Basic Vector Information
Cas9-NAT 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 ℃.
Cas9-NAT vector Sequence
LOCUS 40924_405 10450 bp DNA circular SYN 13-MAY-2021 DEFINITION Cas9 expression cassette carried by a yeast single-copy episome plasmid. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10450) AUTHORS Zhang GC, Kong II, Kim H, Liu JJ, Cate JH, Jin YS TITLE Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease. JOURNAL Appl Environ Microbiol. 2014 Dec;80(24):7694-701. doi: 10.1128/AEM.02310-14. Epub 2014 Oct 3. PUBMED 25281382 REFERENCE 2 (bases 1 to 10450) TITLE Direct Submission REFERENCE 3 (bases 1 to 10450) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1128/AEM.02310-14"; journalName: "Appl Environ Microbiol"; date: "2014-12"; volume: "80"; issue: "24"; pages: "7694-701" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..10450 /mol_type="other DNA" /organism="synthetic DNA construct" primer_bind complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" misc_feature 69..572 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" promoter 609..713 /label=AmpR promoter CDS 714..1571 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 1745..2333 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2487..2504 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2621..2642 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 2657..2687 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2695..2711 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /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)." primer_bind 2700..2722 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2719..2735 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2719..2735 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 2756..2774 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 2791..3190 /label=TEF1 promoter /note="promoter for EF-1-alpha" primer_bind 3194..3210 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 3194..3210 /label=pBluescriptSK /note="For pBluescript vector" CDS 3222..7325 /codon_start=1 /label=Cas9 /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 7338..7358 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 7374..7621 /label=CYC1 terminator /note="transcription terminator for CYC1" promoter complement(7640..7658) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(7668..7685) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(7668..7684) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7677..7699) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 7825..8280 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" gene complement(8961..10080) /label=natMX6 /note="yeast selectable marker conferring nourseothricin resistance" primer_bind complement(10281..10300) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 10400..10422 /label=pGEX 3' /note="pGEX vectors, reverse primer"
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