CN107937429B - Construction method of recombinant sgRNA framework vector in CRIPSR/Cas9 system - Google Patents

Abstract

The invention provides a construction method of a recombinant sgRNA framework vector in a CRIPSR/Cas9 system, which can solve the technical problems that the operation requirement and the reagent preparation requirement of the existing construction method are high, the positive rate of a strain obtained by a common technician is low, and the selection of the positive strain is time-consuming and labor-consuming. A construction method of a recombinant sgRNA framework vector in a CRIPSR/Cas9 system comprises the following steps: (1) preparing a linearized sgRNA empty plasmid; (2) preparing a differential fragment; (3) preparing a recombinant sgRNA framework vector; (4) selecting a recombinant sgRNA framework vector; the method is characterized in that: the distinguishing segment of the step (2) comprisessacBGenes and their Open Reading Frames (ORFs), promoters and terminators.

Description

Construction method of recombinant sgRNA framework vector in CRIPSR/Cas9 system

Technical Field

The invention relates to the field of molecular biology and biotechnology, in particular to a construction method of a recombinant sgRNA framework vector in a CRIPSR/Cas9 system.

Background

The CRIPSR/Cas9 system, called as the third generation gene editing technology, is widely applied to the genetic modification of various in vivo and in vitro systems, the construction of transgenic model animals, and even the field of gene therapy. In gene editing operations using the CRIPSR/Cas9 system, a supply format for sgrnas needs to be selected. Taking gene editing of mammalian cells as an example, sgRNA can be expressed using a plasmid or virus as a vector, or the sgRNA can be produced and purified in vitro and then directly transfected into cells to perform functions. Of course, the form in which the sgRNA is introduced depends on experimental needs and the characteristics of the cell itself, but it is generally more economical and convenient to construct the sgRNA directly on an expression vector. In particular, the emergence of All-in-one vectors capable of simultaneously expressing optimized Cas9 protein and sgRNA enables researchers to more easily realize gene editing based on the CRIPSR/Cas9 system by using a single plasmid [ Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F.

In the construction process of sgRNA backbone vectors, the most important step is the linearization of the plasmid. Almost all sgRNA backbone vectors utilize a type IIS endonuclease with inconsistent recognition and cleavage sites to enable seamless insertion of sgRNA oligonucleotides after single-strand annealing pairing. However, the conventional IIS type endonuclease such as BsmBI has insufficient cleavage effect. To solve this problem, some vectors, such as pX330, are used to add a discrimination fragment between the two reverse type IIS endonuclease cleavage sites, so that the linearized vector can be discriminated from the empty plasmid by gel recovery. Although the method is effective and necessary, the generation of false positive in actual operation cannot be avoided, especially in the glue recovery process, the operation requirement and the reagent preparation requirement are high, the linearized plasmid recovered by common technicians is mixed with an insufficiently digested vector, and after the linearized plasmid is connected with the sgRNA short double-stranded DNA fragment with the sticky end formed by annealing, the transformation efficiency of a few residual empty plasmids is far higher than that of a connection product, so that most of clones grown after the escherichia coli is transformed and plated are false positive transformants containing empty plasmids, the positive rate is low and is often about 10%, and more single clones need to be selected for colony PCR identification to obtain positive clones, which wastes time and labor.

Disclosure of Invention

Aiming at the problems, the invention provides a construction method of a recombinant sgRNA framework vector in a CRIPSR/Cas9 system, which can solve the technical problems that the operation requirement and the reagent preparation requirement of the existing construction method are high, the positive rate of a strain obtained by ordinary technicians is low, and the selection of the positive strain is time-consuming and labor-consuming.

