CN114540400B - Corynebacterium glutamicum CRISPR/Cpf1 genome editing technology - Google Patents

Abstract

The invention provides a corynebacterium glutamicum CRISPR/Cpf1 efficient genome editing technology, which integrates and then induces cutting, separates recombination and cutting events, adopts a double-plasmid system pEC-XK99E derived plasmid to express RecET and Cpf1 to be placed in competent cells, and uses a compatible pXMJ19 plasmid to express crRNA and provide donor DNA molecules, thereby improving the loading capacity of a donor DNA template, solving the difficult problem of large-segment integration of corynebacterium glutamicum, improving genome editing efficiency as a whole, providing possibility for large-segment genome insertion, and providing technical support for the metabolic engineering research and strain construction of corynebacterium glutamicum.

Description

Corynebacterium glutamicum CRISPR/Cpf1 genome editing technology

Technical Field

The invention belongs to the technical field of genome editing, and particularly relates to a corynebacterium glutamicum CRISPR/Cpf1 efficient genome editing technology.

Background

Corynebacterium glutamicum is an industrial strain of important amino acid products, high-efficiency genome editing (genome level gene knockout and integration) can accelerate new strain construction, and has important significance in metabolic engineering research and industrial production. The current common coding method of the corynebacterium glutamicum genes is a non-replicative plasmid (such as pk18 mobsacB) mediated twice homologous recombination technology, and has complex operation flow and low editing efficiency; two problems are highlighted: first, for some gene knockouts that affect growth, the second round of crossover easily restores wild type, resulting in difficulty in obtaining correctly edited strains; secondly, in the case of multicopy integration, the integrated gene sequence is larger than the homologous arm sequence, so that the integrated gene is easy to integrate into the existing copy in the first round of recombination, and the correct single exchange strain is difficult to screen. The recently developed C.glutamicum CRISPR editing technique can avoid the above problems due to the introduction of gRNA/crRNA mediated precise genomic target endonuclease (Cas 9/Cpf 1) cleavage. The genetic components required for CRISPR technology include an endonuclease, a targeting RNA expression element and a donor DNA molecule. After the RNA is combined with Cas9 or Cpf1, a target site of a genome is targeted through a base complementary pairing principle, so that DNA double-strand break is realized, then cells are repaired by taking donor DNA molecules as templates through a homologous recombination mechanism, and meanwhile, genome editing is realized.

The currently reported corynebacterium glutamicum CRISPR genome editing technology has the problems of low gene knockout efficiency and extremely low gene integration efficiency, and especially cannot complete genome integration of large fragment sequences, so that the technology cannot be widely used.

The invention discovers that the important reason of the low efficiency of the current method is that under the condition of CRISPR genome cutting, the efficiency of homologous recombination of donor DNA molecules and genome is extremely low, namely genome breakage affects recombination events, or the homologous recombination of donor DNA and genome is difficult to repair genome breakage with high efficiency.

Disclosure of Invention

The invention aims to solve the technical problem of providing a corynebacterium glutamicum CRISPR/Cpf1 efficient genome editing technology.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a corynebacterium glutamicum CRISPR/Cpf1 efficient genome editing technology comprises the following steps:

(1) Plasmids carrying Cpf1 and RecET expression elements were prepared in competent cells and then electrotransformed with another plasmid carrying only donor DNA and crRNA expression elements (as shown in FIG. 1);

(2) The heterologous recombinase RecET is introduced into the corynebacterium glutamicum, and after the RecET is expressed and the second plasmid is transformed, donor DNA can be recombined on a chromosome with high efficiency under the condition that Cpf1 is not induced, and resuscitating liquid is coated in a solid culture medium added with an inducer, so that most of non-recombined cells are killed by Cpf1 expression, and the editing efficiency is improved.

The efficient genome editing technology of corynebacterium glutamicum CRISPR/Cpf1 can keep the first plasmid in competence for repeated use, and only needs to construct a second plasmid during each operation, and simultaneously, the construction of large-fragment DNA is possible; by introducing heterologous recombinase RecET into corynebacterium glutamicum, recombination efficiency of fragments and genome can be greatly improved under the action of the recombinase RecET, high-efficiency genome integration of large-fragment DNA is realized, and technical bottlenecks in the field are broken through.

Preferably, the corynebacterium glutamicum CRISPR/Cpf1 efficient genome editing technology adopts RAPID genome editing technology, utilizes a dual plasmid system pEC delta per1-Cpf1-RecET (expressed in competent cells, the sequence is shown as SEQ ID: NO 1) and pXMSACB-crRNA-donor (the crRNA and the donor are variable for different gene editing), wherein the pXMSACB-crRNA-donor plasmid is constructed from a framework plasmid pXMSACB-crRNA (the sequence is shown as SEQ ID: NO 2), and a plasmid map and an operation flow are shown as shown in FIG. 1.

Preferably, the above-described Corynebacterium glutamicum CRISPR/Cpf1 efficient genome editing technique employs a stringent Cpf1 inducible expression system, including construction of plasmids pEC-Cpf1-RecET and pXM-crRNA and testing for inducible effects.

Preferably, the corynebacterium glutamicum CRISPR/Cpf1 efficient genome editing technology realizes the efficient loss of the pXMJ19 derived plasmid by applying sacB-mediated sucrose death, realizes the efficient loss of the pEC-XK99E derived plasmid by knocking out the per1, and ensures that the RAPID is applied to iterative genome operation.

Preferably, the efficient genome editing technology of the corynebacterium glutamicum CRISPR/Cpf1 has the cell recovery time of 2-6h after electrotransformation, and simplifies the RAPID operation flow.

Preferably, the efficient genome editing technology of the corynebacterium glutamicum CRISPR/Cpf1 has a cell resuscitating time of 5 hours after electrotransformation.

The beneficial effects are that:

the invention develops a CRISPR/Cpf1 genome editing technology of a two-step method of integrating and then inducing cutting (RecombinAtion Prior to Induced Double-strand-break, RAPID), and realizes high-efficiency gene knockout and large fragment insertion. By utilizing the RNA processing capability of Cpf1, the construction of a crRNA expression frame which does not need to be stopped immediately simplifies plasmid construction. The RAPID technology separates recombination and cleavage events, adopts a double-plasmid system (pEC-XK 99E derived plasmids express RecET and Cpf1 and are placed in competent cells, and compatible pXMJ19 plasmids express crRNA and provide donor DNA molecules), improves the loading capacity of a donor DNA template, solves the problem of large-fragment integration of corynebacterium glutamicum, integrally improves genome editing efficiency, provides possibility for large-fragment genome insertion, and provides technical support for metabolic engineering research and strain construction of corynebacterium glutamicum.

(1) The invention makes recombination and cleavage events separate in corynebacterium glutamicum, and on the basis of no double-strand cleavage of chromosomal DNA, donor DNA is subjected to high-efficiency homologous recombination on a genome; expression of Cpf1 is then induced by the induction system, thus cleaving and killing most of the non-recombinant cells. On the premise that the endonuclease is not expressed, the recombination efficiency of the donor DNA molecules is extremely high, and meanwhile, the recovery time is properly prolonged, so that a large number of correctly edited cells can be obtained, and then the genome is cut to remove non-edited cells, thereby realizing efficient genome editing.

(2) By utilizing the technology, the gene knockout efficiency is improved to 100%, and meanwhile, the high-efficiency genome integration of large-fragment DNA is realized. The competent cells used in the genetic manipulation of the invention contain Cpf1 and RecET expression elements, and the expression of RecET greatly improves the recombination efficiency of donor DNA.

(3) By using the technology of the invention, only one plasmid is needed to be electrically transferred during each step of gene editing operation, and compared with the prior art that all elements are expressed by using one plasmid, the plasmid transformed by the invention only carries donor DNA and crRNA expression elements and can carry larger DNA fragments as an integration sequence. Meanwhile, the crRNA variable sequence is very short (24 bp), can be directly carried at the tail end of the donor DNA through primer design, and has simple plasmid construction flow.

(4) By utilizing the technology of the invention, plasmids can be rapidly lost, and the genome iterative editing of corynebacterium glutamicum can be realized.

Drawings

FIG. 1, schematic of a RAPID genome editing workflow, working with a two plasmid system: pECΔper1-Cpf1-RecET (expressed in competent cells) and pXMSACB-crRNA-donor (containing crRNA expression cassette and donor DNA molecule).

