing: by applying the invention, accurate sequence editing can be realized, and no irrelevant sequence is left;

4. the period is short: by applying the invention, quick iterative editing can be realized, and each round of editing can be finished in 3 days.

Drawings

FIG. 1 is a schematic diagram of the working principle of the present invention in Escherichia coli;

FIG. 2 is a map of plasmid 1 used for E.coli gene editing;

FIG. 3 is a map of plasmid 2 for E.coli sequence insertion;

FIG. 4 is a map of plasmid 2 for E.coli sequence deletion;

FIG. 5 is a map of plasmid 2 for E.coli sequence replacement;

FIG. 6 shows the results of PCR verification in the E.coli sequence insertion experiment;

FIG. 7 shows the results of PCR verification in E.coli sequence deletion experiments.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents, consumables and apparatus used in the present invention are conventional in the art. Unless otherwise specified, reagents and consumables used in the following examples are commercially available.

The primer synthesis and sequencing services used in the invention are provided by Suzhou Jinzhi Biotechnology GmbH.

Unless otherwise indicated, all techniques used in the present invention that relate to the disciplines of chemistry, microbiology, and the like are conventional in the art.

Example 1: a3 kb foreign sequence was inserted into the lacZ gene of E.coli MG1655 strain.

The plasmid 1 used in this example is shown in FIG. 2 (SEQ ID NO.1) and the plasmid 2 used is shown in FIG. 3 (SEQ ID NO. 2).

Plasmid 1 includes a middle copy replication initiation site p15A, a kanamycin resistance gene, a Cas9 protein expression element, a recombinant protein expression element, and a sucrose-sensitive sacB gene. Cas9 protein expression element L-arabinose-induced and glucose-inhibited P_BADThe promoter, the gene encoding SpCas9, the rrnB T1 terminator and the regulatory protein gene araC. The recombinant protein expression element consists of IPTG induced T5 promoter, recombinant protein expression gene (gam, beta, exo), lambda tL3 terminator and regulatory protein gene lacI.

The plasmid 2 comprises a low-copy temperature-sensitive replication initiation site pSC101, an ampicillin resistance gene, a group of sgRNA expression elements and a homologous recombination element. P induced by L-arabinose and glucose inhibited by sgRNA expression element_BADPromoter, sgRNA coding sequence (spacer, gRNA scaffold), and rrnB T1 terminator. The homologous recombination element consists of two sequences (HA1 and HA2) which are homologous with both sides of a target site on the lacZ gene and are about 500bp and an exogenous sequence to be inserted, wherein the exogenous sequence to be inserted is positioned between the two homologous sequences, and two ends of the homologous recombination element are respectively provided with a target site which is the same as the target site on the lacZ gene. The sequence of HA1 is shown in SEQ ID NO.3, the sequence of HA2 is shown in SEQ ID NO.4, the inserted exogenous sequence is shown in SEQ ID NO.5, the sequence of spacer is shown in SEQ ID NO.6, and the sequence of target site is shown in SEQ ID NO. 7.

Preparation work before gene editing:

(1) compounding antibiotics and inducer

a) Ampicillin (1000X): 100g/L

b) Kanamycin (1000 ×): 50g/L

c)IPTG(100X)：100mM

d) L-arabinose (50X): 1M

e) Glucose (50X): 500g/L

f) Sucrose (50X): 500g/L

(2) Preparation of culture Medium

a) LB culture medium: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride

b) SOC culture medium: 20g/L peptone, 5g/L yeast extract, 0.5g/L sodium chloride, 2.5mM potassium chloride, 10mM magnesium sulfate, 20mM glucose

(3) Preparation of the plate

a) Plate a: final kanamycin concentration 1X

b) Plate B: ampicillin 1X final concentration, kanamycin 1X final concentration, glucose 1X

c) Plate C: ampicillin final concentration 1X, kanamycin final concentration 1X, L-arabinose 0.25X

d) Plate D: final concentration of sucrose of 1X

Gene editing process:

(1) transformation editing plasmid

a) Preparing a strain of Escherichia coli MG1655 into chemically competent cells;

b) plasmid 1 was transferred into MG1655 competent cells and plated on plate A;

c) MG 1655/plasmid 1 was prepared as chemically competent cells;

d) plasmid 2 was transferred into MG 1655/plasmid 1 competent cells and plated on plate B.

(2) Editing of official genes

a) Picking single colony from the plate B, inoculating the single colony into 2ml LB culture medium, and culturing for 2 hours at 30 ℃;

b) mu.l ampicillin, 2. mu.l kanamycin and 20. mu.l IPTG were added to the culture, and the culture was continued at 30 ℃ for 1 hour;

c) adding 40 mu l L-arabinose into the culture, and continuing to culture for 3 hours at 30 ℃;

e) 0.1. mu.l and 1. mu.l of the culture were diluted into 100. mu.l of sterile water, respectively, and all coated on plate C;

f) single colonies were picked from plate C for PCR validation. The verification primer is F1/R1, and the sequences are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9.

The PCR verification result is shown in FIG. 6, the band of the negative control group is 1226bp, and the band of the positive result is 4237 bp.

(3) Erasure editing plasmid

a) If the next round of editing is to be performed, only plasmid 2 needs to be eliminated. The method comprises the following steps: the positive clone is inoculated to 2ml LB culture medium, 2 mul kanamycin is added, the culture is carried out for 12 hours at 40 ℃, then 1 mul bacterial liquid is taken to streak on a plate A, and the single colony obtained on the plate is the colony with the plasmid 2 eliminated.

b) If no further rounds of editing are required, plasmid 1 and plasmid 2 can be eliminated simultaneously. The method comprises the following steps: and inoculating the positive clone into 2ml LB culture medium, culturing at 40 ℃ for 12 hours, then taking 1 mu l of bacterial liquid to streak on a plate D, wherein a single colony obtained on the plate is a colony with the plasmid 1 and the plasmid 2 eliminated.

Example 2: a100 kb sequence (1,449,596-plus 1,549,490) was deleted from the genome of the E.coli MG1655 strain.

The plasmid 1 used in this example is shown in FIG. 2 (SEQ ID NO.1) and the plasmid 2 used is shown in FIG. 4 (SEQ ID NO. 10).

Plasmid 1 is as in example 1.

Plasmid 2 comprises a low-copy temperature-sensitive replication initiation site pSC101, an ampicillin resistance gene, two groups of sgRNA expression elements and a homologous recombination element. P induced by L-arabinose and glucose inhibited by sgRNA expression element_BADPromoter, sgRNA coding sequence (spacer1 or spacer2, gRNA scaffold), and rrnB T1 terminator. The two sgrnas target two target sites at both ends of the 100kb sequence, respectively. The homologous recombination element consists of two sequences (HA1 and HA2) which are homologous with sequences of about 500bp on both sides of a 100kb sequence, and both ends of the homologous recombination element are respectively provided with a target site which is the same as the target sites on both sides of the 100kb sequence. HA1 is shown in SEQ ID NO.11, HA2 is shown in SEQ ID NO.12, spacer1 is shown in SEQ ID NO.13, spacer2 is shown in SEQ ID NO.14, and target site 1 is shown in SEQ ID NO.1415, the sequence of the target site 2 is shown as SEQ ID NO. 16.

The preparation and gene editing procedures before gene editing were the same as in example 1.

The verification primers are F2/R2 and F3/R3, and the sequences are respectively shown as SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. 20. Primers F2/R2 flank the two homologous sequences, respectively, and primers F3/R3 flank the 100kb sequence.

The PCR verification result is shown in FIG. 7, the negative control group F2/R2 has no band in amplification, the F3/R3 has an amplification band of 469bp, the positive result F2/R2 has an amplification band of 1181bp, and the F3/R3 has no band in amplification.

Example 3: a100 kb sequence (1,449,596-plus 1,549,490) was deleted from the genome of the E.coli MG1655 strain, and a 7925bp sequence was inserted.

The plasmid 1 used in this example is shown in FIG. 2 (SEQ ID NO.1) and the plasmid 2 used is shown in FIG. 5 (SEQ ID NO. 21).

Plasmid 1 is as in example 1.

Plasmid 2 comprises a low-copy temperature-sensitive replication initiation site pSC101, an ampicillin resistance gene, two groups of sgRNA expression elements and a homologous recombination element. P induced by L-arabinose and glucose inhibited by sgRNA expression element_BADPromoter, sgRNA coding sequence (spacer1 or spacer2, gRNA scaffold), and rrnB T1 terminator. The two sgrnas target two target sites at both ends of the 100kb sequence, respectively. The homologous recombination element consists of two sequences (HA1 and HA2) which are homologous with 100kb sequences at both sides and have the length of about 500bp and an insertion sequence with the length of 7925bp, wherein the insertion sequence is positioned between the two homologous sequences and contains an isobutanol biosynthetic pathway, and the component elements comprise a T7 promoter, an alsS gene, an ilvC gene, an ilvD gene, a kivD gene, an adhA gene and a T7 terminator. The homologous recombination element has a target site at each end, which is identical to the target site on both sides of the 100kb sequence. HA1 is shown in SEQ ID NO.11, HA2 is shown in SEQ ID NO.12, spacer1 is shown in SEQ ID NO.13, spacer2 is shown in SEQ ID NO.14, target site 1 is shown in SEQ ID NO.15, target site 2 is shown in SEQ ID NO.16, and insertion sequence is shown in SEQ ID NO.22Shown in the figure.

The verification primers are F1/R1, F2/R2 and F3/R3, and the sequences are respectively shown as SEQ ID NO.17, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. 20. Primers F1/R1 flank the two homologous sequences, respectively, and primers F2/R2 flank the 100kb sequence.

The PCR verification result is that the negative control group has no band in F1/R1 amplification, no band in F2/R2 amplification, 469bp in F3/R3 amplification band, 1410bp in positive result F1/R1 amplification band, 1422bp in F2/R2 amplification band and no band in F3/R3 amplification band.

