| 质粒名称: | Cas9-T6N |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 199604 |
| 存储实验室: | Junwei Shi |
|
载体骨架基本信息 |
|
| 载体名称: | pC013 - Twinstrep-SUMO-huLwCas13a |
| 载体出品公司: | Clontech |
| 空载体大小: | 6086bp |
| 完整质粒大小: | 11171bp |
| 载体类型: | 细菌表达、 CRISPR |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | Cas9-T6N |
| 插入大小: | 5085bp |
| 克隆方法: | Ligation Independent Cloning |
| 5’测序引物 | SUMO: GGACTCCTTAAGATTCTTG |
| 3’测序引物 | T7-Term: GCTAGTTATTGCTCAGCGG |
|
文献引用方法 |
|
| 材料和方法部分: | Cas9-T6N (pZZ12, TAT-4MycNLS-Cas9-2SV40NLS-sfGFP) was a gift from Junwei Shi (Addgene plasmid # 199604 ; http://n2t.net/addgene:199604 ; RRID:Addgene_199604) |
| 参考文献部分: | Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. Zhang Z, Baxter AE, Ren D, Qin K, Chen Z, Collins SM, Huang H, Komar CA, Bailer PF, Parker JB, Blobel GA, Kohli RM, Wherry EJ, Berger SL, Shi J. Nat Biotechnol. 2023 Apr 24. doi: 10.1038/s41587-023-01756-1. 10.1038/s41587-023-01756-1 PubMed 37095348 |
买家导航
Cas9-T6N载体质粒基本信息
Cas9-T6N质粒图谱载体图谱和Cas9-T6N载体序列质粒序列多克隆位点信息
Cas9-T6N质粒载体简介
Cas9-T6N质粒序列载体序列
LOCUS Cas9-T6N 11171 bp DNA circular SYN 14-APR-2025
DEFINITION Expression of cell permeable spCas9 in E. Coli..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11171)
AUTHORS Zhang Z, Baxter AE, Ren D, Qin K, Chen Z, Collins SM, Huang H, Komar
CA, Bailer PF, Parker JB, Blobel GA, Kohli RM, Wherry EJ, Berger SL,
Shi J
TITLE Efficient engineering of human and mouse primary cells using
peptide-assisted genome editing.
JOURNAL Nat Biotechnol. 2023 Apr 24. doi: 10.1038/s41587-023-01756-1.
PUBMED 37095348
REFERENCE 2 (bases 1 to 11171)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 14, 2025 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2023 Apr 24. doi: 10.1038/s41587-023-01756-1."
COMMENT Sequence Label: Cas9-T6N (pZZ12, TAT-4MycNLS-Cas9-2SV40NLS-sfGFP)
FEATURES Location/Qualifiers
source 1..11171
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(1328..2410)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
primer_bind 2372..2391
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(2411..2488)
/gene="lacI"
/label=lacI promoter
primer_bind 2694..2713
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 2797..2816
/label=T7
/note="T7 promoter, forward primer"
promoter 2797..2815
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 2816..2840
/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)."
RBS 2855..2877
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 2896..2913
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 2923..2940
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS join(2953..2976,2977..3012,3013..3036)
/codon_start=1
/product="two Strep-Tag(R)II moieties connected by a linker
for enhanced binding to Strep-Tactin(R), an engineered form
of streptavidin (Schmidt et al., 2013)"
/label=Twin-Strep-tag
/translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
CDS 3037..3330
/codon_start=1
/gene="S. cerevisiae SMT3 (truncated)"
/product="cleavable ubiquitin-like protein tag"
/label=SUMO
/translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
CDS 3379..3405
/codon_start=1
/product="nuclear localization signal of human c-Myc
proto-oncogene (Dang and Lee, 1988)"
/label=c-myc NLS
/translation="PAAKRVKLD"
CDS 3415..3441
/codon_start=1
/product="nuclear localization signal of human c-Myc
proto-oncogene (Dang and Lee, 1988)"
/label=c-myc NLS
/translation="PAAKRVKLD"
CDS 3451..3477
/codon_start=1
/product="nuclear localization signal of human c-Myc
proto-oncogene (Dang and Lee, 1988)"
/label=c-myc NLS
/translation="PAAKRVKLD"
CDS 3487..3513
/codon_start=1
/product="nuclear localization signal of human c-Myc
proto-oncogene (Dang and Lee, 1988)"
/label=c-myc NLS
/translation="PAAKRVKLD"
CDS 3541..7644
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 3541..7644
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 7651..7671
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7678..7698
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
primer_bind complement(8468..8486)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 8482..8529
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(join(8704..8709,8710..8726,8727..8732))
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
primer_bind complement(8759..8777)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 8845..8949
/gene="bla"
/label=AmpR promoter
CDS join(8950..9018,9019..9810)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(9168..9187)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 9981..10569
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 10470..10489
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 10723..10740
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 10755..10894
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(10820..10839)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 10980..11002
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(join(10996..11171,1..16))
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
61 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
121 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
181 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
241 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
301 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
361 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
421 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
481 gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg
541 cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat
601 tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt
661 cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc
721 ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt
781 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag
841 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc
901 gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa
961 cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc
1021 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
1081 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
1141 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
1201 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
1261 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
1321 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
1381 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
1441 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
1501 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
1561 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
1621 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
1681 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
1741 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
1801 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
1861 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
1921 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
1981 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
2041 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
2101 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
2161 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
2221 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
2281 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
2341 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
2401 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
2461 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
2521 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
2581 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
2641 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
2701 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
2761 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
2821 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
2881 accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc
2941 catatggcta gctggagcca tccgcagttt gaaaaaggtg gtggtagcgg tggtggttca
3001 ggtggtagtg catggtcaca ccctcagttt gagaaaatgt cggactcaga agtcaatcaa
3061 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
3121 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
3181 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
3241 ggtattagaa tccaagctga tcagacccct gaagatttgg acatggagga taacgatatt