FIG. 2 principle of the calicheamicin resistance obtained after the gene editing of the mode strains CgDel and CgInt, wherein the CgDel is 534bp xylA _Eco The strain obtained after knockout gave kanamycin resistance, and the strain obtained after insertion of a fragment containing 193bp C-terminal sequence of kanamycin resistance gene.

FIG. 3 RecET-mediated fragment recombination, recET expression greatly promotes 500bp fragment recombination efficiency compared to control, enabling large fragment (5 kb) integration.

FIG. 4 IPTG induced Cpf1 expression and genomic cleavage, IPTG induced bacterial death with only a few colonies off-target.

FIG. 5 pXMSACB-crRNA-donor plasmid loss using SacB mediated sucrose lethal.

FIG. 6 knockout of per1 promotes loss of pECΔper1-Cpf1-RecET plasmid.

FIG. 7 RAPID technique revives different time gene knockout efficiency, and reviving has an important role in knockout efficiency, and the knockout efficiency is highest at 5h of reviving.

FIG. 8 RAPID technology large fragment gene integration efficiency high efficiency large fragment integration was obtained due to RecET mediated efficient recombination and IPTG induced elimination of non-editing strains.

Detailed Description

The technical scheme of the invention is further described below with reference to specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated.

It should be noted that: the following example protocol uses kanamycin resistance screening as a quick identification of genome editing. When the RAPID technology is applied to metabolic engineering research, the edited strain needs to be identified by means of the existing technologies such as colony PCR and gene sequencing.

Example 1: construction of model strains CgDel and CgInt

For rapid verification of genomic knockdown and integration, two model strains CgDel and CgInt were first constructed, and gene knockdown and integration were identified by kanamycin resistance, respectively (principle see schematic 2).

(1)pk18mobrpsL-kan* _del And pk18mobrps l-kan _int Plasmid construction

The primers used in the present invention are shown in Table 1. The Corynebacterium glutamicum ATCC 13032 genome is used as a template, and the primer pairs P1/P2 and P3/P4 are used for respectively amplifying the upstream and downstream homology arms of the cg1890 gene; two fragments of the kanamycin resistance gene were amplified with primer pairs P5/P6 and P7/P8 using plasmid pk18 mobrpL (Wang et al, an update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum, microbial Biotechnology,2019, 12 (5), 907-919) as a template; a partial fragment of xylA gene (534 bp) was amplified using the E.coli MG1655 genome as template and the primer pair P9/P10. These 5 fragments were ligated together by overlap PCR (primers P1 and P10). The pk18mobrpsL plasmid was digested with Xba I and Kpn I, and the overlapping fragments were ligated to a linear vector by homologous recombination to obtain plasmid pk18mobrpsL-kan _del 。

Amplification of the upstream and downstream homology arms of the G1890 Gene of the Corynebacterium glutamicum genome with primer pair P1/P11 and P12/P6, and amplification of the kanamycin resistance Gene deletion C-terminal 193bp expression cassette (PAM and crRNA) on the pk18 mobrpL with primer pair P13/P14 _xylA Designed on primer P14), 3 fragments were ligated together by overlap PCR and then ligated into the pk18 mobrpL linearization vector by homologous recombination to give plasmid pk18 mobrpL-kan _int 。

(2) Electrotransformation and strain selection

Constructed plasmid pk18mobrps L-kan _del And pk18mobrps l-kan _int Electrotransformation of Corynebacterium glutamicum ATCC 13032rpsL alone ^K43R In competence (Wang et al, an update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum, microbial Biotechnology,2019, 12 (5), 907-919), screening of single and double crossover strains by kanamycin and streptomycin, respectively, resulted in CgDel and CgInt strains.

Example 2: recET expression promotes homologous recombination

(1) pEC-RecET plasmid construction and electrotransformation

Firstly, the calicheamicin resistance gene in pEC-XK99E plasmid is replaced by a grippamycin resistance gene, specifically, plasmids pEC-XK99E and pREDCas9 are used as templates, a pEC-XK99E plasmid frame and a grippamycin resistance gene expression frame are respectively amplified by primer pairs P15/P16 and P17/P18, and then the two fragments are connected by adopting a homologous recombination method, so that the plasmid pECspec is obtained. The plasmid was then linearized by double cleavage with EcoR I and Kpn I. Amplification of P on Corynebacterium glutamicum genome Using primer pair P19/P20 _tuf The promoter, the recET gene on the genome of the escherichia coli is amplified by using a primer pair P21/P22, the two are connected by using an overlap PCR technology (primers P19 and P22), and the connecting fragment is connected with the linearization pECspec to obtain the plasmid pEC-RecET. pEC-RecET and control plasmid pECspec were transformed into CgInt competent cells by electrotransformation.

(2) Construction of donor DNA fragments of different lengths and electrotransformation

The donor fragment consisted of 3 parts, the upstream homology arm was part of the sequence of the kanamycin resistance gene, the middle was the insert sequence, and the downstream homology arm was the downstream sequence of the cgl1890 gene. The template of the upstream and downstream homology arms was pk18mobrps L-kan _del The plasmid, the primer pair for amplifying the upstream homology arm is P23/P24, the upstream primer for amplifying the downstream homology arm is a variable primer, and the downstream primer is P25. The amplification of the insert sequence takes the E.coli genome as a template, the upstream primer is P26, and the downstream primer is a variable primer. Amplifying 500bp,The 1kb, 2kb, 3kb, 4kb, 5kb downstream primers are P27, P28, P29, P30, P31, P32, respectively; the upstream primers corresponding to the amplified downstream homology arms are P33, P34, P35, P36, P37, P38, respectively. The corresponding upstream homology arm, the insertion sequence and the downstream homology arm are respectively connected by using the overlapping PCR technology, and the donor DNA template molecules integrating 500bp, 1kb, 2kb, 3kb, 4kb and 5kb are obtained. These donor DNA fragments were transformed into CgInt competent cells carrying pEC-RecET, respectively, and the 500bp fragment was integrated while competent cells carrying control plasmid were transformed, and furthermore, cells containing pEC-RecET were not transformed as blank.

Since the insertion of the fragment allows correct expression of the kanamycin gene and thus kanamycin resistance, the electrotransformed cells were resuscitated and plated with a plate to which calicheamicin was added, and the number of colonies grown was counted. As a result, as shown in FIG. 3, recET expression greatly promoted homologous recombination of the donor DNA fragment compared to the blank plasmid control. With the aid of this, even the transformation of the 5kb fragment yielded more than 4000 transformants with resistance to carbaryl.

Example 3: IPTG-inducible promoter brings about strict induction expression of Cpf1

(1) Construction of plasmid pEC-Cpf1-RecET and pXM-crRNA

The plasmid pEC-RecET is linearized by NcoI, fncpf1 (the sequence is shown as SEQ ID: NO3, the joint is P86/P87) with the sequence derived from Francisella novicida is synthesized by Jin Wei intelligent company, and the synthesized fragment with the homologous sequence at both ends is connected with a linear vector by homologous recombination technology to obtain the plasmid pEC-Cpf1-RecET. The pXMJ19 was linearized by reverse amplification with primers P39 and P40, and a fragment of the oligonucleotide sequence was synthesized in minigene form (O1, table 1) and ligated to a linear vector by homologous recombination to give the plasmid pXM-crRNA.

(2) Testing for Induction Effect

pEC-Cpf1-RecET was electroporated into CgDel competent cells, again made competent, then electroporated with plasmid pXMJ19-crRNA, plated on kanamycin plates with and without IPTG, respectively, after cell resuscitation, and the number of colonies formed was counted, as shown in FIG. 4. By comparing the colony numbers, it can be known that the genome can be cut efficiently by IPTG-induced Cpf1 expression.

Example 4: establishment of plasmid loss System

(1) Construction of pXMSACB-crRNA and pEC delta per1-Cpf1-RecET plasmid

The pXM-crRNA is reversely amplified by using the primers P41 and P42 to linearize, the sacB expression frame on the plasmid is amplified by using the primer pair P43/P44, and the two are connected by homologous recombination, so that the plasmid pXMSACB-crRNA (the sequence is shown as SEQ ID: NO 2) is obtained. The plasmid pEC-Cpf1-RecET is amplified by using a primer pair P45/P46 and P47/P48, and the two amplified fragments are connected to obtain the plasmid pEC delta per1-Cpf1-RecET (the sequence is shown as SEQ ID: NO 1).

