载体基本信息 | |||
载体名称 | eSpCas9(1.1) | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 8506 bp | 载体类型 | CRISPR Plasmids |
载体来源 | Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. | 拷贝数 | High copy number |
eSpCas9(1.1) 载体载体图谱
eSpCas9(1.1) 载体载体序列
LOCUS Exported 8506 bp ds-DNA circular SYN 03-2-2016 DEFINITION Zhang lab vector for expressing a single guide RNA (sgRNA) together with an S. pyogenes Cas9 variant with improved targeting specificity. ACCESSION . VERSION . KEYWORDS eSpCas9(1.1) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8506) AUTHORS Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. TITLE Rationally engineered Cas9 nucleases with improved specificity. JOURNAL Science 2016;351:84-8. PUBMED 26628643 REFERENCE 2 (bases 1 to 8506) AUTHORS Zhang Lab / Addgene #71814 TITLE Direct Submission COMMENT An error in codon 848 was corrected based on sequencing results from Addgene. FEATURES Location/Qualifiers source 1..8506 /organism="synthetic DNA construct" /lab_host="Mammalian Cells" /mol_type="other DNA" promoter 1..241 /note="U6 promoter" /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 268..343 /note="gRNA scaffold" /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 344..349 /note="polIII terminator" /note="RNA polymerase III transcription terminator" enhancer 440..725 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 727..1004 /note="chicken beta-actin promoter" intron 1005..1233 /note="hybrid intron" /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 1251..1253 /codon_start=1 /product="start codon" /note="ATG" /translation="M" CDS 1254..1319 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /note="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1326..1346 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /protein_id=" " /translation="PKKKRKV" CDS 1371..5471 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Slaymaker et al., 2016)" /note="eSpCas9(1.1)" /note="carries the mutations K848A, K1003A, and R1060A" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5472..5519 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" polyA_signal 5553..5760 /note="bGH poly(A) signal" /note="bovine growth hormone polyadenylation signal" repeat_region 5769..5909 /note="AAV2 ITR" /note="inverted terminal repeat of adeno-associated virus serotype 2" rep_origin 5984..6439 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 6721..6825 /gene="bla" /note="AmpR promoter" CDS 6826..7686 /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" rep_origin 7857..8445 /direction=RIGHT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
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