| 质粒名称: | pHAGE-TO-dCas9 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 75381 |
| 存储实验室: | Thoru Pederson |
|
载体骨架基本信息 |
|
| 载体名称: | pHAGE-DEST |
| 载体抗性: | 氨苄霉素 |
| 空载体大小: | 6000 bp |
| 完整质粒大小: | 10829 bp |
| 载体类型: | 哺乳细胞表达，Lentiviral |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Sp dCas9 |
| 物种来源: | Synthetic |
| 插入大小: | 4101 bp |
| 启动子 | CMV-TO |
| 标签/融合蛋白: | no |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | EcoRI(未破坏) |
| 3' 克隆位点: | XbaI(未破坏) |
| 5' 测序引物 | cgaaggaatagaagaagaaggtgga |
| 3' 测序引物 | CCAAAGGGAGATCCGACTCG |
|
文献引用方法 |
|
| 材料和方法部分: | pHAGE-TO-dCas9 was a gift from Thoru Pederson (爱迪基因 plasmid # 75381) |
| 参考文献部分: | Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526. 10.1038/nbt.3526 PubMed 27088723 |
买家导航
pHAGE-TO-dCas9载体质粒基本信息
pHAGE-TO-dCas9质粒图谱载体图谱和pHAGE-TO-dCas9载体序列质粒序列多克隆位点信息
pHAGE-TO-dCas9质粒载体简介
pHAGE-TO-dCas9质粒序列载体序列
LOCUS pHAGE-TO-dCas9 10835 bp ds-DNA circular SYN 10-JAN-2018
DEFINITION dCas9.
ACCESSION .
VERSION .
KEYWORDS pHAGE-TO-dCas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10835)
AUTHORS Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T
TITLE Multiplexed labeling of genomic loci with dCas9 and engineered
sgRNAs using CRISPRainbow.
JOURNAL Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.
PUBMED 27088723
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT VNTNAME|pHAGE-TO-dCas9|
FEATURES Location/Qualifiers
source 1..10835
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..634
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2058..2175
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
enhancer 2299..2602
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 2603..2806
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 2757..2777
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
protein_bind 2808..2826
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2829..2847
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 2857..2881
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 2974..2983
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 2986..3006
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3025..7125
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 7144..7164
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
misc_feature 7225..7813
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(7278..7298)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(7696..7707)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 7888..8121
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(8240..8258)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 8326..8430
/gene="bla"
/label=AmpR promoter
CDS 8431..9291
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(8649..8668)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 9462..10050
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 9951..9970
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 10204..10221
/label=L4440
/note="L4440 vector, forward primer"
primer_bind 10313..10332
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
protein_bind 10461..10482
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 10497..10527
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 10535..10551
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 10540..10562
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 10559..10575
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 10559..10575
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 10625..10644
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
ORIGIN
1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttgacaaatg
2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagcgat
2221 gctagctgat gcgggcccta gttattaata gtaatcaatt acggggtcat tagttcatag
2281 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
2341 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
2401 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
2461 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
2521 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
2581 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
2641 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
2701 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
2761 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga
2821 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat
2881 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
2941 cctaccggtt aaggtgaacg cgtgcaggct ggcgccacca tggcccctaa aaagaaacgc
3001 aaggtcgaat tcggaagcgg aagcgacaag aagtactcca ttgggctcgc tatcggtacc
3061 aacagcgtcg gctgggccgt cattacggac gagtacaagg tgccgagcaa aaaattcaaa
3121 gttctgggca ataccgatcg ccacagcata aagaagaacc tcattggagc cctcctgttc
3181 gactccgggg agacggccga agccacgcgg ctcaaaagaa cagcacggcg cagatatacc
3241 cgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat ggctaaggtg
3301 gatgactctt tcttccatag gctggaggag tcctttttgg tggaggagga taaaaagcac
3361 gagcgccacc caatctttgg caatatcgtg gacgaggtgg cgtaccatga aaagtaccca
3421 accatatatc atctgaggaa gaagctggta gacagtactg ataaggctga cttgcggttg
3481 atctatctcg cgctggcgca catgatcaaa tttcggggac acttcctcat cgagggggac
3541 ctgaacccag acaacagcga tgtcgacaaa ctctttatcc aactggttca gacttacaat
3601 cagcttttcg aggagaaccc gatcaacgca tccggcgttg acgccaaagc aatcctgagc
3661 gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg cacagctccc tggggagaag
3721 aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc caactttaaa
3781 tctaacttcg acctggccga agatgccaag ctgcaactga gcaaagacac ctacgatgat
3841 gatctcgaca atctgctggc ccagatcggc gaccagtacg cagacctttt tttggcggca
3901 aagaacctgt cagacgccat tctgctgagt gatattctgc gagtgaacac ggagatcacc
3961 aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccacca agacttgact
4021 ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat tttcttcgat
4081 cagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga ggaattttac
4141 aaatttatta agcccatctt ggaaaaaatg gacggcaccg aggagctgct ggtaaagctg
4201 aacagagaag atctgttgcg caaacagcgc actttcgaca atggaagcat cccccaccag
4261 attcacctgg gcgaactgca cgctatcctc aggcggcaag aggatttcta cccctttttg
4321 aaagataaca gggaaaagat tgagaaaatc ctcacatttc ggatacccta ctatgtaggc
4381 cccctcgctc ggggaaattc cagattcgcg tggatgactc gcaaatcaga agagaccatc
4441 actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc cttcatcgaa
4501 aggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa acactctctg
4561 ctgtacgagt acttcacagt ttataacgag ctcaccaagg tcaaatacgt cacagaaggg
4621 atgagaaagc cagcattcct gtctggagag cagaagaaag ctatcgtgga cctcctcttc
4681 aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag actatttcaa aaagattgaa
4741 tgtttcgact ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc cctgggaacg
4801 tatcacgatc tcctgaaaat cattaaagac aaggacttcc tggacaatga ggagaacgag