(2) Plasmid loss rate test

The plasmids pXMSACB-crRNA and pEC.DELTA.per1-Cpf 1-RecET were electroporated into competent cells of Corynebacterium glutamicum ATCC 13032, and after resuscitating the cells, the cells were plated onto plates to which chloramphenicol and spectinomycin were added and screened to give transformants. Transformants were inoculated into a shake tube supplemented with Qixamycin and sucrose (1.5%, m/v) and cultured for 1 generation, three sections were streaked onto Qixamycin-supplemented plates, single colonies were picked up and inoculated onto chloramphenicol-containing plates, colonies were unable to grow, and the loss rate of pXMSACB-crRNA plasmid was found to be 100% (FIG. 5). Cells after loss of pXMSACB-crRNA were inoculated in a non-resistant medium for 1 generation, streaked to a non-resistant plate, and then inoculated with a plate to which spectinomycin was added, and the ratio of colonies grown was counted, resulting in a pEC.DELTA.per 1-Cpf1-RecET plasmid loss rate of 90% (FIG. 6).

Example 5: establishment of RAPID editing system

Based on the high-efficiency recombination and strict cutting and the high-efficiency plasmid losing system. The invention provides a genome editing technology of corynebacterium glutamicum which is used for firstly recombining and then cutting and eliminating non-editing strains.

(1)pXMsacB-crRNA-donor _del Construction of plasmids

The plasmid pXMSACB-crRNA was linearized with Xba I as pk18 mobrpL-kan _del As templates, the upstream and downstream homology arms were amplified with primer pairs P49/P50 and P51/P52, the two fragments were ligated by overlap PCR, and then loaded with pXMSACB-crRNA linearlyLigation to obtain plasmid pXMSACB-crRNA-donor _del 。

(2) Influence of resuscitation time on Gene knockout efficiency

pEC.DELTA.per1-Cpf 1-RecET was electrotransferred into C.glutamicum CgDel competent cells, again competent cells were prepared, and then pXMSACB-crRNA-donor was electrotransferred _del A plasmid. Resuscitating for 2-6h, then coating on a plate added with chloramphenicol, spectinomycin and IPTG, inoculating the single bacterial drop point on a plate added with additional calicheamicin, and calculating the growth rate of bacterial colony, namely the gene knockout efficiency. During resuscitating, homologous recombination of the donor DNA molecule with the genomic target site confers kanamycin resistance on the cells, and IPTG induces Cpf1 expression during plating without cleavage lethality of the recombinant strain. Thus, resuscitation time may have a significant impact on fragment reorganization. As can be seen from FIG. 7, the gene knockout efficiency was highest at 5 hours of resuscitation.

(3) Altering RecET expression patterns

Because RecET has an important role in fragment recombination, theophylline-induced riboswitches were designed to control RecET expression.

The pEC delta per1-Cpf1-RecET plasmid was amplified with primer pairs P53/P54 and P55/P56, and the two fragments were ligated to give a P-deleted _hom Is a linear vector fragment of (a). Amplification of P on Corynebacterium glutamicum genome Using primer pair P57/P58 _gapA Promoter, synthetic oligonucleotide sequences (O2, table 1), P was amplified by overlap PCR _gapA Ligating with the synthetic sequence and then ligating with a linear vector by homologous recombination to obtain plasmid pEC.DELTA.per1-Cpf 1-P _gapA -theoE*-RecET。

With pEC.DELTA.per1-Cpf 1-P _gapA The gene knockout of the CgDel-type strain was performed by replacing the plasmid pecΔper1-Cpf1-RecET with the-RecET, and the procedure was the same. The knockout efficiency can reach 100 percent.

As the RAPID uses CRISPR/Cpf1 as a counter screen, the gene editing efficiency is greatly improved. The linear segment is used as donor DNA molecule, and the gene knockout efficiency also reaches 100%.

Example 6: RAPID for large fragment integration

(1) Construction of template plasmid carrying donor DNA

The previously constructed linear donor DNA molecules were used as templates, and 500bp, 1kb, 2kb, 3kb, 4kb, and 5kb donor DNA templates were amplified and integrated with the primer pair P59/P26, respectively. The plasmid pXMSACB-crRNA was linearized with Xba I and each template molecule was ligated to a linear vector by homologous recombination to obtain pXMSACB-crRNA series plasmids carrying DNA templates.

(2) Genomic integration of fragments of different sizes

pEC.DELTA.per1-Cpf 1-RecET was electroporated into C.glutamicum CgInt competent cells, again to prepare competent cells. Electrotransport pXMSACB-crRNA-donor _int Serial plasmids were resuscitated for 5h, plated with chloramphenicol, spectinomycin, and IPTG, and single colonies grown were inoculated onto plates with additional calicheamicin, and the ratio of colony growth was calculated as gene integration efficiency (fig. 8). As can be seen from FIG. 7, RAPID technology can bring about high efficiency gene integration, the efficiency of integrating 4kb fragments (excluding upstream and downstream homology arms) exceeds 72%, and the efficiency of integrating 5kb sequences reaches 24%.

The primer and oligonucleotide sequences used in the above examples are shown in Table 1 below.

TABLE 1

Note that: lowercase letters indicate homologous sequences for overlap PCR or homologous recombination end joining.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several modifications and alterations without departing from the principles of the present invention, and the steps of constructing the strain of the present invention are not sequential, and those skilled in the art should consider the scope of the present invention as modifications and alterations of the strain according to the method of the present invention or based on the method.

Sequence listing

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gctgaccttc gaaaacatct ccgagtccta catcgattcc gtggtcaacc agggcaagct 3780

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gcacaccctc tactggaagg cactcttcga cgaacgcaac ctgcaggatg tggtctacaa 3900

