LOCUS       sequence-80474-e        4160 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  The expression vector for humanized nickase Cas9 (Cas9D10A) under 
            CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4160)
  TITLE     pCAG-hCas9D10A
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4160)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCAG-hCas9D10A
FEATURES             Location/Qualifiers
     source          1..4160
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      7..16
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(13..39,40..42,43..4116)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /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             13..4116
                     /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="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             4129..4149
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 ggtaccgcca ccatggacaa gaagtactcc attgggctcg ctatcggcac aaacagcgtc
       61 ggctgggccg tcattacgga cgagtacaag gtgccgagca aaaaattcaa agttctgggc
      121 aataccgatc gccacagcat aaagaagaac ctcattggcg ccctcctgtt cgactccggg
      181 gagacggccg aagccacgcg gctcaaaaga acagcacggc gcagatatac ccgcagaaag
      241 aatcggatct gctacctgca ggagatcttt agtaatgaga tggctaaggt ggatgactct
      301 ttcttccata ggctggagga gtcctttttg gtggaggagg ataaaaagca cgagcgccac
      361 ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat
      421 catctgagga agaagcttgt agacagtact gataaggctg acttgcggtt gatctatctc
      481 gcgctggcgc atatgatcaa atttcgggga cacttcctca tcgaggggga cctgaaccca
      541 gacaacagcg atgtcgacaa actctttatc caactggttc agacttacaa tcagcttttc
      601 gaagagaacc cgatcaacgc atccggagtt gacgccaaag caatcctgag cgctaggctg
      661 tccaaatccc ggcggctcga aaacctcatc gcacagctcc ctggggagaa gaagaacggc
      721 ctgtttggta atcttatcgc cctgtcactc gggctgaccc ccaactttaa atctaacttc
      781 gacctggccg aagatgccaa gcttcaactg agcaaagaca cctacgatga tgatctcgac
      841 aatctgctgg cccagatcgg cgaccagtac gcagaccttt ttttggcggc aaagaacctg
      901 tcagacgcca ttctgctgag tgatattctg cgagtgaaca cggagatcac caaagctccg
      961 ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc aagacttgac tttgctgaag
     1021 gcccttgtca gacagcaact gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa
     1081 aatggctacg ccggatacat tgacggcgga gcaagccagg aggaatttta caaatttatt
     1141 aagcccatct tggaaaaaat ggacggcacc gaggagctgc tggtaaagct taacagagaa
     1201 gatctgttgc gcaaacagcg cactttcgac aatggaagca tcccccacca gattcacctg
     1261 ggcgaactgc acgctatcct caggcggcaa gaggatttct accccttttt gaaagataac
     1321 agggaaaaga ttgagaaaat cctcacattt cggataccct actatgtagg ccccctcgcc
     1381 cggggaaatt ccagattcgc gtggatgact cgcaaatcag aagagaccat cactccctgg
     1441 aacttcgagg aagtcgtgga taagggggcc tctgcccagt ccttcatcga aaggatgact
     1501 aactttgata aaaatctgcc taacgaaaag gtgcttccta aacactctct gctgtacgag
     1561 tacttcacag tttataacga gctcaccaag gtcaaatacg tcacagaagg gatgagaaag
     1621 ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg acctcctctt caagacgaac
     1681 cggaaagtta ccgtgaaaca gctcaaagaa gactatttca aaaagattga atgtttcgac
     1741 tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat
     1801 ctcctgaaaa tcattaaaga caaggacttc ctggacaatg aggagaacga ggacattctt
     1861 gaggacattg tcctcaccct tacgttgttt gaagataggg agatgattga agaacgcttg
     1921 aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac agctcaagag gcgccgatat
     1981 acaggatggg ggcggctgtc aagaaaactg atcaatggga tccgagacaa gcagagtgga
     2041 aagacaatcc tggattttct taagtccgat ggatttgcca acaggaactt catgcagttg
     2101 atccatgatg actctctcac ctttaaggag gacatccaga aagcacaagt ttctggccag
     2161 ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat caaaaaggga
     2221 atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag gcataagccc
     2281 gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg acagaagaac
     2341 agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc ccaaatcctt
     2401 aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct gtactacctg
     2461 cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct ctccgactac
     2521 gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga taataaagtg
     2581 ttgacaagat ccgataaaaa tagagggaag agtgataacg tcccctcaga agaagttgtc
     2641 aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac acaacggaag
     2701 ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa agccggcttc
     2761 atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca aattctcgat
     2821 tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt gaaagttatt
     2881 actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta taaggtgaga
     2941 gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt aggcactgca
     3001 cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta taaagtgtac
     3061 gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac cgctaagtac
     3121 ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc caatggagag
     3181 attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag
     3241 ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa
     3301 aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa aaggaacagc
     3361 gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg attcgattct
     3421 cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa
     3481 ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa
     3541 aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt
     3601 aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt
     3661 gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt taatttcttg
     3721 tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga gcagaagcag
     3781 ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat aagcgaattc
     3841 tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc ttacaataag
     3901 cacagggata agcccatcag ggagcaggca gaaaacatta tccacttgtt tactctgacc
     3961 aacttgggcg cgcctgcagc cttcaagtac ttcgacacca ccatagacag aaagcggtac
     4021 acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac ggggctctat
     4081 gaaacaagaa tcgacctctc tcagctcggt ggagacagca gggctgaccc caagaagaag
     4141 aggaaggtgt gagcggccgc
//