The technical scheme is that the construction method of the recombinant sgRNA framework vector in the CRIPSR/Cas9 system comprises the following steps:

(1) preparing a linearized sgRNA empty plasmid, wherein the adopted sgRNA empty plasmid has a pair of symmetrical IIS type endonuclease enzyme cutting sites, the sgRNA empty plasmid is subjected to enzyme cutting by IIS type endonuclease enzyme, and the linearized sgRNA empty plasmid subjected to enzyme cutting is recovered;

(2) preparing a distinguishing segment, wherein both ends of the distinguishing segment are provided with terminal segments corresponding to the IIS type endonuclease after enzyme digestion;

(3) preparing a recombinant sgRNA framework vector, and connecting the linearized sgRNA empty plasmid recovered from the step (1) with the distinguishing fragment prepared in the step (2) to prepare a recombinant sgRNA framework vector;

(4) selecting a recombinant sgRNA framework vector, converting the sgRNA framework vector in the step (3) into escherichia coli, coating a resistant plate to form a monoclonal, and selecting the monoclonal to perform colony PCR identification by using a primer containing the IIS type endonuclease enzyme cutting site joint;

the method is characterized in that:

the distinguishing segment of the step (2) comprisessacBGenes and their Open Reading Frames (ORFs), promoters and terminators, saidsacBThe nucleotide sequence of the gene is shown as SEQ ID No. 1;

and (4) respectively coating the bacteria with positive PCR identification on an LB resistance plate and an LB resistance plate containing 5% -10% of sucrose, and if the bacteria cannot grow on the LB resistance plate containing 5% -10% of sucrose and form lawn on the LB resistance plate, the bacteria on the surface contain the successfully constructed recombinant sgRNA skeleton vector.

Further, preparing a linearized sgRNA empty plasmid in the step (1), if the sgRNA empty plasmid exists, directly carrying out enzyme digestion on the sgRNA empty plasmid by using a corresponding IIS type endonuclease, and recovering glue after enzyme digestion to obtain the linearized sgRNA empty plasmid; or designing a primer to perform whole-plasmid PCR on a plasmid according to the constructed plasmid of a certain sgRNA to obtain an empty sgRNA plasmid containing an IIS type endonuclease enzyme digestion joint, then carrying out enzyme digestion on the empty sgRNA plasmid by using the IIS type endonuclease enzyme digestion joint, and carrying out gel recovery on the linearized sgRNA empty plasmid after enzyme digestion.

Further, the step (2) of preparing the differential fragment, designing a pair of primers containing the type IIS endonuclease cleavage site adaptor according to the vector map and the sequence of pK18mobSacB, wherein the amplicon of the primers containssacBORF of gene and promoter and terminator thereof, PCR is carried out on pK18mobSacB vector by the primer to obtain target fragment, PCR product is recovered by glue, enzyme digestion is carried out on the target fragment by IIS type endonuclease, and distinguished fragment is obtained after column purification.

Further, a recombinant sgRNA framework vector is prepared in step (3), the linearized sgRNA empty plasmid recovered in step (1) and the differentiated fragment prepared in step (2) are mixed uniformly, T4 ligase and T4 ligase Buffer are added, and the mixture is placed in a 16-degree metal bath for overnight connection.

Further, selecting a recombinant sgRNA framework vector in the step (4), transforming the ligation product in the step (3) into escherichia coli, coating a resistant plate, culturing for 12-20 hours until a single clone is formed on the plate, selecting the single clone, carrying out colony PCR identification by using the primer in the step (2), transferring a colony with positive PCR identification into a shake flask for overnight culture, then carrying out plasmid extraction, carrying out enzyme digestion on the plasmid by using IIS type endonuclease, shaking the clone with correct enzyme digestion identification overnight, respectively coating an LB resistant plate and an LB resistant plate containing 5-10% of sucrose after dilution, and if the colony cannot grow on the LB resistant plate containing 5-10% of sucrose and a bacterial lawn is formed on the LB resistant plate, indicating that the bacteria contain the successfully constructed recombinant sgRNA framework vector.

The construction method of the invention can reach two thousand multi-bpsacBThe gene related sequence is constructed in two IIS type endonuclease recognition sites of a traditional sgRNA vector, and one can separate an empty plasmid from a linearized plasmid through electrophoresis; furthermore, a sucrose lethal genesacBCan make Escherichia coli decompose sucrose in environment into glucose and fructose, and catalyze the latter to form fructan, resulting in bacteria death, even if the recovered product still remains in the form of empty plasmid, its Escherichia coli transformant can not form single colony on sucrose-containing resistant plate, because the plate is mostly grown outsacBThe gene is cut by IIS type endonuclease, 5% -10% of sucrose can not die, and the cloning of sgRNA fragments is constructed, and experiments show that the positive rate of the sgRNA framework vector constructed by the method can reach more than 95%.