gctcaacggc gaagcagagc tgttctaccg caagcagtcc atcccaaaga agatcaccca 3960

cccagccaag gaagcaatcg ccaacaagaa caaggataac ccaaagaagg aatccgtgtt 4020

cgagtacgac ctgatcaagg ataagcgctt caccgaggac aagttcttct tccactgccc 4080

aatcaccatc aacttcaagt cctccggcgc caacaagttc aacgatgaaa tcaacctgct 4140

cctgaaggag aaggctaacg acgtgcacat cctgtccatc gatcgcggcg aacgccacct 4200

cgcctactac accctggtcg acggcaaggg caacatcatc aagcaggaca ccttcaacat 4260

catcggcaac gatcgcatga agaccaacta ccacgacaag ctggccgcta tcgagaagga 4320

ccgcgattcc gctcgcaagg attggaagaa gatcaacaac atcaaggaaa tgaaggaagg 4380

ctacctctcc caggtggtcc acgaaatcgc taagctggtg atcgagtaca acgcaatcgt 4440

ggtcttcgaa gacctgaact tcggcttcaa gcgcggccgc ttcaaggtgg agaagcaggt 4500

ctaccagaag ctggaaaaga tgctcatcga gaagctgaac tacctcgtgt tcaaggacaa 4560

cgaattcgat aagaccggcg gcgtcctccg tgcataccag ctgaccgccc cattcgagac 4620

cttcaagaag atgggcaagc agaccggcat catctactac gtgccagctg gcttcacctc 4680

taagatctgc ccagtgaccg gcttcgtcaa ccagctctac ccaaagtacg aatccgtctc 4740

caagtcccag gagttcttct ccaagttcga caagatctgc tacaacctgg ataagggcta 4800

cttcgaattc tccttcgact acaagaactt cggcgataag gcagccaagg gcaagtggac 4860

catcgcatcc ttcggctccc gcctcatcaa cttccgcaac tccgacaaga accacaactg 4920

ggatacccgc gaagtgtacc caaccaagga actggagaag ctcctgaagg attactccat 4980

cgaatacggc cacggcgagt gcatcaaggc tgcaatctgc ggcgaatccg acaagaagtt 5040

cttcgcaaag ctgacctctg tgctcaacac catcctgcag atgcgcaact ccaagaccgg 5100

caccgagctg gattacctca tctccccagt ggccgacgtc aacggcaact tcttcgattc 5160

ccgccaggct ccaaagaaca tgccacagga cgctgatgca aacggcgcct accacatcgg 5220

tctgaagggt ctcatgctcc tgggtcgcat caagaacaac caggaaggca agaagctgaa 5280

tctcgtcatt aagaacgaag aatactttga atttgtccag aaccgcaata actaaggtac 5340

ctgtaaggcc tgcaccaaca atgattgagc gaagctccaa aatgtcctcc ccgggttgat 5400

attagatttc ataaatatac taaaaatctt gagagttttt ccgttgaaaa ctaaaaagct 5460

gggaaggtga atcgaatttc ggggctttaa agcaaaaatg aacagcttgg tctatagtgg 5520

ctaggtaccc tttttgtttt ggacacatgt agggtggccg aaacaaagta ataggacaac 5580

aacgctcgac cgcgattatt tttggagaat catgagcaca aaaccactct tcctgttacg 5640

gaaagcgaaa aaatcatccg gtgaacctga cgtcgtcctg tgggcaagca acgattttga 5700

atcgacctgt gccactctgg actacctgat cgttaagtca ggtaaaaaac tgagcagcta 5760

ttttaaagct gttgccacga attttcctgt cgttaatgac ctgcccgctg aaggtgagat 5820

cgattttacc tggagtgaac gctatcaact cagcaaagac tccatgacat gggaactaaa 5880

accgggagca gcaccagaca acgctcacta tcaaggcaat accaacgtca acggcgaaga 5940

catgactgag attgaggaga atatgctact cccaatttct ggccaggaac tgcccattcg 6000

ttggcttgct caacacggca gcgaaaaacc ggtaacgcac gtttcacgcg acggactcca 6060

ggcattacac attgctcggg ctgaagaact accggctgtt actgccctgg ctgtttccca 6120

caaaaccagc ctgctcgacc cgctggaaat tcgcgaactc cacaaactgg ttcgtgacac 6180

tgacaaagtt ttccctaatc ctggtaattc aaacctggga ctgataactg cttttttcga 6240

agcatacctg aacgctgact acaccgatcg aggactgctg acaaaagagt ggatgaaggg 6300

taatcgtgtt tcacacatca ctcgcacggc ttccggtgct aatgctggcg gcggaaacct 6360

caccgatcgc ggcgaaggtt tcgtacacga tctgacgtca ctggcgcgcg acgtagccac 6420

tggcgtactg gcccgttcaa tggatctgga catctataac cttcatccgg cacacgctaa 6480

acgcattgag gaaattatcg ctgaaaataa accgcccttt tctgttttcc gcgacaaatt 6540

catcaccatg cctggcgggc tggattattc ccgcgccatc gtggttgcgt ccgtaaaaga 6600

agcaccaatt gggatcgagg tcatccccgc gcacgtcact gaatatctga acaaagtact 6660

gactgaaacc gatcatgcca accctgatcc ggaaatcgtg gatattgcct gcggtcgctc 6720

ctctgccccg atgccgcagc gagtaacaga agaaggaaaa caggatgatg aagaaaaacc 6780

gcaaccatct ggaacaacgg cagttgaaca gggagaggct gaaacaatgg aaccggacgc 6840

aactgaacat catcaggaca cgcagccgct ggatgctcag tcacaggtaa attctgttga 6900

tgcgaaatat caggaactgc gggcagaact ccatgaagcc cggaaaaaca ttccatcaaa 6960

aaatcctgtc gatgacgata aattgcttgc tgcatcacgt ggtgaatttg ttgacggaat 7020

tagcgacccg aacgatccga aatgggtaaa ggggatccag actcgcgatt gtgtgtacca 7080

gaaccagcca gaaacggaaa aaaccagccc agatatgaat caacctgagc cagtagtgca 7140

acaggaaccg gaaatagcct gcaatgcctg cggccagact ggcggggata actgccctga 7200

ctgtggtgcg gtgatgggcg acgcaacata ccaggaaaca ttcgatgaag agagtcaggt 7260

tgaagctaag gaaaatgatc cggaggaaat ggaaggcgct gaacatccgc acaatgagaa 7320

tgctggcagc gatccgcatc gcgattgcag tgatgaaact ggcgaagtcg cagatcccgt 7380

aatcgtagaa gacatagagc caggtattta ttacggaatt tcgaatgaga attaccacgc 7440

gggtcccggt atcagtaagt ctcagctcga tgacattgct gatactccgg cactatattt 7500

gtggcgtaaa aatgcccccg tggacaccac aaagacaaaa acgctcgatt taggaactgc 7560

tttccactgc cgggtacttg aaccggaaga attcagtaac cgctttatcg tagcacctga 7620

atttaaccgc cgtacaaacg ccggaaaaga agaagagaaa gcgtttctga tggaatgcgc 7680

aagcacagga aaaacggtta tcactgcgga agaaggccgg aaaattgaac tcatgtatca 7740

aagcgttatg gctttgccgc tggggcaatg gcttgttgaa agcgccggac acgctgaatc 7800

atcaatttac tgggaagatc ctgaaacagg aattttgtgt cggtgccgtc cggacaaaat 7860

tatccctgaa tttcactgga tcatggacgt gaaaactacg gcggatattc aacgattcaa 7920

aaccgcttat tacgactacc gctatcacgt tcaggatgca ttctacagtg acggttatga 7980

agcacagttt ggagtgcagc caactttcgt ttttctggtt gccagcacaa ctattgaatg 8040

cggacgttat ccggttgaaa ttttcatgat gggcgaagaa gcaaaactgg caggtcaaca 8100

ggaatatcac cgcaatctgc gaaccctgtc tgactgcctg aataccgatg aatggccagc 8160

tattaagaca ttatcactgc cccgctgggc taaggaatat gcaaatgact aagcaaccac 8220

caatcgcaaa agccgatctg caaaaaactc agggaaaccg tgcaccagca gcagttaaaa 8280

atagcgacgt gattagtttt attaaccagc catcaatgaa agagcaactg gcagcagctc 8340

ttccacgcca tatgacggct gaacgtatga tccgtatcgc caccacagaa attcgtaaag 8400

ttccggcgtt aggaaactgt gacactatga gttttgtcag tgcgatcgta cagtgttcac 8460

agctcggact tgagccaggt agcgccctcg gtcatgcata tttactgcct tttggtaata 8520

aaaacgaaaa gagcggtaaa aagaacgttc agctaatcat tggctatcgc ggcatgattg 8580

atctggctcg ccgttctggt caaatcgcca gcctgtcagc ccgtgttgtc cgtgaaggtg 8640

acgagtttag cttcgaattt ggccttgatg aaaagttaat acaccgcccg ggagaaaacg 8700

aagatgcccc ggttacccac gtctatgctg tcgcaagact gaaagacgga ggtactcagt 8760

ttgaagttat gacgcgcaaa cagattgagc tggtgcgcag cctgagtaaa gctggtaata 8820

acgggccgtg ggtaactcac tgggaagaaa tggcaaagaa aacggctatt cgtcgcctgt 8880

tcaaatattt gcccgtatca attgagatcc agcgtgcagt atcaatggat gaaaaggaac 8940

cactgacaat cgatcctgca gattcctctg tattaaccgg ggaatacagt gtaatcgata 9000

attcagagga ataatctaga gtcgacctgc aggcatgcaa gcttggctgt tttggcggat 9060

gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac 9120

agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag 9180

tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc 9240

aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt 9300

ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg 9360

aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt 9420

aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct ttttgtttat 9480

ttttctaaat acattcaaat atgtatccgc tcatgaatta attccgctag atgacgtgcg 9540

gcttcgacct cctgggcgtg gcgcttgttg gcgcgctcgc ggctggctgc ggcacgacac 9600

gcgtctgagc agtattttgc gcgccgtcct cgtgggtcag gccggggtgg gatcaggcca 9660

ccgcagtagg cgcagctgat gcgatcctcc accgctgatg cttcaggcca gttttggtac 9720

ttcgtcgtga aggtcatgac accattataa cgaacgttcg ttaaaaattc tagccccaat 9780

tctgataatt tcttccggca ctcctgcgaa aacctgcgag acttcttgcc cagaaaaaac 9840

gccaagcgca gcggttaccg cacttttttt ccaggtgatt tcaccctgac cagcgaagcg 9900

gcactttagt gcatgaggtg tgcccctggt ttcccctctt tggagggttc aacccaaaaa 9960

agcacacaag caaaaatgaa aatcatcatg agcaagttgg tgcgaagcag caacgcgcta 10020

gctccaaaaa ggtctccagg atctcgagga gatttttgag ggggagggag tcgaggaaga 10080

gccagagcag aaggcgggga accgttctct gccgacagcg tgagcccccc ttaaaaatca 10140

ggccggggag gaaccgggga gggatcagag ctaggagcga gacaccctaa agggggggaa 10200

ccgttttctg ctgacggtgt ttcgtttatt agttttcagc ccgtggatag cggagggtga 10260

gggcaagtga gagccagagc aaggacggga cccctaaagg ggggaaccgt tttctgctga 10320

cggtgtttcg tttattagtt ttcagcccgt ggacggccgc gtttagcttc cattccaagt 10380

gcctttctga cttgttggat gcgcctttca ctgacaccta gttcgcctgc aagctcacga 10440

gtcgagggat cagcaaccga ttgagaacgg gcatccagga tcgcagtttt gacgcgaagt 10500

tcgagcaact cgcctgtcat ttctcggcgt ttgtttgctt ccgctaatcg ctgtcgcgtc 10560

tcctgcgcat acttactttc tgggtcagcc catctgcgtg cattcgatgt agctgcgccc 10620

cgtcgcccca tcgtcgctag agctttccgc cctcggctgc tctgcgtttc cacccgacga 10680

gcagggacga ctggctggcc tttagccacg tagccgcgca cacgacgcgc catcgtcagg 10740

cgatcacgca tggcgggaag atccggctcc cggccgtctg caccgaccgc ctgggcaacg 10800

ttgtacgcca cttcatacgc gtcgatgatc ttggcatctt ttaggcgctc accagcagct 10860

ttgagctggt atcccacggt caacgcgtgg cgaaacgcgg tctcgtcgcg cgctcgctct 10920

ggatttgtcc agagcactcg cacgccgtcg atcaggtcgc cggacgcgtc cagggcgctc 10980

ggcaggctcg cgtccaaaat cgctagcgcc ttggcttctg cggtggcgcg ttgtgccgct 11040

tcaatgcggg cgcgtccgct ggaaaagtcc tgctcaatgt actttttcgg cttctgtgat 11100

ccggtcatcg ttcgagcaat ctccattagg tcggccagcc gatccacacg atcatgctgg 11160

cagtgccatt tataggctgt cggatcgtct gagacgtgca gcggccaccg gctcagccta 11220

tgcgaaaaag cctggtcagc gccgaaaaca cgagtcattt cttccgtcgt tgcagccagc 11280

aggcgcatat ttgggctggt tttacctgct gcggcataca ccgggtcaat gagccagatg 11340

agctggcatt tcccgctcag cggattcacg ccgatccaag ccggcgcttt ttctaggcgt 11400

gcccatttct ctaaaatcgc gtagacctgc gggtttacgt gctcaatctt cccgccggcc 11460

tggtggctgg gcacatcgat gtcaagcacg atcaccgcgg catgttgcgc gtgcgtcagc 11520

gcaacgtact ggcaccgcgt cagcgctttt gagccagccc ggtagagctt tggttgggtt 11580

tcgccggtat ccgggttttt aatccaggcg ctcgcgaaat ctcttgtctt gctgccctgg 11640

aagctttcgc gtcccaggtg agcgagcagt tcgcggcgat cttctgccgt ccagccgcgt 11700

gagccgcagc gcatagcttc ggggtgggtg tcgaacagat cggcggacaa tttccacgcg 11760

ctagctgtga ctgtgtcctg cggatcggct agagtcatgt cttgagtgct ttctcccagc 11820

tgatgactgg gggttagccg acgccctgtg agttcccgct cacggggcgt tcaacttttt 11880

caggtatttg tgcagcttat cgtgttttct tcgtaaatga acgcttaact accttgttaa 11940

acgtggcaaa taggcaggat tgatggggat ctagcttcac gctgccgcaa gcactcaggg 12000

cgcaagggct gctaaaggaa gcggaacacg tagaaagcca gtccgcagaa acggtgctga 12060

ccccggatga atgtcgagcc gttccataca gaagctgggc gaacaaacga tgctcgcctt 12120

ccagaaaacc gaggatgcga accacttcat ccggggtcag caccaccggc aagcgccgcg 12180

acggccgagg tcttccgatc tcctgaagcc agggcagatc cgtgcacagc accttgccgt 12240

agaagaacag caaggccgcc aatgcctgac gatgcgtgga gaccgaaacc ttgcgctcgt 12300

tcgccagcca ggacagaaat gcctcgactt cgctgctgcc caaggttgcc gggtgacgca 12360

caccgtggaa acggatgaag gcacgaaccc agtggacata agcctgttcg gttcgtaagc 12420

tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg 12480

gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt 12540

ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat 12600

ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacatcatga 12660

gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc 12720

gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc 12780

tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa 12840

cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga 12900

ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc 12960

cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct 13020

tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata 13080

gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc 13140

tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg 13200

atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa 13260

tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc 13320

ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc 13380

gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg 13440

gcaaataagc gggactctgg ggttcgcgga atcatgacca aaatccctta acgtgagttt 13500

tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 13560

tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 13620

ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 13680

ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 13740

gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 13800

aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 13860

ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 13920

agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 13980

aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 14040

aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 14100

ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 14160

cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 14220

tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 14280

accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc 14340

cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 14400

gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg 14460

cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 14520

ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 14580

catcaccgaa acgcgcgagg cagcagatca attcgcgcgc gaaggcgaag cggcatgcat 14640

ttacgttgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga 14700

agagagtcaa ttcagggtgg tgaat 14725

<210> 2

<211> 7395

<212> DNA

<213> plasmid

<220>

<221> misc_feature

<222> (1)..(7395)