Sequence listing

<110> Shandong Kinghui Biotech Co., Ltd

<120> CRISPR-Cas9 gene editing vector for bacteria and application thereof

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ttaccgaatg gatggatgaa tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa 420

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agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca 4080

aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact 4140

aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa 4200

aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt 4260

actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag 4320

ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct 4380

gaacagcaaa aaaatgaaaa atatcaagtt cctgaattcg attcgtccac aattaaaaat 4440

atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc 4500

actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat 4560

gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc 4620

tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc 4680

ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc 4740

cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca 4800

caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa 4860

tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc 4920

aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc 4980

cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa 5040

tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa 5100

aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg 5160

attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac 5220

acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg 5280

ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac 5340

atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc 5400

cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct 5460

gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc 5520

tacgcagaca aacaatcaac gtttgcgcca agcttcctgc tgaacatcaa aggcaagaaa 5580

acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaaaa 5640

cgcaaaagaa aatgccgata ttgactaccg gaagcagtgc ttcagtcacc tcctagctga 5700

ctcaaatcaa tgcgtgtttc ataaagacca gtgatggatt gatggataag agtggcatct 5760

aaaacttctt ttgtagacgt atatcgttta cgatcaattg ttgtatcaaa atatttaaaa 5820

gcagcgggag ctccaagatt cgtcaacgta aataaatgaa taatattttc tgcttgttca 5880

cgtattggtt tgtctctatg tttgttatat gcactaagaa ctttatctaa attggcatct 5940

gctaaaataa cacgcttaga aaattcactg atttgctcaa taatctcatc taaataatgc 6000

ttatgctgct ccacaaacaa ttgtttttgt tcgttatctt ctggactacc cttcaacttt 6060

tcataatgac tagctaaata taaaaaattc acatatttgc ttggcagagc cagctcattt 6120

cctttttgta attctccggc actagccagc atccgtttac gaccgttttc taactcaaaa 6180

agactatatt taggtagttt aatgattaag tcttttttaa cttccttata tcctttagct 6240

tctaaaaagt caatcggatt tttttcaaag gaacttcttt ccataattgt gatccctagt 6300

aactctttaa cggattttaa cttcttcgat ttcccttttt ccaccttagc aaccactagg 6360

actgaataag ctaccgttgg actatcaaaa ccaccatatt tttttggatc ccagtctttt 6420

ttacgagcaa taagcttgtc cgaatttctt tttggtaaaa ttgactcctt ggagaatccg 6480

cctgtctgta cttctgtttt cttgacaata ttgacttggg gcatggacaa tactttgcgc 6540

actgtggcaa aatctcgccc tttatcccag acaatttctc cagtttcccc attagtttcg 6600

attagagggc gtttgcgaat ctctccattt gcaagtgtaa tttctgtttt gaagaagttc 6660

atgatattag agtaaaagaa atattttgcg gttgctttgc ctatttcttg ctcagactta 6720

gcaatcattt tacgaacatc ataaacttta taatcaccat agacaaactc cgattcaagt 6780

tttggatatt tcttaatcaa agcagttcca acgacggcat ttagatacgc atcatgggca 6840

tgatggtaat tgttaatctc acgtacttta tagaattgga aatcttttcg gaagtcagaa 6900

actaatttag attttaaggt aatcacttta acctctcgaa taagtttatc attttcatcg 6960

tatttagtat tcatgcgact atccaaaatt tgtgccacat gcttagtgat ttggcgagtt 7020

tcaaccaatt ggcgtttgat aaaaccagct ttatcaagtt cactcaaacc tccacgttca 7080

gctttcgtta aattatcaaa cttacgttga gtgattaact tggcgtttag aagttgtctc 7140

caatagtttt tcatcttttt gactacttct tcacttggaa cgttatccga tttaccacga 7200

tttttatcag aacgcgttaa gaccttattg tctattgaat cgtctttaag gaaactttgt 7260

ggaacaatgt gatcgacatc ataatcactt aaacgattaa tatctaattc ttggtccaca 7320

tacatgtctc ttccattttg gagataatag agatagagct tttcattttg caattgagta 7380

ttttcaacag gatgctcttt aagaatctga cttcctaatt ctttgatacc ttcttcgatt 7440

cgtttcatac gctctcgcga atttttctgg cccttttgag ttgtctgatt ttcacgtgcc 7500

atttcaataa cgatattttc tggcttatgc cgccccatta ctttgaccaa ttcatcaaca 7560

acttttacag tctgtaaaat acctttttta atagcagggc taccagctaa atttgcaata 7620

tgttcatgta aactatcgcc ttgtccagac acttgtgctt tttgaatgtc ttctttaaat 7680

gtcaaactat catcatggat cagctgcata aaattgcgat tggcaaaacc atctgatttc 7740

aaaaaatcta atattgtttt gccagattgc ttatccctaa taccattaat caattttcga 7800

gacaaacgtc cccaaccagt ataacggcga cgtttaagct gtttcatcac cttatcatca 7860

aagaggtgag catatgtttt aagtctttcc tcaatcatct ccctatcttc aaataaggtc 7920

aatgttaaaa caatatcctc taagatatct tcattttctt cattatccaa aaaatcttta 7980

tctttaataa tttttagcaa atcatggtag gtacctaatg aagcattaaa tctatcttca 8040

actcctgaaa tttcaacact atcaaaacat tctatttttt tgaaataatc ttcttttaat 8100

tgcttaacgg ttacttttcg atttgttttg aagagtaaat caacaatggc tttcttctgt 8160

tcacctgaaa gaaatgctgg ttttcgcatt ccttcagtaa catatttgac ctttgtcaat 8220

tcgttataaa ccgtaaaata ctcataaagc aaactatgtt ttggtagtac tttttcattt 8280

ggaagatttt tatcaaagtt tgtcatgcgt tcaataaatg attgagctga agcaccttta 8340

tcgacaactt cttcaaaatt ccatggggta attgtttctt cagacttccg agtcatccat 8400

gcaaaacgac tattgccacg cgccaatgga ccaacataat aaggaattcg aaaagtcaag 8460

attttttcaa tcttctcacg attgtctttt aaaaatggat aaaagtcttc ttgtcttctc 8520

aaaatagcat gcagctcacc caagtgaatt tgatggggaa tagagccgtt gtcaaaggtc 8580

cgttgcttgc gcagcaaatc ttcacgattt agtttcacca ataattcctc agtaccatcc 8640

attttttcta aaattggttt gataaattta taaaattctt cttggctagc tcccccatca 8700

atataacctg catatccgtt ttttgattga tcaaaaaaga tttctttata cttttctgga 8760

agttgttgtc gaactaaagc ttttaaaaga gtcaagtctt gatgatgttc atcgtagcgt 8820

ttaatcattg aagctgatag gggagcctta gttatttcag tatttactct taggatatct 8880

gaaagtaaaa tagcatctga taaattctta gctgccaaaa acaaatcagc atattgatct 8940

ccaatttgcg ccaataaatt atctaaatca tcatcgtaag tatcttttga aagctgtaat 9000

ttagcatctt ctgccaaatc aaaatttgat ttaaaattag gggtcaaacc caatgacaaa 9060

gcaatgagat tcccaaataa gccatttttc ttctcaccgg ggagctgagc aatgagattt 9120

tctaatcgtc ttgatttact caatcgtgca gaaagaatcg ctttagcatc tactccactt 9180

gcgttaatag ggttttcttc aaataattga ttgtaggttt gtaccaactg gataaatagt 9240

ttgtccacat cactattatc aggatttaaa tctccctcaa tcaaaaaatg accacgaaac 9300

ttaatcatat gcgctaaggc caaatagatt aagcgcaaat ccgctttatc agtagaatct 9360

accaattttt ttcgcagatg atagatagtt ggatatttct catgataagc aacttcatct 9420

actatatttc caaaaatagg atgacgttca tgcttcttgt cttcttccac caaaaaagac 9480

tcttcaagtc gatgaaagaa actatcatct actttcgcca tctcatttga aaaaatctcc 9540

tgtagataac aaatacgatt cttccgacgt gtataccttc tacgagctgt ccgtttgaga 9600

cgagtcgctt ccgctgtctc tccactgtca aataaaagag cccctataag attttttttg 9660

atactgtggc ggtctgtatt tcccagaacc ttgaactttt tagacggaac cttatattca 9720

tcagtgatca ccgcccatcc gacgctattt gtgccgatat ctaagcctat tgagtatttc 9780

ttatccattt tttataacct ccttagagct cgaattccca aaaaaacggg tatggagaaa 9840

cagtagagag ttgcgataaa aagcgtcagg taggatccgc taatcttatg gataaaaatg 9900

ctatggcata gcaaagtgtg acgccgtgca aataatcaat gtggactttt ctgccgtgat 9960

tatagacact tttgttacgc gtttttgtca tggctttggt cccgctttgt tacagaatgc 10020

ttttaataag cggggttacc ggtttggtta gcgagaagag ccagtaaaag acgcagtgac 10080

ggcaatgtct gatgcaatat ggacaattgg tttcttctct gaatggcggg agtatgaaaa 10140

gtatggctga agcgcaaaat gatcccctgc tgccgggata ctcgtttaat gcccatctgg 10200

tggcgggttt aacgccgatt gaggccaacg gttatctcga tttttttatc gaccgaccgc 10260

tgggaatgaa aggttatatt ctcaatctca ccattcgcgg tcagggggtg gtgaaaaatc 10320

agggacgaga atttgtttgc cgaccgggtg atattttgct gttcccgcca ggagagattc 10380

atcactacgg tcgtcatccg gaggctcgcg aatggtatca ccagtgggtt tactttcgtc 10440

cgcgcgccta ctggcatgaa tggcttaact ggccgtcaat atttgccaat acggggttct 10500

ttcgcccgga tgaagcgcac cagccgcatt tcagcgacct gtttgggcaa atcattaacg 10560

ccgggcaagg ggaagggcgc tattcggagc tgctggcgat aaatctgctt gagcaattgt 10620

tactgcggcg catggaagcg attaacgagt cgctccatcc accgatggat aatcgggtac 10680

gcgaggcttg tcagtacatc agcgatcacc tggcagacag caattttgat atcgccagcg 10740

tcgcacagca tgtttgcttg tcgccgtcgc gtctgtcaca tcttttccgc cagcagttag 10800

ggattagcgt cttaagctgg cgcgaggacc aacgtatcag ccaggcgaag ctgcttttga 10860

gcaccacccg gatgcctatc gccaccgtcg gtcgcaatgt tggttttgac gatcaactct 10920

atttctcgcg ggtatttaaa aaatgcaccg gggccagccc gagcgagttc cgtgccggtt 10980

gtgaagaaaa agtgaatgat gtagccgtca agttgtcata ataaatcgat gcaggtggca 11040

cttttcgggg aaatgtggag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc 11100

tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccgccc 11160

tagacctagg gcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 11220

ttatccacag aatcagggga taacgcagga aagagcatgt gagcaaaagg ccagcaaaag 11280

gccaggaacc gtggatatat tccgcttcct cgctcactga ctcgctacgc tcggtcgttc 11340

gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa gatgccagga 11400

agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc 11460

ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact 11520

ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt 11580

tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac 11640

tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt 11700

ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa 11760

aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc 11820

gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta 11880

cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt 11940

ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt 12000

attaataagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 12060

acgaaaactc acgttaaggg attttggtca tgactagtgc ttggattctc accaataaaa 12120

aacgcccggc ggcaaccgat ttcaagttga taacggacta gccttatttt aacttgctat 12180

gctgttttga atggttccaa caagattatt ttataacttt tataacaaat aatcaaggag 12240

aaattcaaag aaatttatca gccgtgtcgc ccttaattgt gagcggataa caattacgag 12300

cttcatgcac agtgaaatca tgaaaaattt atttgctttg tgagcggata acaattataa 12360

tatgtggaat tgtgagcgct cacaattcca caacggtttc cctctagaaa taattttgtt 12420

taacttttcg agaccttagg aggtaaacat atggatatta atactgaaac tgagatcaag 12480

caaaagcatt cactaacccc ctttcctgtt ttcctaatca gcccggcatt tcgcgggcga 12540

tattttcaca gctatttcag gagttcagcc atgaacgctt attacattca ggatcgtctt 12600

gaggctcaga gctgggcgcg tcactaccag cagctcgccc gtgaagagaa agaggcagaa 12660

ctggcagacg acatggaaaa aggcctgccc cagcacctgt ttgaatcgct atgcatcgat 12720

catttgcaac gccacggggc cagcaaaaaa tccattaccc gtgcgtttga tgacgatgtt 12780

gagtttcagg agcgcatggc agaacacatc cggtacatgg ttgaaaccat tgctcaccac 12840

caggttgata ttgattcaga ggtataa 12867

<210> 2

<211> 7998

<212> DNA

<213> Artificial sequence (Manual sequence)