Drawings

FIG. 1 is a schematic diagram of PCR primer design for preparing linearized pX330 empty plasmid by using pX330-p53 in example 1 as starting vector.

Fig. 2 is a schematic diagram of preparation of a recombinant sgRNA backbone vector using step (3) of example 1.

FIG. 3 is an electrophoretogram of bbsI cleavage assay using step (4) of example 1,

marker: DL10000 Marker; lane1 plasmid 8500 after cutting bbsI enzyme, and the cut sacB gene related fragment 2500.

FIG. 4 is a map of pX330-sacB plasmid of application example 1.

Detailed Description

Example 1

A construction method of a recombinant sgRNA framework vector in a CRIPSR/Cas9 system comprises the following steps:

(1) preparation of linearized sgRNA empty plasmids

The adopted sgRNA empty plasmid has a pair of symmetric IIS type endonuclease enzyme cutting sites, so that a short sgRNA fragment can be seamlessly inserted between a promoter and a scaffold; if the sgRNA empty plasmid exists, directly carrying out enzyme digestion treatment by using a corresponding IIS type endonuclease; and (5) recovering glue after enzyme digestion to obtain linear sgRNA empty plasmids.

If no sgRNA empty plasmid exists and only a plasmid with a certain sgRNA is constructed, designing a primer to perform whole-plasmid PCR on the plasmid so as to eliminate the sgRNA on the plasmid and obtain an sgRNA empty plasmid containing an IIS type endonuclease digestion joint; the initial sgRNA plasmid used cannot have a type IIS endonuclease recognition site designed on the primer; after the PCR is finished, glue is recovered to obtain linear sgRNA empty plasmids containing IIS type endonuclease digestion joints; then carrying out enzyme digestion treatment by using corresponding IIS type endonuclease; and after the enzyme digestion is finished, purifying the enzyme digestion product by a column to obtain a linearized sgRNA empty plasmid.

Enzyme digestion system: plasmid 1ug, 10 Xendonuclease Buffer 5ul, endonuclease 1ul (NEB), and double distilled water to 50 ul.

Enzyme cutting time: 37 ℃ for 1 hour.

And (3) glue recovery: and (4) dropping all the enzyme digestion products into 1% agarose gel, and performing electrophoresis at 120V for half an hour.

Whole plasmid PCR:

and (3) PCR system: 5X Q5 Reaction Buffer 5ul, 10 mdNTPs 0.5ul, 10uM upstream primer 1.25ul, 10uM downstream primer 1.25ul, initial sgRNA plasmid 200ng, Q5 Hi-Fi enzyme 0.5ul, double distilled water to make up to 50 ul;

PCR procedure: pre-denaturation at 98 ℃ for 30 s; an amplification stage: 30s at 98 ℃, 10-30s at 50-72 ℃,

72 ℃ 20-30 s/kb; final extension 72 ℃ 2 min.

(2) Preparation of the differentiating fragments

Differentiating fragment inclusionsacBGenes and their Open Reading Frames (ORFs), promoters and terminators, sacBthe nucleotide sequence of the gene is shown as SEQ ID No.1, and the two ends of the distinguishing segment are provided with terminal segments corresponding to IIS type endonuclease after enzyme digestion; specifically, pK18mobSacB plasmid can be used as a donor pairsacBSubcloning genes and promoters and terminators thereof;sacBthe gene-related fragment may also be obtained from other sources, such as direct preparation; designing primers to ensure that two ends of the obtained fragment contain corresponding IIS type endonuclease digestion linkers; after PCR is finished, glue is recovered to obtain the enzyme cutting adaptor containing IIS type endonucleasesacBA gene-related fragment; then carrying out enzyme digestion treatment by using corresponding IIS type endonuclease; and (4) after enzyme digestion, column-purifying enzyme digestion products to obtain the distinguishing fragments.