3301 attgaggctc acagagaaca gattggtgga tccggccgta agaaacgccg ccagcgtcgc
3361 cgtccgcaag gcggtagccc cgccgcgaag cgtgtaaagc tggatggggg tagcccagcg
3421 gcaaaaaggg tgaaactaga cggcggctcc ccggcagcca agagagtcaa actcgacggg
3481 ggtagccctg ctgctaaacg tgttaagctt gatggtatcc acggagtccc agcagctacc
3541 atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg
3601 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc
3661 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa
3721 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt
3781 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga
3841 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga
3901 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa
3961 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat
4021 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat
4081 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct
4141 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga
4201 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat
4261 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa
4321 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg
4381 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt
4441 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca
4501 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga
4561 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca
4621 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta
4681 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc
4741 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat
4801 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt
4861 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt
4921 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa
4981 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa
5041 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt
5101 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt
5161 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc
5221 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt
5281 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt
5341 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt
5401 ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct
5461 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga
5521 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta
5581 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat
5641 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta
5701 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact
5761 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt
5821 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt
5881 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct
5941 gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga
6001 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac
6061 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct
6121 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa
6181 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta
6241 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa
6301 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat
6361 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct
6421 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat
6481 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa
6541 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa
6601 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct
6661 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc
6721 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt
6781 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta
6841 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt
6901 gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct
6961 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt
7021 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac
7081 tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa
7141 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta
7201 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt
7261 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag
7321 cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt
7381 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa
7441 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct
7501 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa
7561 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt
7621 gatttgagtc agctaggagg tgacgggtca cctaagaaaa aacgaaaagt tgaggatcct
7681 aaaaagaaac gaaaagttga taccggttct aaaggtgaag aactgttcac cggtgttgtt
7741 ccgatcctgg ttgaactgga tggtgatgtt aacggccaca aattctctgt tcgtggtgaa
7801 ggtgaaggtg atgcaaccaa cggtaaactg accctgaaat tcatctgcac taccggtaaa
7861 ctgccggttc catggccgac tctggtgact accctgacct atggtgttca gtgtttttct
7921 cgttacccgg atcacatgaa gcagcatgat ttcttcaaat ctgcaatgcc ggaaggttat
7981 gtacaggagc gcaccatttc tttcaaagac gatggcacct acaaaacccg tgcagaggtt
8041 aaatttgaag gtgatactct ggtgaaccgt attgaactga aaggcattga tttcaaagag
8101 gacggcaaca tcctgggcca caaactggaa tataacttca actcccataa cgtttacatc
8161 accgcagaca aacagaagaa cggtatcaaa gctaacttca aaattcgcca taacgttgaa
8221 gacggtagcg tacagctggc ggaccactac cagcagaaca ctccgatcgg tgatggtccg
8281 gttctgctgc cggataacca ctacctgtcc acccagtctg ttctgtccaa agacccgaac
8341 gaaaagcgcg accacatggt gctgctggag ttcgttactg cagcaggtat cacgcacggc
8401 atggatgagc tctacaaata agcggccgca ctcgaggccc gaaaggaagc tgagttggct
8461 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
8521 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg
8581 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
8641 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
8701 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
8761 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
8821 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
8881 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
8941 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
9001 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
9061 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
9121 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
9181 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
9241 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
9301 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
9361 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
9421 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
9481 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
9541 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
9601 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
9661 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
9721 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
9781 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
9841 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
9901 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
9961 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
10021 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
10081 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
10141 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
10201 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
10261 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
10321 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
10381 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
10441 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
10501 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
10561 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
10621 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
10681 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
10741 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
10801 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
10861 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
10921 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
10981 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
11041 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
11101 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
11161 aagggcggtt t
//
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Cas9-T6N质粒载体应用举例
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