<400> 2

tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag 60

ccatgagaac gaaccattga gatcatactt actttgcatg tcactcaaaa attttgcctc 120

aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt 180

acgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg 240

gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa 300

cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa 360

atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt 420

ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt 480

tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc 540

aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg 600

caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt 660

ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat 720

cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat 780

ctgagcgtat tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat 840

cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc 900

atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa 960

ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat gataattact 1020

agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg ctggaaaact 1080

tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta 1140

tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga 1200

tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc 1260

gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac 1320

cctcgcaagc tcggttgcgg ccgcaatcgg gcaaatcgct gaatattcct tttgtctccg 1380

accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc gctcacggct 1440

ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca tgcaaggaaa 1500

ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat ggcgggtctg 1560

ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga ttttccagtc 1620

tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac ccagtaaggc 1680

agcggtatca tcaacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 1740

ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 1800

taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 1860

tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 1920

cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 1980

gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 2040

cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 2100

ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 2160

aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 2220

atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 2280

tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 2340

gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 2400

aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 2460

acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 2520

ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 2580

tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 2640

aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 2700

catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 2760

atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca gtatccccgt 2820

ttacagggcg gagcgtcaca ctgaggtttt ccgccagacg ccactgctgc caggcgctga 2880

tgtgcccggc ttctgaccat gcggtcgcgt tcggttgcac tacgcgtact gtgagccaga 2940

gttgcccggc gctctccggc tgcggtagtt caggcagttc aatcaactgt ttaccttgtg 3000

gagcgacatc cagaggcact tcaccgcttg ccagcggctt accatccagc gccaccatcc 3060

agtgcaggag ctcgttatcg ctatgacgga acaggtattc gctggtcact tcgatggttt 3120

gcccggataa acggaactgg aaaaactgct gctggtgttt tgcttccgtc agcgctggat 3180

gcggcgtgcg gtcggcaaag accagaccgt tcatacagaa ctggcgatcg ttcggcgtat 3240

cgccaaaatc accgccgtaa gccgaccacg ggttgccgtt ttcatcatat ttaatcagcg 3300

actgatccac ccagtcccag acgaagccgc cctttacgct ccatctgagc ctcatgtaca 3360

cgaagcccca gttcaagaag cattgaagct gttgctgaaa aactgacatc tttttccctt 3420

gccccttcct gtcgacgctt ctcaataatc gcatttattt tttcataggc atcattgctg 3480

atatacaggt tcaccttagc cattagagat tacctttttt gaacataaaa taaagttaat 3540

ttcgaaacac taaaattcat aatgcaaaca gggacgcacc gctagcagcg cccctagcgg 3600

tatcctataa aaaaacacac cgcgccgcta gcagcacccc taatataaaa taatgttttt 3660

tataaaaata gtcagtacca cccctacaaa acggtgtcgg cgcgttgttg tagccgcgcc 3720

gacaccgctt ttttaaatat cataaagaga gtaagagaaa ctaatttttc ataacactct 3780

atttataaag aaaaatcagc aaaaacttgt ttttgcgtgg ggtgtggtgc ttttggtggt 3840

gagaaccacc aacctgttga gcctttttgt ggagtgggtt aaattattta cgaataaagt 3900

caccagaggt ggaaaaatga aaaaatggat gttagccatc tgcctgatgt ttataaatga 3960

gatctgccat gccactgatt gctttgatct tgcaggccgg gattacaaaa tagatcctga 4020

tttactgaga gcaatatcat ggaaagaatc ccgttaccgg gttaatgcca tcggtattaa 4080

tccggtaacg ggatatggca gcggactgat gcaggtagat tcccagcatt ttaacgaact 4140

ggcccgctat ggaattaagc cggaacatct gacaacagat ccctgcatga acatttatac 4200

cggtgcttat tatctggcaa tagcctttaa aaaatggggc gtctcctggg aggccgttgg 4260

tgcatacaat gctggattca ggaagaccga acgccagaac cagagacgtc ttgcctacgc 4320

atcagatgtt taccggattt ataccgggat aaaaagcagt aaaagtatcc ggattccggc 4380

cacgaagaaa tcacttccgg aaattaacag cgtgcagaac aattaatctt acaggcattg 4440

tcagataccg gttatgccgc aaaagcggca tggaaggcca cacacaaaaa tatcatatga 4500

gtgacaatac tgactggccg aagccccgcg aagcagtaac ccatcttata tatacccggt 4560

aactgcgcag cctcctgcca gaccattttt cgcgggcaca atatttacag acaaccgaaa 4620

agccagttga tgaaattgag gtatatcaga cgacacagcg atgccgaaga atgataatat 4680

aagtaaagcc ccgaaatttt ttcggggctt tacttatatt atcatgactt ccactttctg 4740

aactgttcgg caatcaccca caacgccttg ttgattctta tatcaccatc aatacctgta 4800

atggctcttg tcctggtatt tttcccggag gcacttcgcc ccgataatcc accttttatc 4860

atattctcct gaacccgctg ccaggttgtc cagagatcgt tctgtttatc ctcccaacga 4920

cggggagtaa ttatttgttc aggcgtcact ggcgttttat tttcatcttc atacctgacc 4980

atcagtgcag ctctgccaaa taaatgttgc tcgtcactgt taagatgaat ttctttcatc 5040

gcctccatat tatcagtgac cttatcaaag acaccgagca cttcatacgc gccctctata 5100

acctgcccga caatatcacc tttatgtgga acgcggattt cgccaaaatt atttccgcac 5160

accaggccat ttgtgcagac aaaacgaaaa attccgggga tcatctgata actggatgaa 5220

ccatcatgtg aattaagcag gataatttca ggaacttcct gcccgttaat atgcccttcc 5280

ctgcgaagac gtaacatatg tttactgtat tcgcgacgtc ccaaatcacg aacccgactc 5340

tgacaggcaa agaatggctg gaaaccttca tcacgtaact tattgatgat attgattgtt 5400

ggaatatacg tataacgttc actccgggac tcatgtttat caccggaaaa taccgaaggc 5460

acgaactgca ttaattcatc atccgttaaa ggacgttcac ggtggatgga attataccga 5520

ccaaaacggg aagctaatcg cataacagct actcctttat gaaagaataa atttaataaa 5580

aaatttttcc gcttaatatc atcccccctg gtcacaatta acaagattgc aacaatcagg 5640

agggatattc atcacatccg gtcagcagat cgcccggata tcgaggccgg cacgcttaat 5700

ctgacgcagg aactgtttga agtgatcggc ttccgcaatg ttgatatggc ggaactgcgt 5760

atccacaccc cggctgagcg cgtagctata gctgaaggcc atcttttccg ggtggctttt 5820

gcgatatttt ttcagctcag tcaggctgct gaattccagg acgtcccact cttccccggt 5880

gttttttttc ttcccggcaa caagaagcgt gtacgacgcg gtgttaacgt ggatatttcg 5940

ggtactcatc ctgctttctc ctctctctgg ttttcagccc ccatcatctt ttgagtttcg 6000

ggagccgcgg ttttgacgtt ttgcggggac ctgccttttg ttccctccgg tattcaccat 6060

cacctttttg agaaccttcg ccgcaagggg catcactgcc acggccggaa acgactgtcc 6120

gggggcagac cgcaaaactt tttgcctgct tttgggcaaa aagttttgcg ggatgtgcat 6180

ttcacccctg gactggcgga gacggttgtg gctgtctttg cccccggtga caggggctta 6240

aacaggtgaa tggcaccgga cacaacaacg ggcaggcacc cgccgcaacg acgtgaagcg 6300

cggtttgcga aacggcgttg ccccttacat cccggtggcc ttttgtaaac ggggatactg 6360

acgaaacgcc tgccagtatt tagcgaaacc gccaagactg ttacccatcg cgtgggcgta 6420

ttcgcaaagg atcagcgggc gcgtctctcc aggtagcgaa agccattttt tgatggacca 6480

tttcggcaca gccgggaagg gctggtcttc atccacgcgc gcgtacatcg ggcaaataat 6540

atcggtggcc gtggtgtcgg ctccgccgcc ttcatactgc accgggcggg aaggatcgac 6600

agatttgatc cagcgataca gcgcgtcgtg attagcgccg tggcctgatt cattccccag 6660

cgaccagatg atcacactcg ggtgattacg atcgcgctgc accattcgcg ttacgcgttc 6720

gctcatcgcc ggtagccagc gcggatcatc ggtcagacga ttcattggca ccatgccgtg 6780

ggtttcaata ttggcttcat ccaccacata caggccgtag cggtcgcaca gcgtgtacca 6840

cagcggatgg ttcggataat gcgaacagcg ccgccctgta aacggggata ctggcacaga 6900

tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg 6960

gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct 7020

gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 7080

gccagtgcca agcttgcatg cctgcaggtc gactctagag gatccccggg taccgagctc 7140

gaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 7200

cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 7260

ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcataggga 7320

taacagggta atacttttca tactcccgcc attcagagaa gaaaccaatt gtccatattg 7380

catcagacat tgccgtcact gcgtctttta ctggctcttc tcgctaacca aaccggtaac 7440

cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa gccatgacaa aaacgcgtaa 7500