The PCR system and the PCR program are the same as the above, and the enzyme digestion system and the enzyme digestion time are referred to in the step (1).

(3) Preparation of recombinant sgRNA framework vector

And (3) carrying out a connection reaction on the enzyme-digested linearized sgRNA empty plasmid and the column-purified distinguished fragment to obtain a recombinant sgRNA framework vector.

Reaction system: 1ul of T4 DNA ligase (NEB), 2ul of 10X T4 ligase Buffer, molar concentration of linearized sgRNA empty plasmid to distinguished fragment of 1: 5, double distilled water to make up to 20 ul; the reaction was placed in a 16 ° metal bath overnight.

(4) Selecting recombinant sgRNA framework vector

Transforming the sgRNA framework vector in the step (3) into escherichia coli, coating a resistance (vector self resistance gene) flat plate, culturing for 12-20 hours until a monoclonal is formed on the flat plate, and selecting the monoclonal to perform colony PCR identification by using the primer in the step 2; transferring the colony with positive PCR identification into a shake flask for overnight culture, then carrying out plasmid extraction, and carrying out enzyme digestion on the plasmid by using the IIS type endonuclease, wherein the enzyme digestion system is the same as the above; spotting the enzyme-digested product in 2% agarose gel, and performing 120V electrophoresis for 1 hour; shaking the clone with correct enzyme digestion identification overnight, diluting one hundred times, and respectively coating 100ul of the clone to an LB resistance plate (only without sucrose compared with the latter) and an LB resistance plate containing 5-10% of sucrose; if the bacterial lawn cannot grow on the LB resistant plate containing 5% -10% of sucrose and is formed on the LB resistant plate, the bacterial lawn contains the successfully constructed recombinant sgRNA framework vector.

Colony PCR identification:

and (3) PCR system: 5ul of pre-stained Taqmix, 0.5ul of 10uM upstream primer, 0.5ul of 10uM downstream primer, a proper amount of thallus and 4ul of double distilled water; PCR procedure: pre-denaturation at 95 ℃ for 3 min.

An amplification stage: 95 ℃ 30s, 52-58 ℃ 30s, 72 ℃ 1 min/kb; final extension 72 ℃ 2 min.

After the completion, the colony PCR products are respectively spotted into 1% agarose gel, and the gel is seen after 120V electrophoresis for half an hour.

Application example 1

(1) Preparation of linearized px330 sgRNA empty plasmid:

pX330 is one of the most commonly used mammalian cell sgRNA vectors disclosed (Addgene # 42230), the nucleotide sequence of which is shown in SEQ ID No.3, and the empty vector of which contains a pair of symmetric bbsI enzyme cutting sites, and the vector after bbsI enzyme cutting has a sticky end for adding short fragment double chains of the sgRNA. If the pX330 empty plasmid exists, directly using bbsI to perform enzyme digestion treatment, and recovering gel after the enzyme digestion is completed to obtain the linearized pX330 empty plasmid, wherein the nucleotide sequence of the linearized pX330 empty plasmid is shown as SEQ ID No. 4.

Enzyme digestion system: pX330 plasmid 1ug, 10X endonuclease Buffer 5ul, bbsI 1ul (NEB), and double distilled water to make up to 50 ul.

Enzyme cutting time: 37 ℃ for 1 hour.

And (3) glue recovery: and (4) dropping all the enzyme digestion products into 1% agarose gel, and performing electrophoresis at 120V for half an hour.

If no pX330 empty plasmid exists, taking pX330-p53 (Addgene # 59910) inserted with sgRNA as a starting plasmid, and the nucleotide sequence of the starting plasmid is shown in SEQ ID No.5, designing a primer to perform whole plasmid PCR on the plasmid so as to eliminate the sgRNA of the target p53 on the plasmid and obtain an sgRNA empty plasmid containing a bbsI enzyme cutting site joint; after PCR is finished, glue is recovered to obtain a linearized pX330 empty plasmid containing a bbsI enzyme digestion joint; then carrying out enzyme digestion treatment by using bbsI; after the enzyme digestion is finished, purifying the enzyme digestion product by a column to obtain a linearized pX330 empty plasmid.

