载体基本信息 | |||
载体名称 | lentiCas9n(D10A)-Blast | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 12860 bp | 载体类型 | CRISPR Plasmids |
载体来源 | Sanjana NE, Shalem O, Zhang F. | 拷贝数 | High copy number |
lentiCas9n(D10A)-Blast 载体载体图谱
lentiCas9n(D10A)-Blast 载体载体序列
LOCUS Exported 12860 bp ds-DNA circular SYN 06-1-2016 DEFINITION Zhang lab lentiviral vector with a blasticidin resistance marker, for expressing Cas9 with the D10A nickase mutation. ACCESSION . VERSION . KEYWORDS lentiCas9n(D10A)-Blast SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12860) AUTHORS Sanjana NE, Shalem O, Zhang F. TITLE Improved vectors and genome-wide libraries for CRISPR screening. JOURNAL Nat. Methods 2014;11:783-4. PUBMED 25075903 REFERENCE 2 (bases 1 to 12860) AUTHORS Zhang Lab / Addgene #63593 TITLE Direct Submission FEATURES Location/Qualifiers source 1..12860 /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="5' LTR (truncated)" /note="truncated 5' 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 2651..2862 /note="EF-1-alpha core promoter" /note="core promoter for human elongation factor EF-1-alpha" CDS 2887..6990 /codon_start=1 /locus_tag=" " /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="Cas9(D10A)" /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /protein_id=" " /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 6991..7038 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" CDS 7039..7062 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /note="FLAG" /translation="DYKDDDDK" CDS 7072..7128 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /note="P2A" /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 7129..7527 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /note="BSD" /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature 7552..8140 /note="WPRE" /note="woodchuck hepatitis virus posttranscriptional regulatory element" LTR 8665..8845 /note="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" polyA_signal 8877..9101 /note="bGH poly(A) signal" /note="bovine growth hormone polyadenylation signal" rep_origin 9147..9575 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 9589..9918 /note="SV40 promoter" /note="SV40 enhancer and early promoter" rep_origin 9769..9904 /note="SV40 ori" /note="SV40 origin of replication" promoter 9966..10013 /note="EM7 promoter" /note="synthetic bacterial promoter " CDS 10032..10406 /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 10536..10657 /note="SV40 poly(A) signal" /note="SV40 polyadenylation signal" rep_origin complement(11108..11696) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(11867..12727) /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(12728..12832) /gene="bla" /note="AmpR promoter"
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