caaaagtgtc tataatcacg gcagaaaagt ccacattgat tatttgcacg gcgtcacact 7560

ttgctatgcc atagcatttt tatccataag attagcggat cctacctgac gctttttatc 7620

gcaactctct actgtttctc catcagtatc cccgtttaca ggggttttag agctagaaat 7680

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 7740

tagcatccaa actcgagtaa ggatcattaa ggatcccatg gtacgcgtgc tagaggcatc 7800

aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg 7860

tgaacgctct cctgagtagg acaaatccgc cccatgggta tggacagttt tccctttgat 7920

atgtaacggt gaacagttgt tctacttttg tttgttagtc ttgatgcttc actgatagat 7980

acaagagcca taagaacc 7998

<210> 3

<211> 501

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 3

agcgtcacac tgaggttttc cgccagacgc cactgctgcc aggcgctgat gtgcccggct 60

tctgaccatg cggtcgcgtt cggttgcact acgcgtactg tgagccagag ttgcccggcg 120

ctctccggct gcggtagttc aggcagttca atcaactgtt taccttgtgg agcgacatcc 180

agaggcactt caccgcttgc cagcggctta ccatccagcg ccaccatcca gtgcaggagc 240

tcgttatcgc tatgacggaa caggtattcg ctggtcactt cgatggtttg cccggataaa 300

cggaactgga aaaactgctg ctggtgtttt gcttccgtca gcgctggatg cggcgtgcgg 360

tcggcaaaga ccagaccgtt catacagaac tggcgatcgt tcggcgtatc gccaaaatca 420

ccgccgtaag ccgaccacgg gttgccgttt tcatcatatt taatcagcga ctgatccacc 480

cagtcccaga cgaagccgcc c 501

<210> 4

<211> 527

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 4

tgtaaacggg gatactgacg aaacgcctgc cagtatttag cgaaaccgcc aagactgtta 60

cccatcgcgt gggcgtattc gcaaaggatc agcgggcgcg tctctccagg tagcgaaagc 120

cattttttga tggaccattt cggcacagcc gggaagggct ggtcttcatc cacgcgcgcg 180

tacatcgggc aaataatatc ggtggccgtg gtgtcggctc cgccgccttc atactgcacc 240

gggcgggaag gatcgacaga tttgatccag cgatacagcg cgtcgtgatt agcgccgtgg 300

cctgattcat tccccagcga ccagatgatc acactcgggt gattacgatc gcgctgcacc 360

attcgcgtta cgcgttcgct catcgccggt agccagcgcg gatcatcggt cagacgattc 420

attggcacca tgccgtgggt ttcaatattg gcttcatcca ccacatacag gccgtagcgg 480

tcgcacagcg tgtaccacag cggatggttc ggataatgcg aacagcg 527

<210> 5

<211> 3011

<212> DNA

<213> Escherichia coli XL1-blue (Escherichia coli XL1-blue)

<400> 5

tttacgctcc atctgagcct catgtacacg aagccccagt tcaagaagca ttgaagctgt 60

tgctgaaaaa ctgacatctt tttcccttgc cccttcctgt cgacgcttct caataatcgc 120

atttattttt tcataggcat cattgctgat atacaggttc accttagcca ttagagatta 180

ccttttttga acataaaata aagttaattt cgaaacacta aaattcataa tgcaaacagg 240

gacgcaccgc tagcagcgcc cctagcggta tcctataaaa aaacacaccg cgccgctagc 300

agcaccccta atataaaata atgtttttta taaaaatagt cagtaccacc cctacaaaac 360

ggtgtcggcg cgttgttgta gccgcgccga caccgctttt ttaaatatca taaagagagt 420

aagagaaact aatttttcat aacactctat ttataaagaa aaatcagcaa aaacttgttt 480

ttgcgtgggg tgtggtgctt ttggtggtga gaaccaccaa cctgttgagc ctttttgtgg 540

agtgggttaa attatttacg aataaagtca ccagaggtgg aaaaatgaaa aaatggatgt 600

tagccatctg cctgatgttt ataaatgaga tctgccatgc cactgattgc tttgatcttg 660

caggccggga ttacaaaata gatcctgatt tactgagagc aatatcatgg aaagaatccc 720

gttaccgggt taatgccatc ggtattaatc cggtaacggg atatggcagc ggactgatgc 780

aggtagattc ccagcatttt aacgaactgg cccgctatgg aattaagccg gaacatctga 840

caacagatcc ctgcatgaac atttataccg gtgcttatta tctggcaata gcctttaaaa 900

aatggggcgt ctcctgggag gccgttggtg catacaatgc tggattcagg aagaccgaac 960

gccagaacca gagacgtctt gcctacgcat cagatgttta ccggatttat accgggataa 1020

aaagcagtaa aagtatccgg attccggcca cgaagaaatc acttccggaa attaacagcg 1080

tgcagaacaa ttaatcttac aggcattgtc agataccggt tatgccgcaa aagcggcatg 1140

gaaggccaca cacaaaaata tcatatgagt gacaatactg actggccgaa gccccgcgaa 1200

gcagtaaccc atcttatata tacccggtaa ctgcgcagcc tcctgccaga ccatttttcg 1260

cgggcacaat atttacagac aaccgaaaag ccagttgatg aaattgaggt atatcagacg 1320

acacagcgat gccgaagaat gataatataa gtaaagcccc gaaatttttt cggggcttta 1380

cttatattat catgacttcc actttctgaa ctgttcggca atcacccaca acgccttgtt 1440

gattcttata tcaccatcaa tacctgtaat ggctcttgtc ctggtatttt tcccggaggc 1500

acttcgcccc gataatccac cttttatcat attctcctga acccgctgcc aggttgtcca 1560

gagatcgttc tgtttatcct cccaacgacg gggagtaatt atttgttcag gcgtcactgg 1620

cgttttattt tcatcttcat acctgaccat cagtgcagct ctgccaaata aatgttgctc 1680

gtcactgtta agatgaattt ctttcatcgc ctccatatta tcagtgacct tatcaaagac 1740

accgagcact tcatacgcgc cctctataac ctgcccgaca atatcacctt tatgtggaac 1800

gcggatttcg ccaaaattat ttccgcacac caggccattt gtgcagacaa aacgaaaaat 1860

tccggggatc atctgataac tggatgaacc atcatgtgaa ttaagcagga taatttcagg 1920

aacttcctgc ccgttaatat gcccttccct gcgaagacgt aacatatgtt tactgtattc 1980

gcgacgtccc aaatcacgaa cccgactctg acaggcaaag aatggctgga aaccttcatc 2040

acgtaactta ttgatgatat tgattgttgg aatatacgta taacgttcac tccgggactc 2100

atgtttatca ccggaaaata ccgaaggcac gaactgcatt aattcatcat ccgttaaagg 2160

acgttcacgg tggatggaat tataccgacc aaaacgggaa gctaatcgca taacagctac 2220

tcctttatga aagaataaat ttaataaaaa atttttccgc ttaatatcat cccccctggt 2280

cacaattaac aagattgcaa caatcaggag ggatattcat cacatccggt cagcagatcg 2340

cccggatatc gaggccggca cgcttaatct gacgcaggaa ctgtttgaag tgatcggctt 2400

ccgcaatgtt gatatggcgg aactgcgtat ccacaccccg gctgagcgcg tagctatagc 2460

tgaaggccat cttttccggg tggcttttgc gatatttttt cagctcagtc aggctgctga 2520

attccaggac gtcccactct tccccggtgt tttttttctt cccggcaaca agaagcgtgt 2580

acgacgcggt gttaacgtgg atatttcggg tactcatcct gctttctcct ctctctggtt 2640

ttcagccccc atcatctttt gagtttcggg agccgcggtt ttgacgtttt gcggggacct 2700

gccttttgtt ccctccggta ttcaccatca cctttttgag aaccttcgcc gcaaggggca 2760

tcactgccac ggccggaaac gactgtccgg gggcagaccg caaaactttt tgcctgcttt 2820

tgggcaaaaa gttttgcggg atgtgcattt cacccctgga ctggcggaga cggttgtggc 2880

tgtctttgcc cccggtgaca ggggcttaaa caggtgaatg gcaccggaca caacaacggg 2940

caggcacccg ccgcaacgac gtgaagcgcg gtttgcgaaa cggcgttgcc ccttacatcc 3000

cggtggcctt t 3011

<210> 6

<211> 17

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 6

cagtatcccc gtttaca 17

<210> 7

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 7

cagtatcccc gtttacaggg 20

<210> 8

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 8

ctgactggcg gttaaattgc 20

<210> 9

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 9

taaccgtcac gagcatcatc 20

<210> 10

<211> 5285

<212> DNA

<213> Artificial sequence (Manual sequence)

<400> 10

acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg 60

ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa 120

aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta 180

taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat 240

agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac 300

tgtttctcca tgaagtggct aaagagaaca agttttagag ctagaaatag caagttaaaa 360

taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgctta gcatccaaac 420

tcgagtaagg atcattaagg atcccatggt acgcgtgcta gaggcatcaa ataaaacgaa 480

aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc 540

tgagtaggac aaatccgccc catgggtatg gacagttttc cctttgatat gtaacggtga 600

acagttgttc tacttttgtt tgttagtctt gatgcttcac tgatagatac aagagccata 660

agaacctcag atccttccgt atttagccag tatgttctct agtgtggttc gttgtttttg 720

cgtgagccat gagaacgaac cattgagatc atacttactt tgcatgtcac tcaaaaattt 780

tgcctcaaaa ctggtgagct gaatttttgc agttaaagca tcgtgtagtg tttttcttag 840

tccgttatgt aggtaggaat ctgatgtaat ggttgttggt attttgtcac cattcatttt 900

tatctggttg ttctcaagtt cggttacgag atccatttgt ctatctagtt caacttggaa 960

aatcaacgta tcagtcgggc ggcctcgctt atcaaccacc aatttcatat tgctgtaagt 1020

gtttaaatct ttacttattg gtttcaaaac ccattggtta agccttttaa actcatggta 1080

gttattttca agcattaaca tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt 1140

gtgagttttc ttttgtgtta gttcttttaa taaccactca taaatcctca tagagtattt 1200

gttttcaaaa gacttaacat gttccagatt atattttatg aattttttta actggaaaag 1260

ataaggcaat atctcttcac taaaaactaa ttctaatttt tcgcttgaga acttggcata 1320

gtttgtccac tggaaaatct caaagccttt aaccaaagga ttcctgattt ccacagttct 1380

cgtcatcagc tctctggttg ctttagctaa tacaccataa gcattttccc tactgatgtt 1440

catcatctga gcgtattggt tataagtgaa cgataccgtc cgttctttcc ttgtagggtt 1500

ttcaatcgtg gggttgagta gtgccacaca gcataaaatt agcttggttt catgctccgt 1560

taagtcatag cgactaatcg ctagttcatt tgctttgaaa acaactaatt cagacataca 1620

tctcaattgg tctaggtgat tttaatcact ataccaattg agatgggcta gtcaatgata 1680

attactagtc cttttccttt gagttgtggg tatctgtaaa ttctgctaga cctttgctgg 1740

aaaacttgta aattctgcta gaccctctgt aaattccgct agacctttgt gtgttttttt 1800

tgtttatatt caagtggtta taatttatag aataaagaaa gaataaaaaa agataaaaag 1860

aatagatccc agccctgtgt ataactcact actttagtca gttccgcagt attacaaaag 1920

gatgtcgcaa acgctgtttg ctcctctaca aaacagacct taaaacccta aaggcttaag 1980

tagcaccctc gcaagctcgg ttgcggccgc aatcgggcaa atcgctgaat attccttttg 2040

tctccgacca tcaggcacct gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc 2100