The primer design is schematically shown in FIG. 1.

pX330 upstream primer: gaGAAGACggGTTTTAGAGCTAGAAATAGCAAGTT

Downstream primer pX 330: gaGAAGACggGGTGTTTCGTCCTTTCCACAAGATA

(recognition site bbsI underlined)

The linearized plasmid obtained after the primer amplicon (about 8500 bp) is cut by bbsI is completely consistent with the linearized product of the pX330 empty vector after the bbsI is cut by enzyme.

And (3) PCR system: 5X Q5 Reaction Buffer 5ul, 10mMdNTPs 0.5ul, 10uM upstream primer 1.25ul, 10uM downstream primer 1.25ul, pX330 p53 plasmid 200ng, Q5 Hi-Fi enzyme 0.5ul, using double distilled water to make up to 50 ul.

PCR procedure: pre-denaturation at 98 ℃ for 30 s; an amplification stage: 30s at 98 ℃, 20s at 62 ℃ and 4min at 72 ℃; final extension 72 ℃ 2 min.

The enzyme digestion system, time, and gel recovery parameters and time are as above.

(2) Preparation of the differentiating fragments

Designing upstream and downstream primers containing bbsI enzyme digestion joint according to pK18mobSacB complete plasmid sequence, and carrying out sequencing on pK18mobSacBsacBCarrying out PCR amplification on the gene and the related fragments of the promoter and the terminator thereof, wherein the nucleotide sequence of the amplification piece is shown as SEQ ID No. 2; obtaining the linker containing the bbsI restriction enzyme cutting site by glue recovery after PCRsacBA gene-related fragment; then carrying out enzyme digestion treatment by using bbsI; purifying the product after enzyme digestion.

sacB upstream primer: ggCACCGGGTCTTC GCGATGCCTGCTTGCCGAAT

A sacB downstream primer: ggAAACAGGTCTTC CCAGACGAACGAAGAGCGATTGAG

(recognition site bbsI underlined)

The PCR system and the PCR procedure were substantially the same as in step 1, except that the extension time was shortened to 60 s.

The enzyme cutting system and the enzyme cutting time are the same as the step 1.

(3) Preparation of recombinant sgRNA framework vector

As shown in FIG. 2, the plasmid pX330 linearized with bbsI obtained in the first two steps was ligated withsacBAnd carrying out ligation reaction on the purified fragments obtained by gene enzyme digestion.

Reaction system: 1ul T4 DNA ligase (NEB), 2ul 10X T4 ligase Buffer, linearized pX330 plasmid andsacBthe molar concentration of the purified fragment obtained by gene enzyme digestion is 1: 5, and the double distilled water is added to 20 ul.

The reaction was placed in a 16 ° metal bath overnight for ligation.

(4) Selecting recombinant sgRNA framework vector

Transforming escherichia coli by the sgRNA framework vector obtained by connection in the step (3), coating an ampicillin-resistant LB plate (plasmid pX330 has an ampicillin resistant gene), culturing in an incubator at 37 ℃, forming a monoclonal on the plate after 12 hours, and performing colony PCR identification by using the primers in the step 2; transferring the colony identified to be positive by PCR (2400 bp specific strip) into a shake flask for overnight culture, then carrying out plasmid extraction, carrying out enzyme digestion on the plasmid by using bbsI, carrying out 120V electrophoresis on the enzyme digestion product in 2% agarose gel for 1 hour, and then viewing the gel; the clone with correct enzyme restriction identification (having 2400bp and 8500bp bands, and an electrophoretogram thereof is shown in figure 3) is shaken overnight, diluted by one hundred times and respectively coated on 100ul of LB ampicillin plates and LB ampicillin plates containing 5% of sucrose, if the colonies cannot grow on the LB ampicillin plates containing 5% of sucrose and form lawn on the LB ampicillin plates, the construction of the pX330-sacB skeleton vector is successful. The pX330-sacB plasmid map is shown in figure 4, and the plasmid and the corresponding sucrose lethal screening are utilized to carry out traditional sgRNA construction, so that the positive rate can reach more than ninety percent.