<400> 2

aacgtaaatg ccgcttcgcc ttcgcgcgcg aattgcaagc tgatccgggc ttatcgactg 60

cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca 120

ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt 180

ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgg aattccttat 240

cggtaccttg acagctagct cagtcctagg tataatcccg gggaatttct actgttgtag 300

attctagagg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag 360

aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac 420

ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc 480

cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac 540

tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg 600

ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg 660

ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg 720

tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 780

acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 840

tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 900

agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 960

cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 1020

aatgatgagc acttttgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 1080

gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 1140

cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 1200

gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 1260

aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 1320

ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 1380

cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta 1440

ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 1500

cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 1560

agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 1620

gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 1680

gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 1740

tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 1800

agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 1860

agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt tggggtgggc 1920

gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccattcg gggtcgttca 1980

ctggttcccc tttctgattt ctggcataga agaacccccg tgaactgtgt ggttccgggg 2040

gttgctgatt tttgcgagac ttctcgcgca attccctagc ttaggtgaaa acaccatgaa 2100

acactaggga aacacccatg aaacacccat tagggcagta gggcggcttc ttcgtctagg 2160

gcttgcattt gggcggtgat ctggtcttta gcgtgtgaaa gtgtgtcgta ggtggcgtgc 2220

tcaatgcact cgaacgtcac gtcatttacc gggtcacggt gggcaaagag aactagtggg 2280

ttagacattg ttttcctcgt tgtcggtggt ggtgagcttt tctagccgct cggtaaacgc 2340

ggcgatcatg aactcttgga ggttttcacc gttctgcatg cctgcgcgct tcatgtcctc 2400

acgtagtgcc aaaggaacgc gtgcggtgac cacgacgggc ttagcctttg cctgcgcttc 2460

tagtgcttcg atggtggctt gtgcctgcgc ttgctgcgcc tgtagtgcct gttgagcttc 2520

ttgtagttgc tgttctagct gtgccttggt tgccatgctt taagactcta gtagctttcc 2580

tgcgatatgt catgcgcatg cgtagcaaac attgtcctgc aactcattca ttatgtgcag 2640

tgctcctgtt actagtcgta catactcata tttacctagt ctgcatgcag tgcatgcaca 2700

tgcagtcatg tcgtgctaat gtgtaaaaca tgtacatgca gattgctggg ggtgcagggg 2760

gcggagccac cctgtccatg cggggtgtgg ggcttgcccc gccggtacag acagtgagca 2820

ccggggcacc tagtcgcgga taccccccct aggtatcgga cacgtaaccc tcccatgtcg 2880

atgcaaatct ttaacattga gtacgggtaa gctggcacgc atagccaagc taggcggcca 2940

ccaaacacca ctaaaaatta atagtcccta gacaagacaa acccccgtgc gagctaccaa 3000

ctcatatgca cgggggccac ataacccgaa ggggtttcaa ttgacaacca tagcactagc 3060

taagacaacg ggcacaacac ccgcacaaac tcgcactgcg caaccccgca caacatcggg 3120

tctaggtaac actgagtaac actgaaatag aagtgaacac ctctaaggaa ccgcaggtca 3180

atgagggttc taaggtcact cgcgctaggg cgtggcgtag gcaaaacgtc atgtacaaga 3240

tcaccaatag taaggctctg gcggggtgcc ataggtggcg cagggacgaa gctgttgcgg 3300

tgtcctggtc gtctaacggt gcttcgcagt ttgagggtct gcaaaactct cactctcgct 3360

gggggtcacc tctggctgaa ttggaagtca tgggcgaacg ccgcattgag ctggctattg 3420

ctactaagaa tcacttggcg gcgggtggcg cgctcatgat gtttgtgggc actgttcgac 3480

acaaccgctc acagtcattt gcgcaggttg aagcgggtat taagactgcg tactcttcga 3540

tggtgaaaac atctcagtgg aagaaagaac gtgcacggta cggggtggag cacacctata 3600

gtgactatga ggtcacagac tcttgggcga acggttggca cttgcaccgc aacatgctgt 3660

tgttcttgga tcgtccactg tctgacgatg aactcaaggc gtttgaggat tccatgtttt 3720

cccgctggtc tgctggtgtg gttaaggccg gtatggacgc gccactgcgt gagcacgggg 3780

tcaaacttga tcaggtgtct acctggggtg gagacgctgc gaaaatggca acctacctcg 3840

ctaagggcat gtctcaggaa ctgactggct ccgctactaa aaccgcgtct aaggggtcgt 3900

acacgccgtt tcagatgttg gatatgttgg ccgatcaaag cgacgccggc gaggatatgg 3960

acgctgtttt ggtggctcgg tggcgtgagt atgaggttgg ttctaaaaac ctgcgttcgt 4020

cctggtcacg tggggctaag cgtgctttgg gcattgatta catagacgct gatgtacgtc 4080

gtgaaatgga agaagaactg tacaagctcg ccggtctgga agcaccggaa cgggtcgaat 4140

caacccgcgt tgctgttgct ttggtgaagc ccgatgattg gaaactgatt cagtctgatt 4200

tcgcggttag gcagtacgtt ctcgattgcg tggataaggc taaggacgtg gccgctgcgc 4260

aacgtgtcgc taatgaggtg ctggcaagtc tgggtgtgga ttccaccccg tgcatgatcg 4320

ttatggatga tgtggacttg gacgcggttc tgcctactca tggggacgct actaagcgtg 4380

atctgaatgc ggcggtgttc gcgggtaatg agcagactat tcttcgcacc cactaaaagc 4440

ggcataaacc ccgttcgata ttttgtgcga tgaatttatg gtcaatgtcg cgggggcaaa 4500

ctatgatggg tcttgttgtt ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag 4560

aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt 4620

tcgaagggca ccaataactg ccttaaaaaa attacgcccc gccctgccac tcatcgcagt 4680

actgttgtaa ttcattaagc attctgccga catggaagcc atcacagacg gcatgatgaa 4740

cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga 4800

aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca 4860

cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca 4920

ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt 4980

cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac 5040

aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata cggaactccg 5100

gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat 5160

ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac 5220

attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa 5280

cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg 5340

tcgaagctcg gcggatttgt cctactcaag ctgatccgac aaaatccaca cattatccca 5400

ggtgtccgga tcggtcaaat acgctgccag ctcatagacc gtatccaaag catccggggc 5460

tgatccccgg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 5520

cctttatttg ttaactgtta attgtccttg ttcaaggatg ctgtctttga caacagatgt 5580

tttcttgcct ttgatgttca gcaggaagct cggcgcaaac gttgattgtt tgtctgcgta 5640

gaatcctctg tttgtcatat agcttgtaat cacgacattg tttcctttcg cttgaggtac 5700

agcgaagtgt gagtaagtaa aggttacatc gttaggatca agatccattt ttaacacaag 5760

gccagttttg ttcagcggct tgtatgggcc agttaaagaa ttagaaacat aaccaagcat 5820

gtaaatatcg ttagacgtaa tgccgtcaat cgtcattttt gatccgcggg agtcagtgaa 5880

caggtaccat ttgccgttca ttttaaagac gttcgcgcgt tcaatttcat ctgttactgt 5940

gttagatgca atcagcggtt tcatcacttt tttcagtgtg taatcatcgt ttagctcaat 6000

cataccgaga gcgccgtttg ctaactcagc cgtgcgtttt ttatcgcttt gcagaagttt 6060

ttgactttct tgacggaaga atgatgtgct tttgccatag tatgctttgt taaataaaga 6120

ttcttcgcct tggtagccat cttcagttcc agtgtttgct tcaaatacta agtatttgsa 6180

cbtggccttt atcttctacg tagtgaggat ctctcagcgt atggttgtcg cctgagctgt 6240

agttgccttc atcgatgaac tgctgtacat tttgatacgt ttttccgtca ccgtcaaaga 6300

ttgatttata atcctctaca ccgttgatgt tcaaagagct gtctgatgct gatacgttaa 6360

cttgtgcagt tgtcagtgtt tgtttgccgt aatgtttacc ggagaaatca gtgtagaata 6420

aacggatttt tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt gtttggtctt 6480

ttaggataga atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg ccagcgtttt 6540

tccagctgtc aatagaagtt tcgccgactt tttgatagaa catgtaaatc gatgtgtcat 6600

ccgcattttt aggatctccg gctaatgcaa agacgatgtg gtagccgtga tagtttgcga 6660

cagtgccgtc agcgttttgt aatggccagc tgtcccaaac gtccaggcct tttgcagaag 6720

agatattttt aattgtggac gaatcaaatt cagaaacttg atatttttca tttttttgct 6780

gttcagggat ttgcagcata tcatggcgtg taatatggga aatgccgtat gtttccttat 6840

atggcttttg gttcgtttct ttcgcaaacg cttgagttgc gcctcctgcc agcagtgcgg 6900

tagtaaaggt taatactgtt gcttgttttg caaacttttt gatgttcatc gttcatgtct 6960

ccttttttat gtactgtgtt agcggtctgc ttcttccagc cctcctgttt gaagatggca 7020

agttagttac gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc caaagtatac 7080

actttgccct ttacacattt taggtcttgc ctgctttatc agtaacaaac ccgcgcgatt 7140

tacttttcga cctcattcta ttagactctc gtttggattg caactggtct attttcctct 7200

tttgtttgat agaaaatcat aaaaggattt gcagactacg ggcctaaaga actaaaaaat 7260

ctatctgttt cttttcattc tctgtatttt ttatagtttc tgttgcatgg gcataaagtt 7320

gcctttttaa tcacaattca gaaaatatca taatatctca tttcactaaa taatagtgaa 7380

cggcaggtat atgtg 7395

<210> 3

<211> 3943

<212> DNA

<213> Francisella novicida

<220>

<221> misc_feature

<222> (1)..(3943)