4861 gacattcttg aggacattgt cctcaccctt acgttgtttg aagataggga gatgattgaa
4921 gaacgcttga aaacttacgc tcatctcttc gacgacaaag tcatgaaaca gctcaagaga
4981 cgccgatata caggatgggg gcggctgtca agaaaactga tcaatggcat ccgagacaag
5041 cagagtggaa agacaatcct ggattttctt aagtccgatg gatttgccaa ccggaacttc
5101 atgcagttga tccatgatga ctctctcacc tttaaggagg acatccagaa agcacaagtt
5161 tctggccagg gggacagtct tcacgagcac atcgctaatc ttgcaggtag cccagctatc
5221 aaaaagggaa tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt aatgggaagg
5281 cataagcccg agaatatcgt tatcgagatg gcccgagaga accaaactac ccagaaggga
5341 cagaagaaca gtagggaaag gatgaagagg attgaagagg gtataaaaga actggggtcc
5401 caaatcctta aggaacaccc agttgaaaac acccagcttc agaatgagaa gctctacctg
5461 tactacctgc agaacggcag ggacatgtac gtggatcagg aactggacat caaccggttg
5521 tccgactacg acgtggatgc tatcgtgccc caaagctttc tcaaagatga ttctattgat
5581 aataaagtgt tgacaagatc cgataaaaat agagggaaga gtgataacgt cccctcagaa
5641 gaagttgtca agaaaatgaa aaattattgg cggcagctgc tgaacgccaa actgatcaca
5701 caacggaagt tcgataatct gactaaggct gaacgaggtg gcctgtctga gttggataaa
5761 gccggcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca cgtggcccaa
5821 attctcgatt cacgcatgaa caccaagtac gatgaaaatg acaaactgat tcgagaggtg
5881 aaagttatta ctctgaagtc taagctggtc tcagatttca gaaaggactt tcagttttat
5941 aaggtgagag agatcaacaa ttaccaccat gcgcatgatg cctacctgaa tgcagtggta
6001 ggcactgcac ttatcaaaaa atatcccaag ctggaatctg aatttgttta cggagactat
6061 aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaatagg caaggccacc
6121 gctaagtact tcttttacag caatattatg aattttttca agaccgagat tacactggcc
6181 aatggagaga ttcggaagcg accacttatc gaaacaaacg gagaaacagg agaaatcgtg
6241 tgggacaagg gtagggattt cgcgacagtc cgcaaggtcc tgtccatgcc gcaggtgaac
6301 atcgttaaaa agaccgaagt acagaccgga ggcttctcca aggaaagtat cctcccgaaa
6361 aggaacagcg acaagctgat cgcacgcaaa aaagattggg accccaagaa atacggcgga
6421 ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga gaaagggaag
6481 tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatgga gcgatccagc
6541 ttcgagaaaa accccatcga ctttctcgaa gcgaaaggat ataaagaggt caaaaaagac
6601 ctcatcatta agctgcccaa gtactctctc tttgagcttg aaaacggccg gaaacgaatg
6661 ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg cactgccctc taaatacgtt
6721 aatttcttgt atctggccag ccactatgaa aagctcaaag ggtctcccga agataatgag
6781 cagaagcagc tgttcgtgga acaacacaaa cactaccttg atgagatcat cgagcaaata
6841 agcgagttct ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt gctttctgct
6901 tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattat ccacttgttt
6961 actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac catagacaga
7021 aagcggtaca cctctacaaa ggaggtcctg gacgccacac tgattcatca gtcaattacg
7081 gggctctatg aaacaagaat cgacctctct cagctcggtg gagacggctc tactagtggc
7141 tctcccaaga agaagaggaa ggtaggatcc ggatctggcg gccgctctgg catcgatggg
7201 agcctcgagt agggtaatct agataatcaa cctctggatt acaaaatttg tgaaagattg
7261 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
7321 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
7381 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
7441 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
7501 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
7561 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
7621 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
7681 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
7741 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
7801 gccgcctccc cgcctgagat cctttaagac caatgactta caaggcagct gtagatctta
7861 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
7921 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
7981 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
8041 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
8101 cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac
8161 ttgcaaagaa atgaatatca gagagtgaga ggcccgggtt aattaaggaa agggctagat
8221 cattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
8281 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
8341 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
8401 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
8461 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
8521 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
8581 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
8641 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
8701 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
8761 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
8821 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
8881 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
8941 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
9001 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
9061 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
9121 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
9181 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
9241 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
9301 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
9361 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
9421 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
9481 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
9541 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
9601 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
9661 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
9721 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
9781 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
9841 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
9901 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
9961 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
10021 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
10081 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
10141 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
10201 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
10261 gcgcgttggc cgattcatta atgcagcaag ctcatggctg actaattttt tttatttatg
10321 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
10381 gaggcctagg cttttgcaaa aagctccccg tggcacgaca ggtttcccga ctggaaagcg
10441 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
10501 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
10561 ggaaacagct atgacatgat tacgaatttc acaaataaag catttttttc actgcattct
10621 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcaa ctggataact
10681 caagctaacc aaaatcatcc caaacttccc accccatacc ctattaccac tgccaattac
10741 ctgtggtttc atttactcta aacctgtgat tcctctgaat tattttcatt ttaaagaaat
10801 tgtatttgtt aaatatgtac tacaaactta gtagt
//
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pHAGE-TO-dCas9质粒载体应用举例
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