LOCUS       sequence-61359-e        4212 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  natural linear DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4212)
  AUTHORS   Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X
  TITLE     Generation of gene-modified mice via Cas9/RNA-mediated gene 
            targeting.
  JOURNAL   Cell Res. 2013 Apr 2. doi: 10.1038/cr.2013.46.
  PUBMED    23545779
REFERENCE   2  (bases 1 to 4212)
  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: "Cell Res. 
            2013 Apr 2. doi: 10.1038/cr.2013.46."
COMMENT     Sequence Label: pST1374-NLS-flag-linker-Cas9
FEATURES             Location/Qualifiers
     source          1..4212
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             7..27
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             28..48
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             49..69
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             79..102
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             109..4212
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             109..4209
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 atgggaccta agaaaaagag gaaggtgcct aagaaaaaga ggaaggtgcc taagaaaaag
       61 aggaaggtgg cggccgctga ctacaaggat gacgacgata aatctagaga caagaaatac
      121 tctattggac tggatatcgg gacaaactcc gttggctggg ccgtcataac cgacgagtat
      181 aaggtgccaa gcaagaaatt caaggtgctg ggtaatactg accgccattc aatcaagaag
      241 aacctgatcg gagcactcct cttcgactcc ggtgaaaccg ctgaagctac tcggctgaag
      301 cggaccgcaa ggcggagata cacccgccgc aagaatcgga tatgttatct gcaagagatc
      361 tttagcaacg aaatggctaa ggtggacgac tccttctttc accgcctgga agagagcttt
      421 ctggtggagg aggataagaa acacgagagg caccctatat tcggaaatat cgtggatgag
      481 gtggcttacc atgaaaagta tcctacaatc taccatctga ggaagaagct ggtggacagc
      541 accgataaag cagacctgag gctcatctat ctggccctgg ctcatatgat aaagtttaga
      601 ggacactttc tgatcgaggg cgacctgaat cccgataatt ccgatgtgga taaactcttc
      661 attcaactgg tgcagacata taaccaactg ttcgaggaga atcccataaa cgcttctggt
      721 gtggatgcca aggctattct gtccgctcgg ctgtccaagt cacgcagact ggagaatctg
      781 attgcccaac tgccaggaga aaagaagaac ggcctgtttg ggaacctcat cgccctgagc
      841 ctgggcctga cacctaactt caagtccaat tttgatctgg ccgaagatgc taaactccag
      901 ctctccaagg acacctatga cgatgatctg gacaacctgc tcgcacagat aggcgaccag
      961 tacgccgatc tctttctggc tgctaagaat ctctccgacg ccattctgct gagcgacata
     1021 ctccgggtca acactgagat caccaaagca cctctgagcg cctccatgat aaaacgctat
     1081 gatgaacacc atcaagacct gactctgctc aaagccctcg tgaggcaaca gctgccagag
     1141 aagtacaaag agatattctt cgaccagagc aagaatggat atgccggata catcgatggc
     1201 ggagcatcac aggaagaatt ttacaagttc atcaaaccaa tcctcgagaa gatggacggt
     1261 actgaagagc tgctggtgaa gctgaacagg gaggacctgc tgaggaagca gaggaccttt
     1321 gataatggct ccattccaca tcagatacac ctgggagagc tgcatgcaat cctccgcagg
     1381 caggaggatt tctatccttt cctgaaggat aaccgggaga agatagagaa gatcctgacc
     1441 ttcaggatcc cttattacgt cggccctctg gctagaggca actcccgctt cgcttggatg
     1501 accaggaaat ctgaggagac aattactcct tggaacttcg aagaggtcgt ggataagggc
     1561 gcaagcgccc agtcattcat cgaacggatg accaatttcg ataagaacct gcccaacgag