acggctctgg cagtgaatgg gggtaaatgg cactacaggc gccttttatg gattcatgca 2160

aggaaactac ccataataca agaaaagccc gtcacgggct tctcagggcg ttttatggcg 2220

ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt 2280

ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag 2340

taaggcagcg gtatcatcaa cggggtctga cgctcagtgg aacgaaaact cacgttaagg 2400

gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 2460

aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagta gggataacag 2520

ggtaatactt ttcatactcc cgccattcag agaagaaacc aattgtccat attgcatcag 2580

acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg taaccccgct 2640

tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc gtaacaaaag 2700

tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca cactttgcta 2760

tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt tatcgcaact 2820

ctctactgtt tctccatgta aaaccgaata ctgccgggtt ttagagctag aaatagcaag 2880

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttagcat 2940

ccaaactcga gtaaggatca ttaaggatcc catggtacgc gtgctagagg catcaaataa 3000

aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg 3060

ctctcctgag taggacaaat ccgccctgca tgtgtcagag gttttcaccg tcatcaccga 3120

aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 3180

taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgttggcg tttcgtcaaa 3240

gaagttccat ggtttcagca gagtatgtac ccattcggtc ggcataatcg gccactcttc 3300

ggcgcgggcc acatgtgtgg tgccggtggt catccagaca acggcgtcgg tgttgtccag 3360

cgactcgtta tccttactgt attgtccaag accggtgtca tgagtagaac ggttcggata 3420

tttgccttcc gggaaacgct cgccaggatg ataacgcgtt acccagagct gcttgtccat 3480

aaagcttaaa cgatgataga tccactcgtc cggcgcgaac tgggcacctt ttgctaccgg 3540

gtgagtacca cctgcataag gaataatttg ataggaaacc ggattgccca tgcggttctc 3600

tttgttcggg ttactcaaca gacgaatcgt gcccggatca aatttctgtg cggcatcctg 3660

ttcattgccg atgttgtact gattaacttg catggtactg gtgcgtgggc caccgcaacg 3720

gctacgcgct ggaagatctc tatgacgcta atggcgtgct tattgcgaag aaaggtcagt 3780

tgctgagtag ctttgcgcat ctgcgtgatg acggtacaac cgcatcttct tgctggatct 3840

acaccggtag ctggacagag cagggcaacc agatggctaa ccgcgataac tccgacccgt 3900

ccggtctggg gaatacgctg ggatgggcct gggcgtggcc gctcaaccgt cgcgtgctgt 3960

acaaccgtgc ttcggcggat atcaacggta aaccgtggga tccgaaacgg atgctgatcc 4020

agtggaacgg cagcaagtgg acgggtaacg atattcctga cttcggcaat gccgcaccgg 4080

gtacgccaac cgggccgttt atcatgcagc cggaagggat gggacgcctg tttgccatca 4140

acaaaatggc ggaaggtccg ttcccggaac actacgagcc gattgaacgc ggaaccccta 4200

tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 4260

aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 4320

ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 4380

aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 4440

acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 4500

ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 4560

gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 4620

atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 4680

acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 4740

tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 4800

ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 4860

aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 4920

aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 4980

ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 5040

atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 5100

aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 5160

accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 5220

tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 5280

tccac 5285

<210> 11

<211> 491

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 11

ttggcgtttc gtcaaagaag ttccatggtt tcagcagagt atgtacccat tcggtcggca 60

taatcggcca ctcttcggcg cgggccacat gtgtggtgcc ggtggtcatc cagacaacgg 120

cgtcggtgtt gtccagcgac tcgttatcct tactgtattg tccaagaccg gtgtcatgag 180

tagaacggtt cggatatttg ccttccggga aacgctcgcc aggatgataa cgcgttaccc 240

agagctgctt gtccataaag cttaaacgat gatagatcca ctcgtccggc gcgaactggg 300

caccttttgc taccgggtga gtaccacctg cataaggaat aatttgatag gaaaccggat 360

tgcccatgcg gttctctttg ttcgggttac tcaacagacg aatcgtgccc ggatcaaatt 420

tctgtgcggc atcctgttca ttgccgatgt tgtactgatt aacttgcatg gtactggtgc 480

gtgggccacc g 491

<210> 12

<211> 472

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 12

caacggctac gcgctggaag atctctatga cgctaatggc gtgcttattg cgaagaaagg 60

tcagttgctg agtagctttg cgcatctgcg tgatgacggt acaaccgcat cttcttgctg 120

gatctacacc ggtagctgga cagagcaggg caaccagatg gctaaccgcg ataactccga 180

cccgtccggt ctggggaata cgctgggatg ggcctgggcg tggccgctca accgtcgcgt 240

gctgtacaac cgtgcttcgg cggatatcaa cggtaaaccg tgggatccga aacggatgct 300

gatccagtgg aacggcagca agtggacggg taacgatatt cctgacttcg gcaatgccgc 360

accgggtacg ccaaccgggc cgtttatcat gcagccggaa gggatgggac gcctgtttgc 420

catcaacaaa atggcggaag gtccgttccc ggaacactac gagccgattg aa 472

<210> 13

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 13

gaagtggcta aagagaacaa 20

<210> 14

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 14

gtaaaaccga atactgccgg 20

<210> 15

<211> 23

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 15

gtaaaaccga atactgccgg tgg 23

<210> 16

<211> 23

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 16

gaagtggcta aagagaacaa cgg 23

<210> 17

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 17

gtcggtgtgt gtacggtatt 20

<210> 18

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 18

acatacggga actgctcttt 20

<210> 19

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 19

cggtcgatat gcggatgtat 20

<210> 20

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 20

tctcctgctg caggattttg 20

<210> 21

<211> 13256

<212> DNA

<213> Artificial sequence (Manual sequence)

<400> 21

acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg 60

ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa 120

aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta 180

taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat 240

agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac 300

tgtttctcca tgtaaaaccg aatactgccg ggttttagag ctagaaatag caagttaaaa 360

taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgctta gcatccaaac 420

tcgagtaagg atcattaagg atcccatggt acgcgtgcta gaggcatcaa ataaaacgaa 480

aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc 540

tgagtaggac aaatccgccc catgggtatg gacagttttc cctttgatat gtaacggtga 600

acagttgttc tacttttgtt tgttagtctt gatgcttcac tgatagatac aagagccata 660

agaacctcag atccttccgt atttagccag tatgttctct agtgtggttc gttgtttttg 720

cgtgagccat gagaacgaac cattgagatc atacttactt tgcatgtcac tcaaaaattt 780

tgcctcaaaa ctggtgagct gaatttttgc agttaaagca tcgtgtagtg tttttcttag 840

tccgttatgt aggtaggaat ctgatgtaat ggttgttggt attttgtcac cattcatttt 900

tatctggttg ttctcaagtt cggttacgag atccatttgt ctatctagtt caacttggaa 960

aatcaacgta tcagtcgggc ggcctcgctt atcaaccacc aatttcatat tgctgtaagt 1020

gtttaaatct ttacttattg gtttcaaaac ccattggtta agccttttaa actcatggta 1080

gttattttca agcattaaca tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt 1140

gtgagttttc ttttgtgtta gttcttttaa taaccactca taaatcctca tagagtattt 1200

gttttcaaaa gacttaacat gttccagatt atattttatg aattttttta actggaaaag 1260

ataaggcaat atctcttcac taaaaactaa ttctaatttt tcgcttgaga acttggcata 1320

gtttgtccac tggaaaatct caaagccttt aaccaaagga ttcctgattt ccacagttct 1380

cgtcatcagc tctctggttg ctttagctaa tacaccataa gcattttccc tactgatgtt 1440

catcatctga gcgtattggt tataagtgaa cgataccgtc cgttctttcc ttgtagggtt 1500

ttcaatcgtg gggttgagta gtgccacaca gcataaaatt agcttggttt catgctccgt 1560

taagtcatag cgactaatcg ctagttcatt tgctttgaaa acaactaatt cagacataca 1620

tctcaattgg tctaggtgat tttaatcact ataccaattg agatgggcta gtcaatgata 1680

attactagtc cttttccttt gagttgtggg tatctgtaaa ttctgctaga cctttgctgg 1740

aaaacttgta aattctgcta gaccctctgt aaattccgct agacctttgt gtgttttttt 1800

tgtttatatt caagtggtta taatttatag aataaagaaa gaataaaaaa agataaaaag 1860

aatagatccc agccctgtgt ataactcact actttagtca gttccgcagt attacaaaag 1920

gatgtcgcaa acgctgtttg ctcctctaca aaacagacct taaaacccta aaggcttaag 1980

tagcaccctc gcaagctcgg ttgcggccgc aatcgggcaa atcgctgaat attccttttg 2040

tctccgacca tcaggcacct gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc 2100

acggctctgg cagtgaatgg gggtaaatgg cactacaggc gccttttatg gattcatgca 2160

aggaaactac ccataataca agaaaagccc gtcacgggct tctcagggcg ttttatggcg 2220

ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt 2280

ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag 2340

taaggcagcg gtatcatcaa cggggtctga cgctcagtgg aacgaaaact cacgttaagg 2400

gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 2460

aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagta gggataacag 2520

ggtaatactt ttcatactcc cgccattcag agaagaaacc aattgtccat attgcatcag 2580

acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg taaccccgct 2640

tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc gtaacaaaag 2700

tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca cactttgcta 2760

tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt tatcgcaact 2820

ctctactgtt tctccatgaa gtggctaaag agaacaagtt ttagagctag aaatagcaag 2880

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttagcat 2940

ccaaactcga gtaaggatca ttaaggatcc catggtacgc gtgctagagg catcaaataa 3000

aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg 3060

ctctcctgag taggacaaat ccgccctgca tgtgtcagag gttttcaccg tcatcaccga 3120

aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 3180

taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtggtaaaa ccgaatactg 3240

ccggtggttg gcgtttcgtc aaagaagttc catggtttca gcagagtatg tacccattcg 3300

gtcggcataa tcggccactc ttcggcgcgg gccacatgtg tggtgccggt ggtcatccag 3360

acaacggcgt cggtgttgtc cagcgactcg ttatccttac tgtattgtcc aagaccggtg 3420

tcatgagtag aacggttcgg atatttgcct tccgggaaac gctcgccagg atgataacgc 3480

gttacccaga gctgcttgtc cataaagctt aaacgatgat agatccactc gtccggcgcg 3540

aactgggcac cttttgctac cgggtgagta ccacctgcat aaggaataat ttgataggaa 3600

accggattgc ccatgcggtt ctctttgttc gggttactca acagacgaat cgtgcccgga 3660

tcaaatttct gtgcggcatc ctgttcattg ccgatgttgt actgattaac ttgcatggta 3720

ctggtgcgtg ggccaccgga ttcatgcagc tgattaatac gactcactat agggaattca 3780

ttaaagagga gaaaggtaca atgttgacaa aagcaacaaa agaacaaaaa tcccttgtga 3840

aaaacagagg ggcggagctt gttgttgatt gcttagtgga gcaaggtgtc acacatgtat 3900

ttggcattcc aggtgcaaaa attgatgcgg tatttgacgc tttacaagat aaaggacctg 3960

aaattatcgt tgcccggcac gaacaaaacg cagcattcat ggcccaagca gtcggccgtt 4020

taactggaaa accgggagtc gtgttagtca catcaggacc gggtgcctct aacttggcaa 4080

caggcctgct gacagcgaac actgaaggag accctgtcgt tgcgcttgct ggaaacgtga 4140

tccgtgcaga tcgtttaaaa cggacacatc aatctttgga taatgcggcg ctattccagc 4200

cgattacaaa atacagtgta gaagttcaag atgtaaaaaa tataccggaa gctgttacaa 4260

atgcatttag gatagcgtca gcagggcagg ctggggccgc ttttgtgagc tttccgcaag 4320

atgttgtgaa tgaagtcaca aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg 4380

