载体基本信息 | |||
载体名称 | 3xFLAG-dCas9/pCMV-7.1 | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 8848 bp | 载体类型 | CRISPR Plasmids |
载体来源 | Fujita T, Fujii H. | 拷贝数 | High copy number |
3xFLAG-dCas9/pCMV-7.1 载体载体图谱
3xFLAG-dCas9/pCMV-7.1 载体载体序列
LOCUS Exported 8848 bp ds-DNA circular SYN 06-1-2016 DEFINITION Plasmid for expression in mammalian cells of FLAG(R)-tagged catalytically inactive dCas9, for engineered ChIP (enChIP) purification of specific genomic regions. ACCESSION . VERSION . KEYWORDS 3xFLAG-dCas9 pCMV-7.1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8848) AUTHORS Fujita T, Fujii H. TITLE Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. JOURNAL Biochem. Biophys. Res. Commun. 2013;439:132-6. PUBMED 23942116 REFERENCE 2 (bases 1 to 8848) AUTHORS Fujii Lab / Addgene #47948 TITLE Direct Submission FEATURES Location/Qualifiers source 1..8848 /organism="synthetic DNA construct" /lab_host="Mammalian Cells" /mol_type="other DNA" enhancer 318..697 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" promoter 698..901 /note="CMV promoter" /note="human cytomegalovirus (CMV) immediate early promoter" CDS 928..930 /codon_start=1 /product="start codon" /note="ATG" /translation="M" CDS 931..996 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /note="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1000..5103 /codon_start=1 /locus_tag=" " /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="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" /protein_id=" " /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5116..5136 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /protein_id=" " /translation="PKKKRKV" polyA_signal 5189..5811 /note="hGH poly(A) signal" /note="human growth hormone polyadenylation signal" promoter 5840..6169 /note="SV40 promoter" /note="SV40 enhancer and early promoter" rep_origin 6020..6155 /note="SV40 ori" /note="SV40 origin of replication" rep_origin complement(6642..7230) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7401..8261) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(8262..8366) /gene="bla" /note="AmpR promoter" rep_origin 8393..8848 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
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