     1621 aaggtcctgc ccaaacattc actcctgtac gagtatttca ccgtctataa cgagctgact
     1681 aaagtgaagt acgtgaccga gggcatgagg aagcctgcct tcctgtccgg agagcagaag
     1741 aaggctatcg ttgatctgct cttcaagact aatagaaagg tgacagtgaa gcagctcaag
     1801 gaggattact ttaagaagat cgaatgcttt gactcagtgg aaatctctgg cgtggaggac
     1861 cgctttaatg ccagcctggg cacttaccat gatctgctga agataatcaa agacaaagat
     1921 ttcctcgata atgaggagaa cgaggacatc ctggaagata tcgtgctgac cctgactctg
     1981 ttcgaggata gagagatgat cgaagagcgc ctgaagacct atgcccatct gtttgacgat
     2041 aaagtcatga aacagctcaa gcggcggcgc tacactgggt ggggtagact ctccaggaaa
     2101 ctcataaacg gcatccgcga caaacagagc ggaaagacca tcctggattt cctgaaatcc
     2161 gacggattcg ctaacaggaa cttcatgcaa ctgattcacg atgactctct gacatttaaa
     2221 gaggacatcc agaaggcaca ggtgagcggt caaggcgaca gcctgcacga gcacatcgcc
     2281 aacctcgctg gatcacccgc cataaagaag ggaatactgc agacagtcaa ggtcgtggac
     2341 gaactcgtca aagtgatggg tcggcacaag ccagagaata tcgttatcga aatggcaagg
     2401 gagaaccaaa ccacccagaa gggccagaag aactctcggg aacggatgaa aagaatcgaa
     2461 gagggaatta aggagctggg atctcagata ctgaaggagc accctgtgga gaatacacag
     2521 ctccagaacg agaaactcta cctgtactac ctccagaacg ggcgggacat gtacgttgac
     2581 caggaactcg acatcaaccg gctgtccgat tatgacgtgg accatattgt tccacagtcc
     2641 ttcctcaaag atgactccat tgacaacaag gtgctgacca gatccgataa gaatcgcggt
     2701 aagtctgaca atgttccatc agaagaggtg gtcaagaaga tgaagaatta ctggcggcag
     2761 ctcctcaacg ccaaactgat cacccagcgg aagtttgaca atctgactaa ggcagaaaga
     2821 ggaggtctga gcgaactcga caaggccggc tttattaaga ggcaactggt cgaaacacgc
     2881 cagattacca aacacgtggc acaaatcctc gactctagga tgaacactaa gtacgatgag
     2941 aacgataagc tgatcaggga agtgaaagtg ataactctga agagcaagct ggtgtctgac
     3001 ttccggaagg actttcaatt ctacaaagtt cgcgaaataa acaattacca tcatgctcac
     3061 gatgcctatc tcaatgctgt cgttggcacc gccctgatca agaaataccc taaactggag
     3121 tctgagttcg tgtacggtga ctataaagtc tacgatgtga ggaagatgat agcaaagtct
     3181 gagcaagaga ttggcaaagc caccgccaag tacttcttct actctaatat catgaatttc
     3241 tttaagactg agataaccct ggctaacggc gaaatccgga agcgcccact gatcgaaaca
     3301 aacggagaaa caggagaaat cgtgtgggat aaaggcaggg acttcgcaac tgtgcggaag
     3361 gtgctgtcca tgccacaagt caatatcgtg aagaagaccg aagtgcagac cggcggattc
     3421 tcaaaggaga gcatcctgcc aaagcggaac tctgacaagc tgatcgccag gaagaaagat
     3481 tgggacccaa agaagtatgg cggtttcgat tcccctacag tggcttattc cgttctggtc
     3541 gtggcaaaag tggagaaagg caagtccaag aaactcaagt ctgttaagga gctgctcgga
     3601 attactatta tggagagatc cagcttcgag aagaatccaa tcgatttcct ggaagctaag
     3661 ggctataaag aagtgaagaa agatctcatc atcaaactgc ccaagtactc tctctttgag
     3721 ctggagaatg gtaggaagcg gatgctggcc tccgccggag agctgcagaa aggaaacgag
     3781 ctggctctgc cctccaaata cgtgaacttc ctgtatctgg cctcccacta cgagaaactc
     3841 aaaggtagcc ctgaagacaa tgagcagaag caactctttg ttgagcaaca taaacactac
     3901 ctggacgaaa tcattgaaca gattagcgag ttcagcaagc gggttattct ggccgatgca
     3961 aacctcgata aagtgctgag cgcatataat aagcacaggg acaagccaat tcgcgaacaa
     4021 gcagagaata ttatccacct ctttactctg actaatctgg gcgctcctgc tgccttcaag
     4081 tatttcgata caactattga caggaagcgg tacacctcta ccaaagaagt tctcgatgcc
     4141 accctgatac accagtcaat taccggactg tacgagactc gcatcgacct gtctcagctc
     4201 ggcggcgact ag
//
