LOCUS       sequence-364492-        9326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the tracrRNA, the dCas9 catalytic site mutant and a CRISPR
            array designed for the easy cloning of new spacers..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9326)
  AUTHORS   Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA
  TITLE     Programmable repression and activation of bacterial gene expression 
            using an engineered CRISPR-Cas system.
  JOURNAL   Nucleic Acids Res. 2013 Jun 12.
  PUBMED    23761437
REFERENCE   2  (bases 1 to 9326)
  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: "Nucleic 
            Acids Res. 2013 Jun 12."
COMMENT     Sequence Label: pdCas9
FEATURES             Location/Qualifiers
     source          1..9326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    2605..2736
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   2737..2772
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   2803..2838
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     primer_bind     complement(3267..3286)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(5139..5798)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5713..5732
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5799..5901)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(6293..6310)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6427..6972)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(6507..6526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(7084..7089,7090..7106,7107..7112)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     misc_RNA        complement(7430..7508)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(7804..7830,7831..7833,7834..9326,1..994,995..997,
                     998..2584)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 accaaaacat agtttgcttt atgagtattt tacggtttat aacgaattga caaaggtcaa
       61 atatgttact gaaggaatgc gaaaaccagc atttctttca ggtgaacaga agaaagccat
      121 tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt aagcaattaa aagaagatta
      181 tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca ggagttgaag atagatttaa
      241 tgcttcatta ggtacctacc atgatttgct aaaaattatt aaagataaag attttttgga
      301 taatgaagaa aatgaagata tcttagagga tattgtttta acattgacct tatttgaaga
      361 tagggagatg attgaggaaa gacttaaaac atatgctcac ctctttgatg ataaggtgat
      421 gaaacagctt aaacgtcgcc gttatactgg ttggggacgt ttgtctcgaa aattgattaa
      481 tggtattagg gataagcaat ctggcaaaac aatattagat tttttgaaat cagatggttt
      541 tgccaatcgc aattttatgc agctgatcca tgatgatagt ttgacattta aagaagacat
      601 tcaaaaagca caagtgtctg gacaaggcga tagtttacat gaacatattg caaatttagc
      661 tggtagccct gctattaaaa aaggtatttt acagactgta aaagttgttg atgaattggt
      721 caaagtaatg gggcggcata agccagaaaa tatcgttatt gaaatggcac gtgaaaatca
      781 gacaactcaa aagggccaga aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat
      841 caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa
      901 tgaaaagctc tatctctatt atctccaaaa tggaagagac atgtatgtgg accaagaatt
      961 agatattaat cgtttaagtg attatgatgt cgatgccatt gttccacaaa gtttccttaa
     1021 agacgattca atagacaata aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga
     1081 taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac tattggagac aacttctaaa
     1141 cgccaagtta atcactcaac gtaagtttga taatttaacg aaagctgaac gtggaggttt
     1201 gagtgaactt gataaagctg gttttatcaa acgccaattg gttgaaactc gccaaatcac
     1261 taagcatgtg gcacaaattt tggatagtcg catgaatact aaatacgatg aaaatgataa
     1321 acttattcga gaggttaaag tgattacctt aaaatctaaa ttagtttctg acttccgaaa
     1381 agatttccaa ttctataaag tacgtgagat taacaattac catcatgccc atgatgcgta
     1441 tctaaatgcc gtcgttggaa ctgctttgat taagaaatat ccaaaacttg aatcggagtt
     1501 tgtctatggt gattataaag tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga
     1561 aataggcaaa gcaaccgcaa aatatttctt ttactctaat atcatgaact tcttcaaaac
     1621 agaaattaca cttgcaaatg gagagattcg caaacgccct ctaatcgaaa ctaatgggga
     1681 aactggagaa attgtctggg ataaagggcg agattttgcc acagtgcgca aagtattgtc
     1741 catgccccaa gtcaatattg tcaagaaaac agaagtacag acaggcggat tctccaagga
     1801 gtcaatttta ccaaaaagaa attcggacaa gcttattgct cgtaaaaaag actgggatcc
     1861 aaaaaaatat ggtggttttg atagtccaac ggtagcttat tcagtcctag tggttgctaa
     1921 ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa gagttactag ggatcacaat
     1981 tatggaaaga agttcctttg aaaaaaatcc gattgacttt ttagaagcta aaggatataa
     2041 ggaagttaaa aaagacttaa tcattaaact acctaaatat agtctttttg agttagaaaa
     2101 cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa aaaggaaatg agctggctct
     2161 gccaagcaaa tatgtgaatt ttttatattt agctagtcat tatgaaaagt tgaagggtag
     2221 tccagaagat aacgaacaaa aacaattgtt tgtggagcag cataagcatt atttagatga
     2281 gattattgag caaatcagtg aattttctaa gcgtgttatt ttagcagatg ccaatttaga
     2341 taaagttctt agtgcatata acaaacatag agacaaacca atacgtgaac aagcagaaaa
     2401 tattattcat ttatttacgt tgacgaatct tggagctccc gctgctttta aatattttga
     2461 tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa gttttagatg ccactcttat
     2521 ccatcaatcc atcactggtc tttatgaaac acgcattgat ttgagtcagc taggaggtga
     2581 ctgaagtata ttttagatga agattatttc ttaataacta aaaatatggt ataatactct
     2641 taataaatgc agtaatacag gggcttttca agactgaagt ctagctgaga caaatagtgc
     2701 gattacgaaa ttttttagac aaaaatagtc tacgaggttt tagagctatg ctgttttgaa
     2761 tggtcccaaa actgagacca gtctcggaag ctcaaaggtc tcgttttaga gctatgctgt
     2821 tttgaatggt cccaaaactt cagcacactg agacttgttg agttccatgt tttagagcta
     2881 tgctgttttg aatggactcc attcaacatt gccgatgata acttgagaaa gagggttaat
     2941 accagcagtc ggataccttc ctattctttc tgttaaagcg ttttcatgtt ataataggca
     3001 aaagaagagt agtgtgatcg tccattccga cagcatcgcc agtcactatg gcgtgctgct
     3061 agcgctatat gcgttgatgc aatttctatg cgcacccgtt ctcggagcac tgtccgaccg
     3121 ctttggccgc cgcccagtcc tgctcgcttc gctacttgga gccactatcg actacgcgat
     3181 catggcgacc acacccgtcc tgtggatcct ctacgccgga cgcatcgtgg ccggcatcac
     3241 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     3301 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
     3361 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
     3421 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     3481 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     3541 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     3601 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     3661 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     3721 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccgacgcgct
     3781 gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca ttatgattct
     3841 tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca ggcaggtaga
     3901 tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc taacttcgat
     3961 cattggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat ggaacgggtt
     4021 ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc
     4081 atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc
     4141 actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct
     4201 tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc
     4261 agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     4321 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     4381 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     4441 cgtgtttcgt aaagtctgga aacgcggaag tcccctacgt gctgctgaag ttgcccgcaa
     4501 cagagagtgg aaccaaccgg tgataccacg atactatgac tgagagtcaa cgccatgagc
     4561 ggcctcattt cttattctga gttacaacag tccgcaccgc tgccggtagc tccttccggt
     4621 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     4681 taggacaggt gccggcagcg cccaacagtc ccccggccac ggggcctgcc accataccca
     4741 cgccgaaaca agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     4801 taccctgtgg aacacctaca tctgtattaa cgaagcgcta accgttttta tcaggctctg
     4861 ggaggcagaa taaatgatca tatcgtcaat tattacctcc acggggagag cctgagcaaa
     4921 ctggcctcag gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt
     4981 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc
     5041 gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa
     5101 ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca
     5161 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca
     5221 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc
     5281 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg
     5341 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
     5401 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg
     5461 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg
     5521 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg
     5581 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg
     5641 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
     5701 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat
     5761 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa
     5821 aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg
     5881 gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggcccg gggcttcccg
     5941 gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt
     6001 tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg
     6061 tttttgaggt gctccagtgg cttctgtttc tatcagctgt ccctcctgtt cagctactga
     6121 cggggtggtg cgtaacggca aaagcaccgc cggacatcag cgctagcgga gtgtatactg
     6181 gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa
     6241 aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca
     6301 ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg
     6361 gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa
     6421 gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca
     6481 gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct
     6541 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg
     6601 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact
     6661 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg
     6721 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta
     6781 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt
     6841 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc
     6901 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca
     6961 aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa
     7021 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctaa
     7081 tgtttgacag cttatcatcg ataagcttta atgcggtagt ttatcacagt taaattgcta
     7141 acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc tcggcaccgt
     7201 caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc tcttgcggga
     7261 ttacgaaatc atcctgtgga gcttagtagg tttagcaaga tggcagcgcc taaatgtaga
     7321 atgataaaag gattaagaga ttaatttccc taaaaatgat aaaacaagcg ttttgaaagc
     7381 gcttgttttt ttggtttgca gtcagagtag aatagaagta tcaaaaaaag caccgactcg
     7441 gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat gctgttttga
     7501 atggttccaa caagattatt ttataacttt tataacaaat aatcaaggag aaattcaaag
     7561 aaatttatca gccataaaac aatacttaat actatagaat gataacaaaa taaactactt
     7621 tttaaaagaa ttttgtgtta taatctattt attattaagt attgggtaat attttttgaa
     7681 gagatatttt gaaaaagaaa aattaaagca tattaaacta atttcggagg tcattaaaac
     7741 tattattgaa atcatcaaac tcattatgga tttaatttaa actttttatt ttaggaggca
     7801 aaaatggata agaaatactc aataggctta gctatcggca caaatagcgt cggatgggcg
     7861 gtgatcactg atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac
     7921 cgccacagta tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg
     7981 gaagcgactc gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt
     8041 tgttatctac aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat
     8101 cgacttgaag agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt
     8161 ggaaatatag tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga
     8221 aaaaaattgg tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg
     8281 catatgatta agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt
     8341 gatgtggaca aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac
     8401 cctattaacg caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca
     8461 agacgattag aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg
     8521 aatctcattg ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca
     8581 gaagatgcta aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg
     8641 gcgcaaattg gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct
     8701 attttacttt cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct
     8761 tcaatgatta aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt
     8821 cgacaacaac ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat
     8881 gcaggttata ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt
     8941 ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg
     9001 cgcaagcaac ggacctttga caacggctct attccccatc aaattcactt gggtgagctg
     9061 catgctattt tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag
     9121 attgaaaaaa tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat
     9181 agtcgttttg catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa
     9241 gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat
     9301 aaaaatcttc caaatgaaaa agtact
//