<400> 3

acacaggaaa cagaccatgg atgtccatct accaagagtt tgtgaataaa tactccctgt 60

ccaagaccct ccgttttgag ctgatccccc aaggcaagac cctcgaaaac atcaaggcac 120

gcggcctcat cctggatgac gaaaagcgcg ctaaggatta caagaaggca aagcagatca 180

tcgacaagta ccaccagttc ttcatcgaag agatcctgtc ctccgtgtgc atctccgagg 240

acctgctcca gaactactcc gatgtctact tcaagctcaa gaagtccgat gacgataacc 300

tgcagaagga cttcaagtcc gctaaggata ccatcaagaa gcagatctcc gaatacatca 360

aggattccga gaagttcaag aacctcttca accagaacct gatcgacgca aagaagggcc 420

aggaatccga tctcatcctg tggctcaagc agtccaagga taacggcatc gagctcttca 480

aggccaactc cgacatcacc gacatcgatg aagctctgga gatcatcaag tccttcaagg 540

gctggaccac ctacttcaag ggcttccacg aaaaccgcaa gaacgtgtac tcctccaacg 600

atatcccaac ctctatcatc taccgcatcg tcgacgataa cctgccaaag ttcctcgaaa 660

acaaggcaaa gtacgagtcc ctgaaggata aggccccaga agctatcaac tacgagcaga 720

tcaagaagga cctggccgaa gagctcacct tcgacatcga ttacaagacc tctgaagtga 780

accagcgcgt cttctccctc gatgaagtgt tcgagatcgc caacttcaac aactacctga 840

accagtccgg catcaccaag ttcaacacca tcatcggcgg caagttcgtc aacggcgaaa 900

acaccaagcg caagggcatc aacgagtaca tcaacctcta ctcccagcag atcaacgata 960

agaccctgaa gaagtacaag atgtccgtgc tcttcaagca gatcctgtcc gacaccgaat 1020

ccaagtcctt cgtcatcgac aagctggagg acgattccga tgtggtcacc accatgcagt 1080

ccttctacga acagatcgca gccttcaaga ccgtggaaga gaagtccatc aaggagaccc 1140

tctccctgct cttcgacgat ctgaaggctc agaagctgga tctctccaag atctacttca 1200

agaacgacaa gtccctgacc gatctctccc agcaggtctt cgacgattac tccgtgatcg 1260

gcaccgcagt cctggaatac atcacccagc agatcgcccc aaagaacctc gataacccat 1320

ccaagaagga acaggagctg atcgccaaga agaccgaaaa ggctaagtac ctgtccctcg 1380

agaccatcaa gctggctctc gaagagttca acaagcaccg cgacatcgat aagcagtgcc 1440

gcttcgaaga gatcctcgca aacttcgctg caatcccaat gatcttcgac gaaatcgcac 1500

agaacaagga taacctggcc cagatctcca tcaagtacca gaaccagggc aagaaggatc 1560

tgctccaggc ctccgctgag gacgatgtga aggcaatcaa ggacctgctc gatcagacca 1620

acaacctgct ccacaagctg aagatcttcc acatctccca gtccgaagac aaggccaaca 1680

tcctcgacaa ggatgagcac ttctacctgg tgttcgaaga gtgctacttc gaactcgcta 1740

acatcgtccc actgtacaac aagatccgca actacatcac ccagaagcca tactccgatg 1800

aaaagttcaa gctcaacttc gagaactcca ccctggcaaa cggctgggac aagaacaagg 1860

aaccagataa caccgccatc ctcttcatca aggacgataa gtactacctg ggcgtgatga 1920

acaagaagaa caacaagatc ttcgacgata aggccatcaa ggaaaacaag ggcgagggct 1980

acaagaagat cgtgtacaag ctgctcccag gcgctaacaa gatgctccca aaggtcttct 2040

tctccgcaaa gtccatcaag ttctacaacc catccgaaga tatcctgcgc atccgcaacc 2100

actccaccca caccaagaac ggctccccac agaagggcta cgaaaagttc gagttcaaca 2160

tcgaagactg ccgcaagttc atcgatttct acaagcagtc catctccaag cacccagagt 2220

ggaaggactt cggcttccgc ttctccgata cccagcgcta caactccatc gatgaattct 2280

accgcgaagt ggagaaccag ggctacaagc tgaccttcga aaacatctcc gagtcctaca 2340

tcgattccgt ggtcaaccag ggcaagctgt acctcttcca gatctacaac aaggacttct 2400

ccgcttactc caagggccgc ccaaacctgc acaccctcta ctggaaggca ctcttcgacg 2460

aacgcaacct gcaggatgtg gtctacaagc tcaacggcga agcagagctg ttctaccgca 2520

agcagtccat cccaaagaag atcacccacc cagccaagga agcaatcgcc aacaagaaca 2580

aggataaccc aaagaaggaa tccgtgttcg agtacgacct gatcaaggat aagcgcttca 2640

ccgaggacaa gttcttcttc cactgcccaa tcaccatcaa cttcaagtcc tccggcgcca 2700

acaagttcaa cgatgaaatc aacctgctcc tgaaggagaa ggctaacgac gtgcacatcc 2760

tgtccatcga tcgcggcgaa cgccacctcg cctactacac cctggtcgac ggcaagggca 2820

acatcatcaa gcaggacacc ttcaacatca tcggcaacga tcgcatgaag accaactacc 2880

acgacaagct ggccgctatc gagaaggacc gcgattccgc tcgcaaggat tggaagaaga 2940

tcaacaacat caaggaaatg aaggaaggct acctctccca ggtggtccac gaaatcgcta 3000

agctggtgat cgagtacaac gcaatcgtgg tcttcgaaga cctgaacttc ggcttcaagc 3060

gcggccgctt caaggtggag aagcaggtct accagaagct ggaaaagatg ctcatcgaga 3120

agctgaacta cctcgtgttc aaggacaacg aattcgataa gaccggcggc gtcctccgtg 3180

cataccagct gaccgcccca ttcgagacct tcaagaagat gggcaagcag accggcatca 3240

tctactacgt gccagctggc ttcacctcta agatctgccc agtgaccggc ttcgtcaacc 3300

agctctaccc aaagtacgaa tccgtctcca agtcccagga gttcttctcc aagttcgaca 3360

agatctgcta caacctggat aagggctact tcgaattctc cttcgactac aagaacttcg 3420

gcgataaggc agccaagggc aagtggacca tcgcatcctt cggctcccgc ctcatcaact 3480

tccgcaactc cgacaagaac cacaactggg atacccgcga agtgtaccca accaaggaac 3540

tggagaagct cctgaaggat tactccatcg aatacggcca cggcgagtgc atcaaggctg 3600

caatctgcgg cgaatccgac aagaagttct tcgcaaagct gacctctgtg ctcaacacca 3660

tcctgcagat gcgcaactcc aagaccggca ccgagctgga ttacctcatc tccccagtgg 3720

ccgacgtcaa cggcaacttc ttcgattccc gccaggctcc aaagaacatg ccacaggacg 3780

ctgatgcaaa cggcgcctac cacatcggtc tgaagggtct catgctcctg ggtcgcatca 3840

agaacaacca ggaaggcaag aagctgaatc tcgtcattaa gaacgaagaa tactttgaat 3900

ttgtccagaa ccgcaataac taagaattct gtaaggcctg cac 3943

<210> 4

<211> 44

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(44)

<400> 4

tgcctgcagg tcgactctag agactagtgg gggtttctgc tgtt 44

<210> 5

<211> 45

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(45)

<400> 5

gttccgcttc ctttagcagc cctggcgata ggtgtcaaga attcg 45

<210> 6

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 6

gaggtcgaaa agtaaatcgc gaacttcgcc aaccacgcta t 41

<210> 7

<211> 45

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(45)

<400> 7

tacgaattcg agcgcggtac caatggtagt gaactaccgt ccctt 45

<210> 8

<211> 45

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(45)

<400> 8

cgaattcttg acacctatcg ccagggctgc taaaggaagc ggaac 45

<210> 9

<211> 44

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(44)

<400> 9

gctggtcaaa ataggcttgc acatgatatt cggcaagcag gcat 44

<210> 10

<211> 40

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(40)

<400> 10

agcgatggaa gcaacccata gtggaaaatg gccgcttttc 40

<210> 11

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 11

atagcgtggt tggcgaagtt cgcgatttac ttttcgacct c 41

<210> 12

<211> 44

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(44)

<400> 12

atgcctgctt gccgaatatc atgtgcaagc ctattttgac cagc 44

<210> 13

<211> 40

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(40)

<400> 13

gaaaagcggc cattttccac tatgggttgc ttccatcgct 40

<210> 14

<211> 47

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(47)

<400> 14

ctttctacgt gttccgcttc ctcctggcga taggtgtcaa gaattcg 47

<210> 15

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 15

ctggcgttgg cgaagcgtaa aaacttcgcc aaccacgcta t 41

<210> 16

<211> 47

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(47)

<400> 16

cgaattcttg acacctatcg ccaggaggaa gcggaacacg tagaaag 47

<210> 17

<211> 70

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(70)

<400> 17

atagcgtggt tggcgaagtt tttacgcttc gccaacgcca gtgcctcacc attttccacc 60

atgatattcg 70

<210> 18

<211> 20

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(20)

