LOCUS       sequence-249648-        5769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes dspCAS9 as a Type 3b part to be used in the MTK system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5769)
  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 5769)
  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: MTK3b_018
FEATURES             Location/Qualifiers
     source          1..5769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     500..519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(714..737,738..740,741..3296,3297..3299,3300..4814)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(714..737,738..740,741..3227,3228..3230,3231..4814)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     terminator      complement(4834..4868)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(4939..5598)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5513..5532
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
       61 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
      121 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
      181 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
      241 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
      301 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
      361 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      421 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      481 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      541 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      601 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      661 tctttcctgc gttatcccct gattctgtgg ataaccgtag tcggtctcat tctgacaaga
      721 agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg
      781 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
      841 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
      901 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
      961 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1021 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1081 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1141 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1201 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1261 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1321 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1381 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac ctgattgccc
     1441 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     1501 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     1561 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     1621 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     1681 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     1741 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     1801 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     1861 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     1921 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     1981 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2041 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2101 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2161 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2221 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2281 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2341 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     2401 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     2461 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     2521 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     2581 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     2641 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     2701 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     2761 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     2821 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     2881 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     2941 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3001 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3061 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3121 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3181 tggaccagga actggacatc aaccggctgt ccgactacga tgtggacgcc atcgtgcctc
     3241 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3301 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3361 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     3421 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     3481 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     3541 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     3601 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     3661 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     3721 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     3781 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     3841 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     3901 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     3961 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4021 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4081 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4141 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4201 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4261 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4321 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     4381 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     4441 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     4501 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     4561 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     4621 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     4681 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     4741 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     4801 agctgggagg cgacggatcc tgagaccaga ccaataaaaa acgcccggcg gcaaccgagc
     4861 gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca
     4921 agcgagctcg atatcaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc
     4981 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     5041 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     5101 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     5161 tgaaacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     5221 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     5281 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     5341 atcccatatc accagctcac cgtctttcat tgccatacga aattccggat gagcattcat
     5401 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     5461 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     5521 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     5581 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa
     5641 tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgcccgat
     5701 caatcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5761 aaaagatca
//
