Basic Vector Information
- Vector Name:
- AAVS1-idCas9-vpr
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13040 bp
- Type:
- Protein expression, Tetracycline inducible
- Replication origin:
- ori
- Host:
- Mammalian cells
- Selection Marker:
- Puro
- Promoter:
- tight TRE
- 5' Primer:
- T7
- Growth Temperature:
- 37℃
AAVS1-idCas9-vpr vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
AAVS1-idCas9-vpr vector Sequence
LOCUS 62056_246 13040 bp DNA circular SYN 01-JAN-1980 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13040) AUTHORS . TITLE Direct Submission FEATURES Location/Qualifiers source 1..13040 /mol_type="other DNA" /organism="synthetic DNA construct" CDS complement(453..506) /codon_start=1 /label=T2A /note="2A peptide from Thosea asigna virus capsid protein" /translation="EGRGSLLTCGDVEENPGP" misc_feature complement(530..555) /label=SA /note="splice acceptor site" misc_feature complement(562..1365) /label=HA-L /note="left homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" promoter complement(1400..1418) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(1439..1455) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(1463..1479) /label=lac operator /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(1487..1517) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(1532..1553) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(1841..2429) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS 2553..3410 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" CDS complement(4813..5112) /codon_start=1 /label=ccdB /note="CcdB, a bacterial toxin that poisons DNA gyrase" /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" promoter 5273..5291 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_feature complement(5326..6162) /label=HA-R /note="right homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" promoter 6179..6493 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 6510..6534 /label=attB1 /note="recombination site for the Gateway(R) BP reaction" CDS 6596..10699 /codon_start=1 /label=dCas9 /note="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /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 10760..10909 /codon_start=1 /label=VP64 /note="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 11348..11704 /codon_start=1 /label=RelA (p65) AD /note="transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)" /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD EDFSSIADMDFSALL" CDS 11723..12292 /codon_start=1 /label=Rta AD /note="transcriptional activation domain from the human herpesvirus 4 (Epstein-Barr virus) replication and transcription activator Rta/BRLF1 (Hardwick et al., 1992; Chavez et al., 2015)" /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL DTFLNDECLLHAMHISTGFSIFDTSLF" protein_bind complement(12326..12350) /label=attB2 /note="recombination site for the Gateway(R) BP reaction" polyA_signal 12534..12615 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal complement(12622..12846) /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" CDS complement(join(12887..13040,1..443)) /codon_start=1 /label=PuroR /note="puromycin N-acetyltransferase" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
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