载体基本信息 | |||
载体名称 | dCAS9-VP64_GFP | 载体抗性 | Ampicillin |
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载体长度 | 14382 bp | 载体类型 | CRISPR Plasmids |
载体来源 | Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena | 拷贝数 | High copy number |
dCAS9-VP64_GFP 载体载体图谱
dCAS9-VP64_GFP 载体载体序列
LOCUS Exported 14382 bp ds-DNA circular SYN 06-1-2016 DEFINITION Zhang lab lentiviral vector encoding enhanced GFP (EGFP) plus catalytically inactive dCas9 fused to the VP64 transcriptional activation domain. ACCESSION . VERSION . KEYWORDS dCAS9-VP64_GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14382) AUTHORS Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. TITLE Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. JOURNAL Nature 2015;517:583-8. PUBMED 25494202 REFERENCE 2 (bases 1 to 14382) AUTHORS Zhang Lab / Addgene #61422 TITLE Direct Submission FEATURES Location/Qualifiers source 1..14382 /organism="synthetic DNA construct" /lab_host="Mammalian Cells" /mol_type="other DNA" enhancer 238..617 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" promoter 619..817 /note="CMV promoter" /note="human cytomegalovirus (CMV) immediate early promoter" LTR 835..1015 /note="LTR (truncated)" /note="truncated long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /note="HIV-1 Psi" /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1680..1913 /note="RRE" /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." misc_feature 2440..2557 /note="cPPT/CTS" /note="central polypurine tract and central termination sequence of HIV-1" promoter 2682..3859 /note="EF-1-alpha promoter" /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2913..3850 /note="EF-1-alpha intron A" /note="intron upstream of the start codon of human EF-1-alpha" CDS 3872..3874 /codon_start=1 /product="start codon" /note="ATG" /translation="M" CDS 3878..3898 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /protein_id=" " /translation="PKKKRKV" CDS 3908..8008 /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="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 8063..8083 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /protein_id=" " /translation="PKKKRKV" CDS 8108..8257 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /note="VP64" /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 8279..8332 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /note="T2A" /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /protein_id=" " /translation="EGRGSLLTCGDVEENPGP" CDS 8333..9049 /codon_start=1 /product="enhanced GFP" /note="EGFP" /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" misc_feature 9074..9662 /note="WPRE" /note="woodchuck hepatitis virus posttranscriptional regulatory element" LTR 10187..10367 /note="LTR (truncated)" /note="truncated long terminal repeat (LTR) from HIV-1" polyA_signal 10399..10623 /note="bGH poly(A) signal" /note="bovine growth hormone polyadenylation signal" rep_origin 10669..11097 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 11111..11440 /note="SV40 promoter" /note="SV40 enhancer and early promoter" rep_origin 11291..11426 /note="SV40 ori" /note="SV40 origin of replication" promoter 11488..11535 /note="EM7 promoter" /note="synthetic bacterial promoter " CDS 11554..11928 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /note="BleoR" /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 12058..12179 /note="SV40 poly(A) signal" /note="SV40 polyadenylation signal" rep_origin complement(12630..13218) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(13389..14249) /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(14250..14354) /gene="bla" /note="AmpR promoter"
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