Colony PCR identification:

and (3) PCR system: 5ul of pre-stained Taqmix, 0.5ul of 10uM upstream primer, 0.5ul of 10uM downstream primer, a proper amount of thallus and 4ul of double distilled water.

PCR procedure: pre-denaturation at 95 ℃ for 3 min; an amplification stage: 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 3 min; final extension 72 ℃ 2 min.

After the completion, the colony PCR products are respectively spotted into 1% agarose gel, and the gel is seen after 120V electrophoresis for half an hour.

The enzyme cutting system and the enzyme cutting time are the same as the step 1.

Sequence listing

<110> university of south of the Yangtze river

Construction method of recombinant sgRNA framework vector in <120> CRIPSR/Cas9 system

<130> 2017

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1422

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

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gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca 120

tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat 180

gaaaaatatc aagtttctga atttgattcg tccacaatta aaaatatctc ttctgcaaaa 240

ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat 300

cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg 360

atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc 420

cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca 480

caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact 540

gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca 600

gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt 660

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gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc 900

gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat 960

gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa 1020

attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc 1080

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ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa 1320

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gcggacgtgc tcatagtcca cgacgcccgt gattttgtag ccctggccga cggccagcag 2340

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

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atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360

agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420

aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480

ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720

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ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840

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gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 960

ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 1020

tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1080

accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1140

ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1200

ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 1260

aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1320

tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1380

acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1440

acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1500

agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1560

agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1620

tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1680

tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1740

aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1800

gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1860

ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1920

tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 1980

gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 2040

tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 2100

gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 2160

agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 2220

agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 2280

tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 2340

acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2400

agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2460

gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2520

gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2580

tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2640

ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2700

ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2760

tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 2820

gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 2880

agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 2940

ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 3000

aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 3060

aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 3120

atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 3180

acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 3240

tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3300

acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 3360

agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3420

ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 3480

aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3540

ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3600

acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3660

gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3720

aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3780

agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3840

accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 3900

gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 3960

gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4020

agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 4080

gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 4140

ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 4200

agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 4260

atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4320

acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4380

aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4440

gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4500

ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4560

caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4620

aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4680

tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4740

actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4800

tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4860

ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4920

agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 4980

agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 5040

aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 5100

tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 5160

acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 5220

ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 5280

aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5340

agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5400

ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5460

tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 5520

aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5580

tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 5640

ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 5700

tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 5760

gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 5820

cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 5880

gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca 5940

tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc 6000

gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 6060

ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 6120

cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 6180

gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 6240

gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 6300

ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 6360

tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 6420

atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag 6480

ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 6540

ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 6600

tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 6660

gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 6720

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 6780

caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 6840

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 6900

gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 6960

gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 7020

atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 7080

caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 7140

gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 7200

accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 7260

ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 7320

gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 7380

acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 7440

atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 7500

ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca 7560

gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 7620

gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 7680

tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 7740

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 7800

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 7860

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 7920

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 7980

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 8040

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 8100

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 8160

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 8220

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 8280

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 8340

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 8400

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 8460

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt 8506

<210> 4

<211> 8508

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gagaagacgg gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac 60

ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa 120

ataaggctag tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagaggta 180

cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 240

ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 300

cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 360

gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac 420

tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc 480

cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta 540

tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca 600

ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc 660

aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc 720

tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga cgctgccttc gccccgtgcc 780

ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca 840

ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg 900

gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga 960

aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca cgacggagac 1020

tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag 1080

cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac 1140

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 1200

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 1260

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 1320

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 1380

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1440

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1500

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1560

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1620

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1680

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1740

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1800

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1860

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1920

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1980

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 2040

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 2100

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 2160

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 2220

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 2280

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 2340

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 2400

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2460

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2520

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2580

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2640

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2700

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2760

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2820

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2880

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2940

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 3000

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 3060

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 3120

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 3180

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 3240

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 3300

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 3360

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3420

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3480

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3540

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3600

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3660

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3720

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3780

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3840

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3900

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3960

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 4020

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 4080

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 4140

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 4200

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 4260

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 4320

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 4380

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4440

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4500

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4560

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4620

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4680

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4740

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4800

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4860

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4920

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4980

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 5040

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 5100

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 5160

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaaaagg 5220

ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt cctagagctc 5280

gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg 5340

tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 5400

ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 5460

gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc 5520

tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 5580

caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 5640

gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 5700

accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 5760

tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 5820

cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 5880

ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5940

tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 6000

gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 6060

tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 6120

aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 6180

tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 6240

cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 6300

acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 6360

acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 6420

aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 6480

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 6540

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 6600

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 6660

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 6720

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 6780

agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 6840

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 6900

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 6960

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 7020

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 7080

accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 7140

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 7200

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 7260

taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg 7320

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 7380

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 7440

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 7500

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 7560

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 7620

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 7680

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 7740

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 7800

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 7860

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 7920

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 7980

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 8040

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 8100

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 8160

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 8220

ggccttttgc tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt 8280

tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa 8340

agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt 8400

taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt 8460

tcttggcttt atatatcttg tggaaaggac gaaacacccc gtcttctc 8508

<210> 5

<211> 8487

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg cctcgagctc cctctgagcc gttttagagc tagaaatagc aagttaaaat 300

aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga 360

gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca 420

gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac 480

cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac 540

gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 600

tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc 660

agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 720

ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 780

cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 840

gggggggggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca 900

atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct 960

ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc 1020

gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg 1080

tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt 1140

ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa 1200

tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta 1260

caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg 1320

gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat 1380

cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa 1440

attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct 1500

gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag 1560

atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc 1620

caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa 1680

gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa 1740

gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct 1800

gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga 1860

gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac 1920

ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat 1980

cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg 2040

cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa 2100

cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta 2160

cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct 2220

ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga 2280

gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga 2340

cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt 2400

cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga 2460

gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt 2520

gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc 2580

ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc 2640

attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta 2700

cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga 2760

aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt 2820

catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca 2880

cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac 2940

cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct 3000

gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa 3060

aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct 3120

gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga 3180

aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat 3240

gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct 3300

gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg 3360

ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag 3420

aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc 3480

ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc 3540

cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat 3600

gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca 3660

gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct 3720

gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct 3780

gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa 3840

ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc 3900

catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc 3960

ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct 4020

gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact 4080

ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt 4140

ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg 4200

ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca 4260

gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc 4320

cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg 4380

cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa 4440

ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac 4500

cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga 4560

gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca 4620

agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct 4680

gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta 4740

cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa 4800

gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag 4860

aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa 4920

aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa 4980

gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa 5040

atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga 5100

taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga 5160

gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct 5220

gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca 5280

cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat 5340

cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag 5400

catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc 5460

ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taagaattcc tagagctcgc 5520

tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 5580

ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 5640

gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 5700

aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta 5760

gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca 5820

aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc 5880

tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 5940

cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg 6000

tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg 6060

ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 6120

ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 6180

tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 6240

tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta 6300

tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa 6360

atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 6420

tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 6480

cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 6540

aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 6600

gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 6660

tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 6720

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 6780

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 6840

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 6900

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 6960

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 7020

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 7080

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 7140

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 7200

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 7260

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 7320

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 7380

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 7440

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 7500

aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta 7560

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 7620

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 7680

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 7740

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 7800

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 7860

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 7920

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 7980

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 8040

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 8100

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 8160

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 8220

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 8280

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 8340

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 8400

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 8460

ccttttgctg gccttttgct cacatgt 8487

Claims (4)

1. A method for constructing a recombinant sgRNA backbone vector in CRIPSR/Cas9 system, comprising the steps of:

   (1) preparing a linearized sgRNA empty plasmid, wherein the adopted sgRNA empty plasmid has a pair of symmetrical IIS type endonuclease enzyme cutting sites, the sgRNA empty plasmid is subjected to enzyme cutting by IIS type endonuclease enzyme, and the linearized sgRNA empty plasmid subjected to enzyme cutting is recovered;

