LOCUS       sequence-249073-        5826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes spCAS9 tagged with NLS as a Type 3a part to be used in the 
            MTK system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5826)
  AUTHORS   Fonseca JP, Bonny AR, Kumar GR, Ng AH, Town J, Wu QC, Aslankoohi E, 
            Chen SY, Dods G, Harrigan P, Osimiri LC, Kistler AL, El-Samad H
  TITLE     A Toolkit for Rapid Modular Construction of Biological Circuits in 
            Mammalian Cells.
  JOURNAL   ACS Synth Biol. 2019 Nov 15;8(11):2593-2606. doi: 
            10.1021/acssynbio.9b00322. Epub 2019 Nov 5.
  PUBMED    31686495
REFERENCE   2  (bases 1 to 5826)
  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"; doi: 
            "10.1021/acssynbio.9b00322"; journalName: "ACS Synth Biol"; date: 
            "2019-11-15- 15"; volume: "8"; issue: "11"; pages: "2593-2606"
COMMENT     Sequence Label: MTK3a_024
FEATURES             Location/Qualifiers
     source          1..5826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             11..4114
                     /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             11..4114
                     /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             4127..4147
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4151..4171
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      complement(4191..4225)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(4296..4955)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4870..4889
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      5137..5725
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5626..5645
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcggtctcat atggacaaga agtattctat cggactggat atcgggacta atagcgtcgg
       61 gtgggccgtg atcactgacg agtacaaggt gccctctaag aagttcaagg tgctcgggaa
      121 caccgaccgg cattccatca agaaaaatct gatcggagct ctcctctttg attcagggga
      181 aaccgctgaa gcaacccgcc tcaagcggac tgctagacgg cggtacacca ggaggaagaa
      241 ccggatttgt taccttcaag agatattctc caacgaaatg gcaaaggtcg acgacagctt
      301 cttccatagg ctggaagaat cattcctcgt ggaagaggat aagaagcatg aacggcatcc
      361 catcttcggt aatatcgtcg acgaggtggc ctatcacgag aaatacccaa ccatctacca
      421 tcttcgcaaa aagctggtgg actcaaccga caaggcagac ctccggctta tctacctggc
      481 cctggcccac atgatcaagt tcagaggcca cttcctgatc gagggcgacc tcaatcctga
      541 caatagcgat gtggataaac tgttcatcca gctggtgcag acttacaacc agctctttga
      601 agagaacccc atcaatgcaa gcggagtcga tgccaaggcc attctgtcag cccggctgtc
      661 aaagagccgc agacttgaga atcttatcgc tcagctgccg ggtgaaaaga aaaatggact
      721 gttcgggaac ctgattgctc tttcacttgg gctgactccc aatttcaagt ctaatttcga
      781 cctggcagag gatgccaagc tgcaactgtc caaggacacc tatgatgacg atctcgacaa
      841 cctcctggcc cagatcggtg accaatacgc cgaccttttc cttgctgcta agaatctttc
      901 tgacgccatc ctgctgtctg acattctccg cgtgaacact gaaatcacca aggcccctct
      961 ttcagcttca atgattaagc ggtatgatga gcaccaccag gacctgaccc tgcttaaggc
     1021 actcgtccgg cagcagcttc cggagaagta caaggaaatc ttctttgacc agtcaaagaa
     1081 tggatacgcc ggctacatcg acggaggtgc ctcccaagag gaattttata agtttatcaa
     1141 acctatcctt gagaagatgg acggcaccga agagctcctc gtgaaactga atcgggagga
     1201 tctgctgcgg aagcagcgca ctttcgacaa tgggagcatt ccccaccaga tccatcttgg
     1261 ggagcttcac gccatccttc ggcgccaaga ggacttctac ccctttctta aggacaacag
     1321 ggagaagatt gagaaaattc tcactttccg catcccctac tacgtgggac ccctcgccag
     1381 aggaaatagc cggtttgctt ggatgaccag aaagtcagaa gaaactatca ctccctggaa
     1441 cttcgaagag gtggtggaca agggagccag cgctcagtca ttcatcgaac ggatgactaa
     1501 cttcgataag aacctcccca atgagaaggt cctgccgaaa cattccctgc tctacgagta
     1561 ctttaccgtg tacaacgagc tgaccaaggt gaaatatgtc accgaaggga tgaggaagcc
     1621 cgcattcctg tcaggcgaac aaaagaaggc aattgtggac cttctgttca agaccaatag
     1681 aaaggtgacc gtgaagcagc tgaaggagga ctatttcaag aaaattgaat gcttcgactc
     1741 tgtggagatt agcggggtcg aagatcggtt caacgcaagc ctgggtactt accatgatct
     1801 gcttaagatc atcaaggaca aggattttct ggacaatgag gagaacgagg acatccttga
     1861 ggacattgtc ctgactctca ctctgttcga ggaccgggaa atgatcgagg agaggcttaa
     1921 gacctacgcc catctgttcg acgataaagt gatgaagcaa cttaaacgga gaagatatac
     1981 cggatgggga cgccttagcc gcaaactcat caacggaatc cgggacaaac agagcggaaa
     2041 gaccattctt gatttcctta agagcgacgg attcgctaat cgcaacttca tgcaacttat