<400> 18

gcgaaacgat cctcatcctg 20

<210> 19

<211> 18

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(18)

<400> 19

gcgggactct ggggttcg 18

<210> 20

<211> 38

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(38)

<400> 20

caggatgagg atcgtttcgc atgcgctcac gcaactgg 38

<210> 21

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 21

cgaaccccag agtcccgctt atttgccgac taccttggtg a 41

<210> 22

<211> 42

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(42)

<400> 22

aggaaacaga ccatggaatt ctgtaaggcc tgcaccaaca at 42

<210> 23

<211> 42

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(42)

<400> 23

aagagtggtt ttgtgctcat gattctccaa aaataatcgc gg 42

<210> 24

<211> 42

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(42)

<400> 24

ccgcgattat ttttggagaa tcatgagcac aaaaccactc tt 42

<210> 25

<211> 47

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(47)

<400> 25

tagaggatcc ccgggtacct tattcctctg aattatcgat tacactg 47

<210> 26

<211> 45

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(45)

<400> 26

gtggtttttc ttttcaccag tgagacaaca gacaatcggc tgctc 45

<210> 27

<211> 43

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(43)

<400> 27

ggaagcaata aaatggcaca tgatcctcta gcgaacccca gag 43

<210> 28

<211> 50

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(50)

<400> 28

gagctcggta cccggggatc ctctagaatg gtagtgaact accgtccctt 50

<210> 29

<211> 43

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(43)

<400> 29

ctctggggtt cgctagagga tcatgtgcca ttttattgct tcc 43

<210> 30

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 30

atagcgtggt tggcgaagtt gtttcgtaac cttcacgacc g 41

<210> 31

<211> 42

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(42)

<400> 31

atagcgtggt tggcgaagtt gccgatatga ccgtaaaaca ga 42

<210> 32

<211> 42

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(42)

<400> 32

atagcgtggt tggcgaagtt tctggcttgc ttaagaaccc tt 42

<210> 33

<211> 43

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(43)

<400> 33

atagcgtggt tggcgaagtt gcgtttaaaa caccgataaa caa 43

<210> 34

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 34

atagcgtggt tggcgaagtt tggtatcttt actgtgggcc g 41

<210> 35

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 35

atagcgtggt tggcgaagtt ggtgttataa cgttttgccg c 41

<210> 36

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 36

cggtcgtgaa ggttacgaaa caacttcgcc aaccacgcta t 41

<210> 37

<211> 43

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(43)

<400> 37

atctgtttta cggtcatatc ggcaacttcg ccaaccacgc tat 43

<210> 38

<211> 42

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(42)

<400> 38

aagggttctt aagcaagcca gaaacttcgc caaccacgct at 42

<210> 39

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 39

cggcccacag taaagatacc aaacttcgcc aaccacgcta t 41

<210> 40

<211> 43

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(43)

<400> 40

ttgtttatcg gtgttttaaa cgcaacttcg ccaaccacgc tat 43

<210> 41

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 41

gcggcaaaac gttataacac caacttcgcc aaccacgcta t 41

<210> 42

<211> 24

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(24)

<400> 42

ctcactggtg aaaagaaaaa ccac 24

<210> 43

<211> 26

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(26)

<400> 43

ctagaggatc cccgggtacc gagctc 26

<210> 44

<211> 20

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(20)

<400> 44

aacgtaaatg ccgcttcgcc 20

<210> 45

<211> 20

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(20)

<400> 45

agggcaatca gctgttgccc 20

<210> 46

<211> 20

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(20)

<400> 46

aacgtaaatg ccgcttcgcc 20

<210> 47

<211> 20

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(20)

<400> 47

agggcaatca gctgttgccc 20

<210> 48

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 48

tcgctcaagg cgcactcccg ttctggataa tgttttttgc g 41

<210> 49

<211> 38

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(38)

<400> 49

tggcctgaag catcagcggt ggaggatcgc atcagctg 38

<210> 50

<211> 38

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(38)

<400> 50

cagctgatgc gatcctccac cgctgatgct tcaggcca 38

<210> 51

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 51

cgcaaaaaac attatccaga acgggagtgc gccttgagcg a 41

<210> 52

<211> 42

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(42)

<400> 52

cgcttcgcca acgccagtgc ctcattgcac gcaggttctc cg 42

<210> 53

<211> 40

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(40)

<400> 53

gaaaagcggc cattttccac catgatattc ggcaagcagg 40

<210> 54

<211> 40

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(40)

<400> 54

cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc 40

<210> 55

<211> 42

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(42)

<400> 55

ctctcatccg ccaaaacagc ccgcgattta cttttcgacc tc 42

<210> 56

<211> 44

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(44)

<400> 56

tcgccctaaa acaaagttaa acatcatgag ggaagcggtg atcg 44

<210> 57

<211> 25

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(25)

<400> 57

ggtaccttag ttattgcggt tctgg 25

<210> 58

<211> 24

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(24)

<400> 58

atgagcacaa aaccactctt cctg 24

<210> 59

<211> 44

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(44)

<400> 59

cgatcaccgc ttccctcatg atgtttaact ttgttttagg gcga 44

<210> 60

<211> 42

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(42)

<400> 60

accgcaataa ctaaggtacc gaagccagtg tgagttgcat ca 42

<210> 61

<211> 41

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(41)

<400> 61

aagacgatgc tggtatcacc tgtcctcaac tttagcgggg a 41

<210> 62

<211> 45

<212> DNA

<213> primer

<220>

<221> primer_bind

<222> (1)..(45)

<400> 62

cgcttcgcca acgccagtgc ctcaacaaca gacaatcggc tgctc 45

<210> 63

<211> 129

<212> DNA

<213> oligonucleotide

<220>

<221> misc_feature

<222> (1)..(129)

<400> 63

gtggtttttc ttttcaccag tgagttgaca gctagctcag tcctaggtat aatcccgggg 60

aatttctact gttgtagatc gcttcgccaa cgccagtgcc tcatctagag gatccccggg 120

taccgagct 129

<210> 64

<211> 81

<212> DNA

<213> oligonucleotide

<220>

<221> misc_feature

<222> (1)..(81)

<400> 64

ggtgatacca gcatcgtctt gatgcccttg gcagcaccct gctaaggagg caacaagatg 60

agcacaaaac cactcttcct g 81

Claims (5)

1. A corynebacterium glutamicum CRISPR/Cpf1 genome editing method, characterized in that: the method comprises the following steps:

(1) Preparing a plasmid carrying Cpf1 and RecET expression elements in competent cells, and then electrotransferring another plasmid carrying donor DNA and crRNA expression elements;

(2) The heterologous recombinase RecET is introduced into the corynebacterium glutamicum, after the second plasmid is transformed due to the expression of RecET, donor DNA can be recombined on a chromosome with high efficiency under the condition that Cpf1 is not induced, and then the corynebacterium glutamicum resuscitator is coated into a solid culture medium added with an inducer, so that the genome of a non-recombined cell is cut and killed due to the expression of Cpf 1.

2. The method for editing the corynebacterium glutamicum CRISPR/Cpf1 genome according to claim 1, wherein: adopts RAPID genome editing method, is beneficialpECdelta with the Dual plasmid Systemper1Cpf1-RecET and pXMsacB-crRNA-donor, wherein plasmid pecΔper1The sequence of the-Cpf 1-RecET is shown as SEQ ID NO 1; the pXMsacBThe crRNA-donor plasmid is obtained by ligating the donor DNA with the framework plasmid pXMSACB-crRNA, the sequence of which is shown in SEQ ID NO 2.

3. The method for editing the corynebacterium glutamicum CRISPR/Cpf1 genome according to claim 2, wherein: the sacB-mediated sucrose lethal is used for realizing the high-efficiency loss of the pXMJ19 derived plasmid, and the per1 is knocked out for realizing the high-efficiency loss of the pEC-XK99E derived plasmid, so that the iterative genome operation by using RAPID is ensured.

4. The method for editing the corynebacterium glutamicum CRISPR/Cpf1 genome according to claim 1, wherein: the cell resuscitating time after electrotransformation is 2-6h.

5. The method for editing the corynebacterium glutamicum CRISPR/Cpf1 genome according to claim 1 or 4, wherein: cell resuscitation time after electrotransformation was 5h.