   (2) preparing a distinguishing segment, wherein both ends of the distinguishing segment are provided with terminal segments corresponding to the IIS type endonuclease after enzyme digestion;

   (3) preparing a recombinant sgRNA framework vector, and connecting the linearized sgRNA empty plasmid recovered from the step (1) with the distinguishing fragment prepared in the step (2) to prepare a recombinant sgRNA framework vector;

   (4) selecting a recombinant sgRNA framework vector, converting the sgRNA framework vector in the step (3) into escherichia coli, coating a resistant plate to form a monoclonal, and selecting the monoclonal to perform colony PCR identification by using a primer containing the IIS type endonuclease enzyme cutting site joint;

   the method is characterized in that:

   the distinguishing segment of the step (2) comprisessacBGenes and their Open Reading Frames (ORFs), promoters and terminators, saidsacBThe nucleotide sequence of the gene is shown as SEQ ID No. 1;

   preparing a distinguishing fragment in the step (2), designing a pair of primers containing the IIS type endonuclease enzyme cutting site joint according to the vector map and the sequence of the pK18mobSacB, wherein an amplicon of the primers containssacBORF of gene and its promoter and terminator, using said primer to make PCR of pK18mobSacB vector to obtain target fragment, recovering PCR product, using IIS type endonuclease to make itPerforming enzyme digestion, and performing column purification to obtain a distinguishing fragment;

   and (4) respectively coating the bacteria with positive PCR identification on an LB resistance plate and an LB resistance plate containing 5% -10% of sucrose, and if the bacteria cannot grow on the LB resistance plate containing 5% -10% of sucrose and form lawn on the LB resistance plate, the bacteria on the surface contain the successfully constructed recombinant sgRNA skeleton vector.
2. 2. The method for constructing a recombinant sgRNA framework vector in the CRIPSR/Cas9 system according to claim 1, wherein the method comprises the following steps: preparing a linearized sgRNA empty plasmid in the step (1), if the sgRNA empty plasmid exists, directly carrying out enzyme digestion on the sgRNA empty plasmid by using a corresponding IIS type endonuclease, and recovering glue after enzyme digestion to obtain the linearized sgRNA empty plasmid; or designing a primer to perform whole-plasmid PCR on a plasmid according to the constructed plasmid of a certain sgRNA to obtain an empty sgRNA plasmid containing an IIS type endonuclease enzyme digestion joint, then carrying out enzyme digestion on the empty sgRNA plasmid by using the IIS type endonuclease enzyme digestion joint, and carrying out gel recovery on the linearized sgRNA empty plasmid after enzyme digestion.
3. 3. The method for constructing a recombinant sgRNA framework vector in the CRIPSR/Cas9 system according to claim 1, wherein the method comprises the following steps: and (3) preparing a recombinant sgRNA framework vector, uniformly mixing the linearized sgRNA empty plasmid recovered in the step (1) with the distinguished fragments prepared in the step (2), adding T4 ligase and T4 ligase Buffer, and placing in a 16-degree metal bath for overnight connection.
4. 4. The method for constructing a recombinant sgRNA framework vector in the CRIPSR/Cas9 system according to claim 1, wherein the method comprises the following steps: and (4) selecting a recombinant sgRNA framework vector, transforming the ligation product obtained in the step (3) into escherichia coli, coating a resistant plate, culturing for 12-20 hours until a single clone is formed on the plate, selecting the single clone, carrying out colony PCR identification by using the primer obtained in the step (2), transferring a colony identified by PCR positive identification into a shake flask for overnight culture, then carrying out plasmid extraction, carrying out enzyme digestion on the plasmid by using IIS type endonuclease, shaking the clone identified by enzyme digestion to be correct overnight, diluting, and respectively coating an LB resistant plate and an LB resistant plate containing 5-10% of sucrose, wherein if the colony cannot grow on the LB resistant plate containing 5-10% of sucrose and forms a bacterial lawn on the LB resistant plate, the bacterial containing the successfully constructed recombinant sgRNA framework vector is indicated.

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