gtcctgcagc agatgatgca atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc 4440

ctgtcgtttt ggtcggcatg aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc 4500

ttttgaaaaa ggttcagctt ccatttgttg aaacatatca agctgccggt accctttcta 4560

gagatttaga ggatcaatat tttggccgta tcggtttgtt ccgcaaccag cctggcgatt 4620

tactgctaga gcaggcagat gttgttctga cgatcggcta tgacccgatt gaatatgatc 4680

cgaaattctg gaatatcaat ggagaccgga caattatcca tttagacgag attatcgctg 4740

acattgatca tgcttaccag cctgatcttg aattgatcgg tgacattccg tccacgatca 4800

atcatatcga acacgatgct gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt 4860

ctgatttaaa acaatatatg catgaaggtg agcaggtgcc tgcagattgg aaatcagaca 4920

gagcgcaccc tcttgaaatc gttaaagagt tgcgtaatgc agtcgatgat catgttacag 4980

taacttgcga tatcggttcg cacgccattt ggatgtcacg ttatttccgc agctacgagc 5040

cgttaacatt aatgatcagt aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa 5100

tcggcgcttc attggtgaaa ccgggagaaa aagtggtttc tgtctctggt gacggcggtt 5160

tcttattctc agcaatggaa ttagagacag cagttcgact aaaagcacca attgtacaca 5220

ttgtatggaa cgacagcaca tatgacatgg ttgcattcca gcaattgaaa aaatataacc 5280

gtacatctgc ggtcgatttc ggaaatatcg atatcgtgaa atatgcggaa agcttcggag 5340

caactggctt gcgcgtagaa tcaccagacc agctggcaga tgttctgcgt caaggcatga 5400

acgctgaagg tcctgtcatc atcgatgtcc cggttgacta cagtgataac attaatttag 5460

caagtgacaa gcttccgaaa gaattcgggg aactcatgaa aacgaaagct ctctaggtcg 5520

acgaggaatc accatggcta actacttcaa tacactgaat ctgcgccagc agctggcaca 5580

gctgggcaaa tgtcgcttta tgggccgcga tgaattcgcc gatggcgcga gctaccttca 5640

gggtaaaaaa gtagtcatcg tcggctgtgg cgcacagggt ctgaaccagg gcctgaacat 5700

gcgtgattct ggtctcgata tctcctacgc tctgcgtaaa gaagcgattg ccgagaagcg 5760

cgcgtcctgg cgtaaagcga ccgaaaatgg ttttaaagtg ggtacttacg aagaactgat 5820

cccacaggcg gatctggtga ttaacctgac gccggacaag cagcactctg atgtagtgcg 5880

caccgtacag ccactgatga aagacggcgc ggcgctgggc tactcgcacg gtttcaacat 5940

cgtcgaagtg ggcgagcaga tccgtaaaga tatcaccgta gtgatggttg cgccgaaatg 6000

cccaggcacc gaagtgcgtg aagagtacaa acgtgggttc ggcgtaccga cgctgattgc 6060

cgttcacccg gaaaacgatc cgaaaggcga aggcatggcg attgccaaag cctgggcggc 6120

tgcaaccggt ggtcaccgtg cgggtgtgct ggaatcgtcc ttcgttgcgg aagtgaaatc 6180

tgacctgatg ggcgagcaaa ccatcctgtg cggtatgttg caggctggct ctctgctgtg 6240

cttcgacaag ctggtggaag aaggtaccga tccagcatac gcagaaaaac tgattcagtt 6300

cggttgggaa accatcaccg aagcactgaa acagggcggc atcaccctga tgatggaccg 6360

tctctctaac ccggcgaaac tgcgtgctta tgcgctttct gaacagctga aagagatcat 6420

ggcacccctg ttccagaaac atatggacga catcatctcc ggcgaattct cttccggtat 6480

gatggcggac tgggccaacg atgataagaa actgctgacc tggcgtgaag agaccggcaa 6540

aaccgcgttt gaaaccgcgc cgcagtatga aggcaaaatc ggcgagcagg agtacttcga 6600

taaaggcgta ctgatgattg cgatggtgaa agcgggcgtt gaactggcgt tcgaaaccat 6660

ggtcgattcc ggcatcattg aagagtctgc atattatgaa tcactgcacg agctgccgct 6720

gattgccaac accatcgccc gtaagcgtct gtacgaaatg aacgtggtta tctctgatac 6780

cgctgagtac ggtaactatc tgttctctta cgcttgtgtg ccgttgctga aaccgtttat 6840

ggcagagctg caaccgggcg acctgggtaa agctattccg gaaggcgcgg tagataacgg 6900

gcaactgcgt gatgtgaacg aagcgattcg cagccatgcg attgagcagg taggtaagaa 6960

actgcgcggc tatatgacag atatgaaacg tattgctgtt gcgggttaac ccggaaggag 7020

atataccatg cctaagtacc gttccgccac caccactcat ggtcgtaata tggcgggtgc 7080

tcgtgcgctg tggcgcgcca ccggaatgac cgacgccgat ttcggtaagc cgattatcgc 7140

ggttgtgaac tcgttcaccc aatttgtacc gggtcacgtc catctgcgcg atctcggtaa 7200

actggtcgcc gaacaaattg aagcggctgg cggcgttgcc aaagagttca acaccattgc 7260

ggtggatgat gggattgcca tgggccacgg ggggatgctt tattcactgc catctcgcga 7320

actgatcgct gattccgttg agtatatggt caacgcccac tgcgccgacg ccatggtctg 7380

catctctaac tgcgacaaaa tcaccccggg gatgctgatg gcttccctgc gcctgaatat 7440

tccggtgatc tttgtttccg gcggcccgat ggaggccggg aaaaccaaac tttccgatca 7500

gatcatcaag ctcgatctgg ttgatgcgat gatccagggc gcagacccga aagtatctga 7560

ctcccagagc gatcaggttg aacgttccgc gtgtccgacc tgcggttcct gctccgggat 7620

gtttaccgct aactcaatga actgcctgac cgaagcgctg ggcctgtcgc agccgggcaa 7680

cggctcgctg ctggcaaccc acgccgaccg taagcagctg ttccttaatg ctggtaaacg 7740

cattgttgaa ttgaccaaac gttattacga gcaaaacgac gaaagtgcac tgccgcgtaa 7800

tatcgccagt aaggcggcgt ttgaaaacgc catgacgctg gatatcgcga tgggtggatc 7860

gactaacacc gtacttcacc tgctggcggc ggcgcaggaa gcggaaatcg acttcaccat 7920

gagtgatatc gataagcttt cccgcaaggt tccacagctg tgtaaagttg cgccgagcac 7980

ccagaaatac catatggaag atgttcaccg tgctggtggt gttatcggta ttctcggcga 8040

actggatcgc gcggggttac tgaaccgtga tgtgaaaaac gtacttggcc tgacgttgcc 8100

gcaaacgctg gaacaatacg acgttatgct gacccaggat gacgcggtaa aaaatatgtt 8160

ccgcgcaggt cctgcaggca ttcgtaccac acaggcattc tcgcaagatt gccgttggga 8220

tacgctggac gacgatcgcg ccaatggctg tatccgctcg ctggaacacg cctacagcaa 8280

agacggcggc ctggcggtgc tctacggtaa ctttgcggaa aacggctgca tcgtgaaaac 8340

ggcaggcgtc gatgacagca tcctcaaatt caccggcccg gcgaaagtgt acgaaagcca 8400

ggacgatgcg gtagaagcga ttctcggcgg taaagttgtc gccggagatg tggtagtaat 8460

tcgctatgaa ggcccgaaag gcggtccggg gatgcaggaa atgctctacc caaccagctt 8520

cctgaaatca atgggtctcg gcaaagcctg tgcgctgatc accgacggtc gtttctctgg 8580

tggcacctct ggtctttcca tcggccacgt ctcaccggaa gcggcaagcg gcggcagcat 8640

tggcctgatt gaagatggtg acctgatcgc tatcgacatc ccgaaccgtg gcattcagtt 8700

acaggtaagc gatgccgaac tggcggcgcg tcgtgaagcg caggacgctc gaggtgacaa 8760

agcctggacg ccgaaaaatc gtgaacgtca ggtctccttt gccctgcgtg cttatgccag 8820

cctggcaacc agcgccgaca aaggcgcggt gcgcgataaa tcgaaactgg ggggttaaac 8880

gcgtgctagg aattcattaa agaggagaaa ggtaccatgt atacagtagg agattaccta 8940

ttagaccgat tacacgagtt aggaattgaa gaaatttttg gagtccctgg agactataac 9000

ttacaatttt tagatcaaat tatttcccgc aaggatatga aatgggtcgg aaatgctaat 9060

gaattaaatg cttcatatat ggctgatggc tatgctcgta ctaaaaaagc tgccgcattt 9120

cttacaacct ttggagtagg tgaattgagt gcagttaatg gattagcagg aagttacgcc 9180

gaaaatttac cagtagtaga aatagtggga tcacctacat caaaagttca aaatgaagga 9240

aaatttgttc atcatacgct ggctgacggt gattttaaac actttatgaa aatgcacgaa 9300

cctgttacag cagctcgaac tttactgaca gcagaaaatg caaccgttga aattgaccga 9360

gtactttctg cactattaaa agaaagaaaa cctgtctata tcaacttacc agttgatgtt 9420

gctgctgcaa aagcagagaa accctcactc cctttgaaaa aagaaaactc aacttcaaat 9480

acaagtgacc aagagatctt gaacaaaatt caagaaagct tgaaaaatgc caaaaaacca 9540

atcgtgatta caggacatga aataattagt tttggcttag aaaaaacagt ctctcaattt 9600

atttcaaaga caaaactacc tattacgaca ttaaactttg gaaaaagttc agttgatgaa 9660

gctctccctt catttttagg aatctataat ggtaaactct cagagcctaa tcttaaagaa 9720

ttcgtggaat cagccgactt catcctgatg cttggagtta aactcacaga ctcttcaaca 9780

ggagccttca ctcatcattt aaatgaaaat aaaatgattt cactgaatat agatgaagga 9840

aaaatattta acgaaagcat ccaaaatttt gattttgaat ccctcatctc ctctctctta 9900

gacctaagcg aaatagaata caaaggaaaa tatatcgata aaaagcaaga agactttgtt 9960

ccatcaaatg cgcttttatc acaagaccgc ctatggcaag cagttgaaaa cctaactcaa 10020

agcaatgaaa caatcgttgc tgaacaaggg acatcattct ttggcgcttc atcaattttc 10080

ttaaaaccaa agagtcattt tattggtcaa cccttatggg gatcaattgg atatacattc 10140

ccagcagcat taggaagcca aattgcagat aaagaaagca gacacctttt atttattggt 10200

gatggttcac ttcaacttac ggtgcaagaa ttaggattag caatcagaga aaaaattaat 10260

ccaatttgct ttattatcaa taatgatggt tatacagtcg aaagagaaat tcatggacca 10320

aatcaaagct acaatgatat tccaatgtgg aattactcaa aattaccaga atcatttgga 10380

gcaacagaag aacgagtagt ctcgaaaatc gttagaactg aaaatgaatt tgtgtctgtc 10440

atgaaagaag ctcaagcaga tccaaataga atgtactgga ttgagttaat tttggcaaaa 10500

gaagatgcac caaaagtact gaaaaaaatg ggcaaactat ttgctgaaca aaataaatca 10560

taagcatgca ggagatatac catgaaagca gcagtagtaa gacacaatcc agatggttat 10620

gcggaccttg ttgaaaagga acttcgagca atcaaaccta atgaagcttt gcttgacatg 10680

gagtattgtg gagtctgtca taccgatttg cacgttgcag caggtgatta tggcaacaaa 10740

gcagggactg ttcttggtca tgaaggaatt ggaattgtca aagaaattgg agctgatgta 10800

agctcgcttc aagttggtga tcgggtttca gtggcttggt tctttgaagg atgtggtcac 10860

tgtgaatact gtgtatctgg taatgaaact ttttgtcgag aagttaaaaa tgcaggatat 10920

tcagttgatg gcggaatggc tgaagaagca attgttgttg ccgattatgc tgtcaaagtt 10980

cctgacggac ttgacccaat tgaagctagc tcaattactt gtgctggagt aacaacttac 11040

aaagcaatca aagtatcagg agtaaaacct ggtgattggc aagtaatttt tggtgctgga 11100

ggacttggaa atttagcaat tcaatatgct aaaaatgttt ttggagcaaa agtaattgct 11160

gttgatatta atcaagataa attaaattta gctaaaaaaa ttggagctga tgtgattatc 11220

aattctggtg atgtaaatcc agttgatgaa attaaaaaaa taactggcgg cttaggggtg 11280

caaagtgcaa tagtttgtgc tgttgcaagg attgcttttg aacaagcggt tgcttctttg 11340

aaacctatgg gcaaaatggt tgctgtggca cttcccaata ctgagatgac tttatcagtt 11400

ccaacagttg tttttgacgg agtggaggtt gcaggttcac ttgtcggaac aagacttgac 11460

ttggcagaag cttttcaatt tggagcagaa ggtaaggtaa aaccaattgt tgcgacacgc 11520

aaactggaag aaatcaatga tattattgat gaaatgaagg caggaaaaat tgaaggccga 11580

atggtcattg attttactaa ataatctaga ggcatcaaaa aacccctcaa gacccgttta 11640

gaggccccaa ggggttatgc tagcaacggc tacgcgctgg aagatctcta tgacgctaat 11700

ggcgtgctta ttgcgaagaa aggtcagttg ctgagtagct ttgcgcatct gcgtgatgac 11760

ggtacaaccg catcttcttg ctggatctac accggtagct ggacagagca gggcaaccag 11820

atggctaacc gcgataactc cgacccgtcc ggtctgggga atacgctggg atgggcctgg 11880

gcgtggccgc tcaaccgtcg cgtgctgtac aaccgtgctt cggcggatat caacggtaaa 11940

ccgtgggatc cgaaacggat gctgatccag tggaacggca gcaagtggac gggtaacgat 12000

attcctgact tcggcaatgc cgcaccgggt acgccaaccg ggccgtttat catgcagccg 12060

gaagggatgg gacgcctgtt tgccatcaac aaaatggcgg aaggtccgtt cccggaacac 12120

tacgagccga ttgaaccgtt gttctcttta gccacttccg cggaacccct atttgtttat 12180

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 12240

aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 12300

tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 12360

atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 12420

agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 12480

tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 12540

tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 12600

atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 12660

ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 12720

tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 12780

acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 12840

ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 12900

aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 12960

ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 13020

cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 13080

gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 13140

actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 13200

agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccac 13256

<210> 22

<211> 7925

<212> DNA

<213> Artificial sequence (Manual sequence)

