CN114540400B - Corynebacterium glutamicum CRISPR/Cpf1 genome editing technology - Google Patents
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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
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
<110> university of Tianjin science and technology
<120> Corynebacterium glutamicum CRISPR/Cpf1 efficient genome editing technique
<130> 2022
<160> 64
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14725
<212> DNA
<213> plasmid
<220>
<221> misc_feature
<222> (1)..(14725)
<400> 1
gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt 60
tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg 120
gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag 180
tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc 240
gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa 300
cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt 360
gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc 420
actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt 480
ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag 540
caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc 600
tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg 660
agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact 720
gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc 780
gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca 840
tgttatatcc cgccgtcaac caccatcaaa caggattttc gcctgctggg gcaaaccagc 900
gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc 960
gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc 1020
gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 1080
tgagcgcaac gcaattaatg tgagttagcg cgaattgatc tggtttgaca gcttatcatc 1140
gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct 1200
gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata 1260
atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca 1320
attaatcatc cggctcgtat aatgtgtgga attgtgagcg gataacaatt tcacacagga 1380
aacagaccat gagcggataa caatttcaca caggaaacag accatggaat tcatgtccat 1440
ctaccaagag tttgtgaata aatactccct gtccaagacc ctccgttttg agctgatccc 1500
ccaaggcaag accctcgaaa acatcaaggc acgcggcctc atcctggatg acgaaaagcg 1560
cgctaaggat tacaagaagg caaagcagat catcgacaag taccaccagt tcttcatcga 1620
agagatcctg tcctccgtgt gcatctccga ggacctgctc cagaactact ccgatgtcta 1680
cttcaagctc aagaagtccg atgacgataa cctgcagaag gacttcaagt ccgctaagga 1740
taccatcaag aagcagatct ccgaatacat caaggattcc gagaagttca agaacctctt 1800
caaccagaac ctgatcgacg caaagaaggg ccaggaatcc gatctcatcc tgtggctcaa 1860
gcagtccaag gataacggca tcgagctctt caaggccaac tccgacatca ccgacatcga 1920
tgaagctctg gagatcatca agtccttcaa gggctggacc acctacttca agggcttcca 1980
cgaaaaccgc aagaacgtgt actcctccaa cgatatccca acctctatca tctaccgcat 2040
cgtcgacgat aacctgccaa agttcctcga aaacaaggca aagtacgagt ccctgaagga 2100
taaggcccca gaagctatca actacgagca gatcaagaag gacctggccg aagagctcac 2160
cttcgacatc gattacaaga cctctgaagt gaaccagcgc gtcttctccc tcgatgaagt 2220
gttcgagatc gccaacttca acaactacct gaaccagtcc ggcatcacca agttcaacac 2280
catcatcggc ggcaagttcg tcaacggcga aaacaccaag cgcaagggca tcaacgagta 2340
catcaacctc tactcccagc agatcaacga taagaccctg aagaagtaca agatgtccgt 2400
gctcttcaag cagatcctgt ccgacaccga atccaagtcc ttcgtcatcg acaagctgga 2460
ggacgattcc gatgtggtca ccaccatgca gtccttctac gaacagatcg cagccttcaa 2520
gaccgtggaa gagaagtcca tcaaggagac cctctccctg ctcttcgacg atctgaaggc 2580
tcagaagctg gatctctcca agatctactt caagaacgac aagtccctga ccgatctctc 2640
ccagcaggtc ttcgacgatt actccgtgat cggcaccgca gtcctggaat acatcaccca 2700
gcagatcgcc ccaaagaacc tcgataaccc atccaagaag gaacaggagc tgatcgccaa 2760
gaagaccgaa aaggctaagt acctgtccct cgagaccatc aagctggctc tcgaagagtt 2820
caacaagcac cgcgacatcg ataagcagtg ccgcttcgaa gagatcctcg caaacttcgc 2880
tgcaatccca atgatcttcg acgaaatcgc acagaacaag gataacctgg cccagatctc 2940
catcaagtac cagaaccagg gcaagaagga tctgctccag gcctccgctg aggacgatgt 3000
gaaggcaatc aaggacctgc tcgatcagac caacaacctg ctccacaagc tgaagatctt 3060
ccacatctcc cagtccgaag acaaggccaa catcctcgac aaggatgagc acttctacct 3120
ggtgttcgaa gagtgctact tcgaactcgc taacatcgtc ccactgtaca acaagatccg 3180
caactacatc acccagaagc catactccga tgaaaagttc aagctcaact tcgagaactc 3240
caccctggca aacggctggg acaagaacaa ggaaccagat aacaccgcca tcctcttcat 3300
caaggacgat aagtactacc tgggcgtgat gaacaagaag aacaacaaga tcttcgacga 3360
taaggccatc aaggaaaaca agggcgaggg ctacaagaag atcgtgtaca agctgctccc 3420
aggcgctaac aagatgctcc caaaggtctt cttctccgca aagtccatca agttctacaa 3480
cccatccgaa gatatcctgc gcatccgcaa ccactccacc cacaccaaga acggctcccc 3540
acagaagggc tacgaaaagt tcgagttcaa catcgaagac tgccgcaagt tcatcgattt 3600
ctacaagcag tccatctcca agcacccaga gtggaaggac ttcggcttcc gcttctccga 3660
tacccagcgc tacaactcca tcgatgaatt ctaccgcgaa gtggagaacc agggctacaa 3720
gctgaccttc gaaaacatct ccgagtccta catcgattcc gtggtcaacc agggcaagct 3780
gtacctcttc cagatctaca acaaggactt ctccgcttac tccaagggcc gcccaaacct 3840
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.
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Citations (5)
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CN107119067A (en) * | 2017-04-25 | 2017-09-01 | 华南理工大学 | A kind of method of the continuous traceless knockout of gene in Corynebacterium glutamicum |
CN108676809A (en) * | 2018-05-11 | 2018-10-19 | 天津科技大学 | A kind of Corynebacterium glutamicum gene group edit methods |
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