CN107937429B - Construction method of recombinant sgRNA framework vector in CRIPSR/Cas9 system - Google Patents
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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
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
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tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat 180
gaaaaatatc aagtttctga atttgattcg tccacaatta aaaatatctc ttctgcaaaa 240
ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat 300
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gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga aaaacgctgg 1140
ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa aagaccaaac 1200
acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt tattctacac 1260
tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag ttaacgtatc 1320
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cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca aatacttagt 1500
atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt tatttaacaa 1560
agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac ttctgcaaag 1620
cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg agctaaacga 1680
tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag taacagatga 1740
aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca ctgactcccg 1800
cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc ttggttatgt 1860
ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg tgttaaaaat 1920
ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac ctcaagcgaa 1980
aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg cagacaaaca 2040
atcaacgttt gcgccgagct tcctgctgaa catcaaaggc aagaaaacat ctgttgtcaa 2100
agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca aaagaaaatg 2160
ccgatggtac cgagcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc 2220
gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccctc 2280
gcggacgtgc tcatagtcca cgacgcccgt gattttgtag ccctggccga cggccagcag 2340
gtaggccgac aggctcatgc cggccgccgc cgccttttcc tcaatcgctc ttcgttcgtc 2400
tgg 2403
<210> 3
<211> 8506
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
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 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
ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780
ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840
ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 900
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)
- 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. 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. 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. 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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