载体基本信息 | |||
载体名称 | 3xFLAG-dCas9/pMXs-neo | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 10444 bp | 载体类型 | CRISPR Plasmids |
载体来源 | Fujita T, Fujii H. | 筛选标记 | Neomycin/G418(Geneticin) |
拷贝数 | High copy number |
3xFLAG-dCas9/pMXs-neo 载体载体图谱
3xFLAG-dCas9/pMXs-neo 载体载体序列
LOCUS Exported 10444 bp ds-DNA circular SYN 06-1-2016 DEFINITION Retroviral vector with a G418 resistance marker, encoding FLAG(R)-tagged dCas9 for engineered ChIP (enChIP) purification of specific genomic regions. ACCESSION . VERSION . KEYWORDS 3xFLAG-dCas9 pMXs-neo SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10444) AUTHORS Fujita T, Fujii H. TITLE Identification of proteins associated with an IFN-gamma-responsive promoter by a retroviral expression system for enChIP using CRISPR. JOURNAL PLoS ONE 2014;9:e103084. PUBMED 25051498 REFERENCE 2 (bases 1 to 10444) AUTHORS Fujii Lab / Addgene #51260 TITLE Direct Submission FEATURES Location/Qualifiers source 1..10444 /organism="synthetic DNA construct" /lab_host="Mammalian Cells" /mol_type="other DNA" LTR 22..615 /note="long terminal repeat from Moloney murine leukemia virus" misc_feature 678..1035 /note="MMLV Psi" /note="packaging signal of Moloney murine leukemia virus (MMLV)" CDS 1094..1510 /note="gag (truncated)" /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" misc_feature 1520..1894 /note="pol region" /note="Moloney murine leukemia virus (MMLV) pol region containing the splice acceptor site" CDS 1948..1950 /codon_start=1 /product="start codon" /note="ATG" /translation="M" CDS 1951..2016 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /note="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 2020..6123 /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 6136..6156 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /protein_id=" " /translation="PKKKRKV" promoter 6341..6670 /note="SV40 promoter" /note="SV40 enhancer and early promoter" rep_origin 6521..6656 /note="SV40 ori" /note="SV40 origin of replication" CDS 7027..7821 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /note="NeoR/KanR" /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" LTR 7889..8482 /note="long terminal repeat from Moloney murine leukemia virus" rep_origin complement(8719..9307) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(9471..10331) /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(10332..10436) /gene="bla" /note="AmpR promoter"
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