<400> 22

gattcatgca gctgattaat acgactcact atagggaatt cattaaagag gagaaaggta 60

caatgttgac aaaagcaaca aaagaacaaa aatcccttgt gaaaaacaga ggggcggagc 120

ttgttgttga ttgcttagtg gagcaaggtg tcacacatgt atttggcatt ccaggtgcaa 180

aaattgatgc ggtatttgac gctttacaag ataaaggacc tgaaattatc gttgcccggc 240

acgaacaaaa cgcagcattc atggcccaag cagtcggccg tttaactgga aaaccgggag 300

tcgtgttagt cacatcagga ccgggtgcct ctaacttggc aacaggcctg ctgacagcga 360

acactgaagg agaccctgtc gttgcgcttg ctggaaacgt gatccgtgca gatcgtttaa 420

aacggacaca tcaatctttg gataatgcgg cgctattcca gccgattaca aaatacagtg 480

tagaagttca agatgtaaaa aatataccgg aagctgttac aaatgcattt aggatagcgt 540

cagcagggca ggctggggcc gcttttgtga gctttccgca agatgttgtg aatgaagtca 600

caaatacgaa aaacgtgcgt gctgttgcag cgccaaaact cggtcctgca gcagatgatg 660

caatcagtgc ggccatagca aaaatccaaa cagcaaaact tcctgtcgtt ttggtcggca 720

tgaaaggcgg aagaccggaa gcaattaaag cggttcgcaa gcttttgaaa aaggttcagc 780

ttccatttgt tgaaacatat caagctgccg gtaccctttc tagagattta gaggatcaat 840

attttggccg tatcggtttg ttccgcaacc agcctggcga tttactgcta gagcaggcag 900

atgttgttct gacgatcggc tatgacccga ttgaatatga tccgaaattc tggaatatca 960

atggagaccg gacaattatc catttagacg agattatcgc tgacattgat catgcttacc 1020

agcctgatct tgaattgatc ggtgacattc cgtccacgat caatcatatc gaacacgatg 1080

ctgtgaaagt ggaatttgca gagcgtgagc agaaaatcct ttctgattta aaacaatata 1140

tgcatgaagg tgagcaggtg cctgcagatt ggaaatcaga cagagcgcac cctcttgaaa 1200

tcgttaaaga gttgcgtaat gcagtcgatg atcatgttac agtaacttgc gatatcggtt 1260

cgcacgccat ttggatgtca cgttatttcc gcagctacga gccgttaaca ttaatgatca 1320

gtaacggtat gcaaacactc ggcgttgcgc ttccttgggc aatcggcgct tcattggtga 1380

aaccgggaga aaaagtggtt tctgtctctg gtgacggcgg tttcttattc tcagcaatgg 1440

aattagagac agcagttcga ctaaaagcac caattgtaca cattgtatgg aacgacagca 1500

catatgacat ggttgcattc cagcaattga aaaaatataa ccgtacatct gcggtcgatt 1560

tcggaaatat cgatatcgtg aaatatgcgg aaagcttcgg agcaactggc ttgcgcgtag 1620

aatcaccaga ccagctggca gatgttctgc gtcaaggcat gaacgctgaa ggtcctgtca 1680

tcatcgatgt cccggttgac tacagtgata acattaattt agcaagtgac aagcttccga 1740

aagaattcgg ggaactcatg aaaacgaaag ctctctaggt cgacgaggaa tcaccatggc 1800

taactacttc aatacactga atctgcgcca gcagctggca cagctgggca aatgtcgctt 1860

tatgggccgc gatgaattcg ccgatggcgc gagctacctt cagggtaaaa aagtagtcat 1920

cgtcggctgt ggcgcacagg gtctgaacca gggcctgaac atgcgtgatt ctggtctcga 1980

tatctcctac gctctgcgta aagaagcgat tgccgagaag cgcgcgtcct ggcgtaaagc 2040

gaccgaaaat ggttttaaag tgggtactta cgaagaactg atcccacagg cggatctggt 2100

gattaacctg acgccggaca agcagcactc tgatgtagtg cgcaccgtac agccactgat 2160

gaaagacggc gcggcgctgg gctactcgca cggtttcaac atcgtcgaag tgggcgagca 2220

gatccgtaaa gatatcaccg tagtgatggt tgcgccgaaa tgcccaggca ccgaagtgcg 2280

tgaagagtac aaacgtgggt tcggcgtacc gacgctgatt gccgttcacc cggaaaacga 2340

tccgaaaggc gaaggcatgg cgattgccaa agcctgggcg gctgcaaccg gtggtcaccg 2400

tgcgggtgtg ctggaatcgt ccttcgttgc ggaagtgaaa tctgacctga tgggcgagca 2460

aaccatcctg tgcggtatgt tgcaggctgg ctctctgctg tgcttcgaca agctggtgga 2520

agaaggtacc gatccagcat acgcagaaaa actgattcag ttcggttggg aaaccatcac 2580

cgaagcactg aaacagggcg gcatcaccct gatgatggac cgtctctcta acccggcgaa 2640

actgcgtgct tatgcgcttt ctgaacagct gaaagagatc atggcacccc tgttccagaa 2700

acatatggac gacatcatct ccggcgaatt ctcttccggt atgatggcgg actgggccaa 2760

cgatgataag aaactgctga cctggcgtga agagaccggc aaaaccgcgt ttgaaaccgc 2820

gccgcagtat gaaggcaaaa tcggcgagca ggagtacttc gataaaggcg tactgatgat 2880

tgcgatggtg aaagcgggcg ttgaactggc gttcgaaacc atggtcgatt ccggcatcat 2940

tgaagagtct gcatattatg aatcactgca cgagctgccg ctgattgcca acaccatcgc 3000

ccgtaagcgt ctgtacgaaa tgaacgtggt tatctctgat accgctgagt acggtaacta 3060

tctgttctct tacgcttgtg tgccgttgct gaaaccgttt atggcagagc tgcaaccggg 3120

cgacctgggt aaagctattc cggaaggcgc ggtagataac gggcaactgc gtgatgtgaa 3180

cgaagcgatt cgcagccatg cgattgagca ggtaggtaag aaactgcgcg gctatatgac 3240

agatatgaaa cgtattgctg ttgcgggtta acccggaagg agatatacca tgcctaagta 3300

ccgttccgcc accaccactc atggtcgtaa tatggcgggt gctcgtgcgc tgtggcgcgc 3360

caccggaatg accgacgccg atttcggtaa gccgattatc gcggttgtga actcgttcac 3420

ccaatttgta ccgggtcacg tccatctgcg cgatctcggt aaactggtcg ccgaacaaat 3480

tgaagcggct ggcggcgttg ccaaagagtt caacaccatt gcggtggatg atgggattgc 3540

catgggccac ggggggatgc tttattcact gccatctcgc gaactgatcg ctgattccgt 3600

tgagtatatg gtcaacgccc actgcgccga cgccatggtc tgcatctcta actgcgacaa 3660

aatcaccccg gggatgctga tggcttccct gcgcctgaat attccggtga tctttgtttc 3720

cggcggcccg atggaggccg ggaaaaccaa actttccgat cagatcatca agctcgatct 3780

ggttgatgcg atgatccagg gcgcagaccc gaaagtatct gactcccaga gcgatcaggt 3840

tgaacgttcc gcgtgtccga cctgcggttc ctgctccggg atgtttaccg ctaactcaat 3900

gaactgcctg accgaagcgc tgggcctgtc gcagccgggc aacggctcgc tgctggcaac 3960

ccacgccgac cgtaagcagc tgttccttaa tgctggtaaa cgcattgttg aattgaccaa 4020

acgttattac gagcaaaacg acgaaagtgc actgccgcgt aatatcgcca gtaaggcggc 4080

gtttgaaaac gccatgacgc tggatatcgc gatgggtgga tcgactaaca ccgtacttca 4140

cctgctggcg gcggcgcagg aagcggaaat cgacttcacc atgagtgata tcgataagct 4200

ttcccgcaag gttccacagc tgtgtaaagt tgcgccgagc acccagaaat accatatgga 4260

agatgttcac cgtgctggtg gtgttatcgg tattctcggc gaactggatc gcgcggggtt 4320

actgaaccgt gatgtgaaaa acgtacttgg cctgacgttg ccgcaaacgc tggaacaata 4380

cgacgttatg ctgacccagg atgacgcggt aaaaaatatg ttccgcgcag gtcctgcagg 4440

cattcgtacc acacaggcat tctcgcaaga ttgccgttgg gatacgctgg acgacgatcg 4500

cgccaatggc tgtatccgct cgctggaaca cgcctacagc aaagacggcg gcctggcggt 4560

gctctacggt aactttgcgg aaaacggctg catcgtgaaa acggcaggcg tcgatgacag 4620

catcctcaaa ttcaccggcc cggcgaaagt gtacgaaagc caggacgatg cggtagaagc 4680

gattctcggc ggtaaagttg tcgccggaga tgtggtagta attcgctatg aaggcccgaa 4740

aggcggtccg gggatgcagg aaatgctcta cccaaccagc ttcctgaaat caatgggtct 4800

cggcaaagcc tgtgcgctga tcaccgacgg tcgtttctct ggtggcacct ctggtctttc 4860

catcggccac gtctcaccgg aagcggcaag cggcggcagc attggcctga ttgaagatgg 4920

tgacctgatc gctatcgaca tcccgaaccg tggcattcag ttacaggtaa gcgatgccga 4980

actggcggcg cgtcgtgaag cgcaggacgc tcgaggtgac aaagcctgga cgccgaaaaa 5040

tcgtgaacgt caggtctcct ttgccctgcg tgcttatgcc agcctggcaa ccagcgccga 5100

