载体基本信息 | |||
载体名称 | pCLIP-Cas9-Nickase-TRE3G-tRFP | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 10893 bp | 载体类型 | CRISPR Plasmids |
载体来源 | transOMIC | 拷贝数 | High copy number |
pCLIP-Cas9-Nickase-TRE3G-tRFP 载体载体图谱
pCLIP-Cas9-Nickase-TRE3G-tRFP 载体载体序列
LOCUS Exported 10893 bp ds-DNA circular SYN 05-1-2016 DEFINITION Lentiviral CRISPR vector encoding a Cas9 nickase mutant and the red fluorescent protein TurboRFP expressed from a Tet-responsive promoter. ACCESSION . VERSION . KEYWORDS pCLIP-Cas9(D10a)-Nickase-TRE3G-tRFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10893) AUTHORS transOMIC TITLE Direct Submission FEATURES Location/Qualifiers source 1..10893 /organism="synthetic DNA construct" /lab_host="Mammalian Cells" /mol_type="other DNA" enhancer 27..404 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" LTR 411..668 /note="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 715..840 /note="HIV-1 Psi" /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1337..1570 /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 2062..2177 /note="cPPT/CTS" /note="central polypurine tract and central termination sequence of HIV-1 (lacking the first T)" promoter 2244..2622 /note="TRE3G promoter" /note="3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On(R) 3G" CDS 2640..6743 /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 6744..6791 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" CDS 6792..6815 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /note="FLAG" /translation="DYKDDDDK" CDS 6825..6881 /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 6882..7577 /codon_start=1 /product="red fluorescent protein from Entacmaea quadricolor" /note="TurboRFP" /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC DLPSKLGHR" misc_feature 7602..8190 /note="WPRE" /note="woodchuck hepatitis virus posttranscriptional regulatory element" LTR 8395..8628 /note="3' LTR (Delta-U3)" /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" promoter 8724..9053 /note="SV40 promoter" /note="SV40 enhancer and early promoter" rep_origin complement(9142..9727) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(9898..10758) /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(10759..10863) /gene="bla" /note="AmpR promoter"
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