LOCUS       sequence-82982-e        4173 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses N-terminal His tag fused human codon-optimized Cas9 
            nuclease having C-terminal Cysteine .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4173)
  AUTHORS   Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H
  TITLE     Gene disruption by cell-penetrating peptide-mediated delivery of 
            Cas9 protein and guide RNA.
  JOURNAL   Genome Res. 2014 Apr 2.
  PUBMED    24696462
REFERENCE   2  (bases 1 to 4173)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Genome Res.
            2014 Apr 2."
COMMENT     Sequence Label: pET28a/Cas9-Cys
FEATURES             Location/Qualifiers
     source          1..4173
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             1..4104
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /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             1..4104
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /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             4120..4140
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4141..4167
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             4141..4167
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
ORIGIN
        1 atggacaaga agtacagcat cggcctggac atcggtacca acagcgtggg ctgggccgtg
       61 atcaccgacg agtacaaggt gcccagcaag aagttcaagg tgctgggcaa caccgaccgc
      121 cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggcga gaccgccgag
      181 gccacccgcc tgaagcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc
      241 tacctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt cttccaccgc
      301 ctggaggaga gcttcctggt ggaggaggac aagaagcacg agcgccaccc catcttcggc
      361 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgcgcaag
      421 aagctggtgg acagcaccga caaggccgac ctgcgcctga tctacctggc cctggcccac
      481 atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
      541 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc
      601 atcaacgcca gcggcgtgga cgccaaggcc atcctgagcg cccgcctgag caagagccgc
      661 cgcctggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct gttcggcaac
      721 ctgatcgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
      781 gacgccaagc tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
      841 cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgag cgacgccatc
      901 ctgctgagcg acatcctgcg cgtgaacacc gagatcacca aggcccccct gagcgccagc
      961 atgatcaagc gctacgacga gcaccaccag gacctgaccc tgctgaaggc cctggtgcgc
     1021 cagcagctgc ccgagaagta caaggagatc ttcttcgacc agagcaagaa cggctacgcc
     1081 ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa gcccatcctg
     1141 gagaagatgg acggcaccga ggagctgctg gtgaagctga accgcgagga cctgctgcgc
     1201 aagcagcgca ccttcgacaa cggcagcatc ccccaccaga tccacctggg cgagctgcac
     1261 gccatcctgc gccgccagga ggacttctac cccttcctga aggacaaccg cgagaagatc
     1321 gagaagatcc tgaccttccg catcccctac tacgtgggcc ccctggcccg cggcaacagc
     1381 cgcttcgcct ggatgacccg caagagcgag gagaccatca ccccctggaa cttcgaggag
     1441 gtggtggaca agggcgccag cgcccagagc ttcatcgagc gcatgaccaa cttcgacaag
     1501 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     1561 tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgcgcaagcc cgccttcctg
     1621 agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaaccg caaggtgacc
     1681 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag cgtggagatc
     1741 agcggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct gctgaagatc
     1801 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtg
     1861 ctgaccctga ccctgttcga ggaccgcgag atgatcgagg agcgcctgaa gacctacgcc
     1921 cacctgttcg acgacaaggt gatgaagcag ctgaagcgcc gccgctacac cggctggggc
     1981 cgcctgagcc gcaagcttat caacggcatc cgcgacaagc agagcggcaa gaccatcctg
     2041 gacttcctga agagcgacgg cttcgccaac cgcaacttca tgcagctgat ccacgacgac
     2101 agcctgacct tcaaggagga catccagaag gcccaggtga gcggccaggg cgacagcctg
     2161 cacgagcaca tcgccaacct ggccggcagc cccgccatca agaagggcat cctgcagacc
     2221 gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc acaagcccga gaacatcgtg
     2281 atcgagatgg cccgcgagaa ccagaccacc cagaagggcc agaagaacag ccgcgagcgc
     2341 atgaagcgca tcgaggaggg catcaaggag ctgggcagcc agatcctgaa ggagcacccc
     2401 gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggccgc
     2461 gacatgtacg tggaccagga gctggacatc aaccgcctga gcgactacga cgtggaccac
     2521 atcgtgcccc agagcttcct gaaggacgac agcatcgaca acaaggtgct gacccgcagc
     2581 gacaagaacc gcggcaagag cgacaacgtg cccagcgagg aggtggtgaa gaagatgaag
     2641 aactactggc gccagctgct gaacgccaag ctgatcaccc agcgcaagtt cgacaacctg
     2701 accaaggccg agcgcggcgg cctgagcgag ctggacaagg ccggcttcat caagcgccag
     2761 ctggtggaga cccgccagat caccaagcac gtggcccaga tcctggacag ccgcatgaac
     2821 accaagtacg acgagaacga caagctgatc cgcgaggtga aggtgatcac cctgaagagc
     2881 aagctggtga gcgacttccg caaggacttc cagttctaca aggtgcgcga gatcaacaac
     2941 taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct gatcaagaag
     3001 taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcgcaag
     3061 atgatcgcca agagcgagca ggagatcggc aaggccaccg ccaagtactt cttctacagc
     3121 aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat ccgcaagcgc
     3181 cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg ccgcgacttc
     3241 gccaccgtgc gcaaggtgct gagcatgccc caggtgaaca tcgtgaagaa gaccgaggtg
     3301 cagaccggcg gcttcagcaa ggagagcatc ctgcccaagc gcaacagcga caagctgatc
     3361 gcccgcaaga aggactggga ccccaagaag tacggcggct tcgacagccc caccgtggcc
     3421 tacagcgtgc tggtggtggc caaggtggag aagggcaaga gcaagaagct gaagagcgtg
     3481 aaggagctgc tgggcatcac catcatggag cgcagcagct tcgagaagaa ccccatcgac
     3541 ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctgcccaag
     3601 tacagcctgt tcgagctgga gaacggccgc aagcgcatgc tggccagcgc cggcgagctg
     3661 cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta cctggccagc
     3721 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag
     3781 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttcag caagcgcgtg
     3841 atcctggccg acgccaacct ggacaaggtg ctgagcgcct acaacaagca ccgcgacaag
     3901 cccatccgcg agcaggccga gaacatcatc cacctgttca ccctgaccaa cctgggcgcc
     3961 cccgccgcct tcaagtactt cgacaccacc atcgaccgca agcgctacac cagcaccaag
     4021 gaggtgctgg acgccaccct gatccaccag agcatcaccg gtctgtacga gacccgcatc
     4081 gacctgagcc agctgggcgg cgacggcggc tccggacctc caaagaaaaa gagaaaagta
     4141 tacccctacg acgtgcccga ctacgcctgt taa
//