     2101 ccatgatgat tccctgacct ttaaggagga catccagaag gcccaagtgt ctggacaagg
     2161 tgactcactg cacgagcata tcgcaaatct ggctggttca cccgctatta agaagggtat
     2221 tctccagacc gtgaaagtcg tggacgagct ggtcaaggtg atgggtcgcc ataaaccaga
     2281 gaacattgtc atcgagatgg ccagggaaaa ccagactacc cagaagggac agaagaacag
     2341 cagggagcgg atgaaaagaa ttgaggaagg gattaaggag ctcgggtcac agatccttaa
     2401 agagcacccg gtggaaaaca cccagcttca gaatgagaag ctctatctgt actaccttca
     2461 aaatggacgc gatatgtatg tggaccaaga gcttgatatc aacaggctct cagactacga
     2521 cgtggaccat atcgtccctc agagcttcct caaagacgac tcaattgaca ataaggtgct
     2581 gactcgctca gacaagaacc ggggaaagtc agataacgtg ccctcagagg aagtcgtgaa
     2641 aaagatgaag aactattggc gccagcttct gaacgcaaag ctgatcactc agcggaagtt
     2701 cgacaatctc actaaggctg agaggggcgg actgagcgaa ctggacaaag caggattcat
     2761 taaacggcaa cttgtggaga ctcggcagat tactaaacat gtcgcccaaa tccttgactc
     2821 acgcatgaat accaagtacg acgaaaacga caaacttatc cgcgaggtga aggtgattac
     2881 cctgaagtcc aagctggtca gcgatttcag aaaggacttt caattctaca aagtgcggga
     2941 gatcaataac tatcatcatg ctcatgacgc atatctgaat gccgtggtgg gaaccgccct
     3001 gatcaagaag tacccaaagc tggaaagcga gttcgtgtac ggagactaca aggtctacga
     3061 cgtgcgcaag atgattgcca aatctgagca ggagatcgga aaggccaccg caaagtactt
     3121 cttctacagc aacatcatga atttcttcaa gaccgaaatc acccttgcaa acggtgagat
     3181 ccggaagagg ccgctcatcg agactaatgg ggagactggc gaaatcgtgt gggacaaggg
     3241 cagagatttc gctaccgtgc gcaaagtgct ttctatgcct caagtgaaca tcgtgaagaa
     3301 aaccgaggtg caaaccggag gcttttctaa ggaatcaatc ctccccaagc gcaactccga
     3361 caagctcatt gcaaggaaga aggattggga ccctaagaag tacggcggat tcgattcacc
     3421 aactgtggct tattctgtcc tggtcgtggc taaggtggaa aaaggaaagt ctaagaagct
     3481 caagagcgtg aaggaactgc tgggtatcac cattatggag cgcagctcct tcgagaagaa
     3541 cccaattgac tttctcgaag ccaaaggtta caaggaagtc aagaaggacc ttatcatcaa
     3601 gctcccaaag tatagcctgt tcgaactgga gaatgggcgg aagcggatgc tcgcctccgc
     3661 tggcgaactt cagaagggta atgagctggc tctcccctcc aagtacgtga atttcctcta
     3721 ccttgcaagc cattacgaga agctgaaggg gagccccgag gacaacgagc aaaagcaact
     3781 gtttgtggag cagcataagc attatctgga cgagatcatt gagcagattt ccgagttttc
     3841 taaacgcgtc attctcgctg atgccaacct cgataaagtc cttagcgcat acaataagca
     3901 cagagacaaa ccaattcggg agcaggctga gaatatcatc cacctgttca ccctcaccaa
     3961 tcttggtgcc cctgccgcat tcaagtactt cgacaccacc atcgaccgga aacgctatac
     4021 ctccaccaaa gaagtgctgg acgccaccct catccaccag agcatcaccg gactttacga
     4081 aactcggatt gacctctcac agctcggagg ggatgaggga gctgatccaa aaaagaagag
     4141 aaaggtagat ccaaaaaaga agagaaaggt aggttcttga gaccagacca ataaaaaacg
     4201 cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat
     4261 ctatcaacag gagtccaagc gagctcgata tcaaattacg ccccgccctg ccactcatcg
     4321 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
     4381 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
     4441 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
     4501 ctcacccagg gattggctga aacgaaaaac atattctcaa taaacccttt agggaaatag
     4561 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
     4621 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
     4681 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacgaaat
     4741 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
     4801 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
     4861 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
     4921 tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
     4981 ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa
     5041 cctcttacgt gcccgatcaa tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5101 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5161 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5221 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5281 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5341 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5401 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5461 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5521 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5581 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5641 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5701 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5761 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5821 ccgtag
//