caaaggcgcg gtgcgcgata aatcgaaact ggggggttaa acgcgtgcta ggaattcatt 5160

aaagaggaga aaggtaccat gtatacagta ggagattacc tattagaccg attacacgag 5220

ttaggaattg aagaaatttt tggagtccct ggagactata acttacaatt tttagatcaa 5280

attatttccc gcaaggatat gaaatgggtc ggaaatgcta atgaattaaa tgcttcatat 5340

atggctgatg gctatgctcg tactaaaaaa gctgccgcat ttcttacaac ctttggagta 5400

ggtgaattga gtgcagttaa tggattagca ggaagttacg ccgaaaattt accagtagta 5460

gaaatagtgg gatcacctac atcaaaagtt caaaatgaag gaaaatttgt tcatcatacg 5520

ctggctgacg gtgattttaa acactttatg aaaatgcacg aacctgttac agcagctcga 5580

actttactga cagcagaaaa tgcaaccgtt gaaattgacc gagtactttc tgcactatta 5640

aaagaaagaa aacctgtcta tatcaactta ccagttgatg ttgctgctgc aaaagcagag 5700

aaaccctcac tccctttgaa aaaagaaaac tcaacttcaa atacaagtga ccaagagatc 5760

ttgaacaaaa ttcaagaaag cttgaaaaat gccaaaaaac caatcgtgat tacaggacat 5820

gaaataatta gttttggctt agaaaaaaca gtctctcaat ttatttcaaa gacaaaacta 5880

cctattacga cattaaactt tggaaaaagt tcagttgatg aagctctccc ttcattttta 5940

ggaatctata atggtaaact ctcagagcct aatcttaaag aattcgtgga atcagccgac 6000

ttcatcctga tgcttggagt taaactcaca gactcttcaa caggagcctt cactcatcat 6060

ttaaatgaaa ataaaatgat ttcactgaat atagatgaag gaaaaatatt taacgaaagc 6120

atccaaaatt ttgattttga atccctcatc tcctctctct tagacctaag cgaaatagaa 6180

tacaaaggaa aatatatcga taaaaagcaa gaagactttg ttccatcaaa tgcgctttta 6240

tcacaagacc gcctatggca agcagttgaa aacctaactc aaagcaatga aacaatcgtt 6300

gctgaacaag ggacatcatt ctttggcgct tcatcaattt tcttaaaacc aaagagtcat 6360

tttattggtc aacccttatg gggatcaatt ggatatacat tcccagcagc attaggaagc 6420

caaattgcag ataaagaaag cagacacctt ttatttattg gtgatggttc acttcaactt 6480

acggtgcaag aattaggatt agcaatcaga gaaaaaatta atccaatttg ctttattatc 6540

aataatgatg gttatacagt cgaaagagaa attcatggac caaatcaaag ctacaatgat 6600

attccaatgt ggaattactc aaaattacca gaatcatttg gagcaacaga agaacgagta 6660

gtctcgaaaa tcgttagaac tgaaaatgaa tttgtgtctg tcatgaaaga agctcaagca 6720

gatccaaata gaatgtactg gattgagtta attttggcaa aagaagatgc accaaaagta 6780

ctgaaaaaaa tgggcaaact atttgctgaa caaaataaat cataagcatg caggagatat 6840

accatgaaag cagcagtagt aagacacaat ccagatggtt atgcggacct tgttgaaaag 6900

gaacttcgag caatcaaacc taatgaagct ttgcttgaca tggagtattg tggagtctgt 6960

cataccgatt tgcacgttgc agcaggtgat tatggcaaca aagcagggac tgttcttggt 7020

catgaaggaa ttggaattgt caaagaaatt ggagctgatg taagctcgct tcaagttggt 7080

gatcgggttt cagtggcttg gttctttgaa ggatgtggtc actgtgaata ctgtgtatct 7140

ggtaatgaaa ctttttgtcg agaagttaaa aatgcaggat attcagttga tggcggaatg 7200

gctgaagaag caattgttgt tgccgattat gctgtcaaag ttcctgacgg acttgaccca 7260

attgaagcta gctcaattac ttgtgctgga gtaacaactt acaaagcaat caaagtatca 7320

ggagtaaaac ctggtgattg gcaagtaatt tttggtgctg gaggacttgg aaatttagca 7380

attcaatatg ctaaaaatgt ttttggagca aaagtaattg ctgttgatat taatcaagat 7440

aaattaaatt tagctaaaaa aattggagct gatgtgatta tcaattctgg tgatgtaaat 7500

ccagttgatg aaattaaaaa aataactggc ggcttagggg tgcaaagtgc aatagtttgt 7560

gctgttgcaa ggattgcttt tgaacaagcg gttgcttctt tgaaacctat gggcaaaatg 7620

gttgctgtgg cacttcccaa tactgagatg actttatcag ttccaacagt tgtttttgac 7680

ggagtggagg ttgcaggttc acttgtcgga acaagacttg acttggcaga agcttttcaa 7740

tttggagcag aaggtaaggt aaaaccaatt gttgcgacac gcaaactgga agaaatcaat 7800

gatattattg atgaaatgaa ggcaggaaaa attgaaggcc gaatggtcat tgattttact 7860

aaataatcta gaggcatcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat 7920

gctag 7925

<210> 23

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 23

ccggcagctt gatatgtttc 20

<210> 24

<211> 20

<212> DNA

<213> Escherichia coli MG1655(Escherichia coli MG1655)

<400> 24

ggtttcagtg gcttggttct 20

Claims

1. A CRISPR-Cas9 gene editing vector for bacteria, comprising plasmid 1 and plasmid 2;

2. The CRISPR-Cas9 gene editing vector according to claim 1, wherein the replication initiation site of plasmid 1 is a medium copy replication initiation site, and the replication initiation site of plasmid 2 is a temperature sensitive replication initiation site.

3. The CRISPR-Cas9 gene editing vector according to claim 1, which isIs characterized in that the Cas9 protein expression element consists of a high-stringency inducible promoter, a Cas9 encoding gene, a terminator and a regulatory protein gene; the high stringency inducible promoter includes but is not limited to P_BADPromoter, rhaP_BADPromoters and tetA promoters.

4. The CRISPR-Cas9 gene editing vector according to claim 1, wherein the recombinant protein expression element consists of inducible promoter, recombinant protein expression gene, terminator and regulatory protein; the inducible promoter may be a promoter with high stringency or a promoter with low stringency.

5. The CRISPR-Cas9 gene editing vector according to claim 1, wherein the sgRNA expression element consists of a strongly stringent inducible promoter, a sgRNA coding sequence and a terminator; the sgRNA expression element comprises the same inducible promoter as the Cas9 protein expression element.

6. The CRISPR-Cas9 gene editing vector according to claim 1, wherein the sgRNA recognizes the target DNA of recipient bacterium containing 5' - (N)_X-NGG-3' structure, (N)_XDenotes X N, N is A, G, C or T, X is a natural number greater than or equal to 12.

7. The CRISPR-Cas9 gene editing vector of claim 1, wherein the homologous recombination element consists of two sequences homologous to genome or two sequences homologous to genome and an insertion sequence, the insertion sequence is located between the two homologous sequences, one or both ends of the homologous recombination element contain 5' - (N) which can be recognized by sgRNA_X-NGG-3' structure.

8. The CRISPR-Cas9 gene editing vector according to claim 1,

the nucleotide sequence of the plasmid 1 is SEQ ID NO. 1;

the nucleotide sequence of the plasmid 2 is SEQ ID NO.2 or SEQ ID NO.10 or SEQ ID NO. 21.

9. A gene editing kit for bacteria, comprising the CRISPR-Cas9 gene editing vector for bacteria of any one of claims 1-8.

10. Use of a CRISPR-Cas9 gene editing vector for bacteria, wherein the CRISPR-Cas9 gene editing vector for bacteria is used for gene editing in bacteria.