LOCUS       sequence-308630-        7368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dxCas9(3.7), MCP-SoxS(R93A/S101A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7368)
  AUTHORS   Fontana J, Dong C, Kiattisewee C, Chavali VP, Tickman BI, Carothers 
            JM, Zalatan JG
  TITLE     Effective CRISPRa-mediated control of gene expression in bacteria 
            must overcome strict target site requirements.
  JOURNAL   Nat Commun. 2020 Apr 1;11(1):1618. doi: 10.1038/s41467-020-15454-y.
  PUBMED    32238808
REFERENCE   2  (bases 1 to 7368)
  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: "Nat 
            Commun."; date: "2020-04-1"; volume: "11(1):1618. doi"; pages: " 
            10.1038/s41467-020-15454-y"
COMMENT     Sequence Label: pCD564
FEATURES             Location/Qualifiers
     source          1..7368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(181..259)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             555..4658
                     /codon_start=1
                     /product="catalytically dead Streptococcus pyogenes Cas9 
                     mutant with broad PAM compatibility (Hu et al., 2018)"
                     /label=dxCas9(3.7)
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      4685..4756
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4772..4799
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4852..4886
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             4896..4907
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(5675..5692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5809..6354)
                     /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(5889..5908)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6468..6562)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6583..7242)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7157..7176
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7243..7345)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcttaa gttacgaaat catcctgtgg agcttagtag gtttagcaag atggcagcgc
       61 ctaaatgtag aatgataaaa ggattaagag attaatttcc ctaaaaatga taaaacaagc
      121 gttttgaaag cgcttgtttt tttggtttgc agtcagagta gaatagaagt atcaaaaaaa
      181 gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta
      241 tgctgttttg aatggttcca acaagattat tttataactt ttataacaaa taatcaagga
      301 gaaattcaaa gaaatttatc agccataaaa caatacttaa tactatagaa tgataacaaa
      361 ataaactact ttttaaaaga attttgtgtt ataatctatt tattattaag tattgggtaa
      421 tattttttga agagatattt tgaaaaagaa aaattaaagc atattaaact aatttcggag
      481 gtcattaaaa ctattattga aatcatcaaa ctcattatgg atttaattta aactttttat
      541 tttaggaggc aaaaatggac aagaagtact ccattgggct cgctatcggc acaaacagcg
      601 tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg
      661 gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg
      721 gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa
      781 agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact
      841 ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc
      901 acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat
      961 atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc
     1021 tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc
     1081 cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt
     1141 tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc
     1201 tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg
     1261 gcctgtttgg taatcttatc gccctgtccc tcgggctgac ccccaacttt aaatctaact
     1321 tcgacctggc cgaagatacc aagcttcaac tgagcaaaga cacctacgat gatgatctcg
     1381 acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc
     1441 tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc
     1501 cgctgagcgc tagtatgatc aagctctatg atgagcacca ccaagacttg actttgctga
     1561 aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta
     1621 aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta
     1681 ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag
     1741 aagatctgtt gcgcaaacag cgcactttcg acaatggaat catcccccac cagattcacc
     1801 tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata
     1861 acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg
     1921 cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct
     1981 ggaacttcga gaaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga
     2041 ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg
     2101 agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa
     2161 agccagcatt cctgtctgga gatcagaaga aagctattgt ggacctcctc ttcaagacga
     2221 accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg
     2281 actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg
     2341 atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc
     2401 ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct
     2461 tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa gcagctcaag aggcgccgat
     2521 atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg
     2581 gaaagacaat cctggatttt cttaagtccg atggatttgc caaccggaac ttcattcagt
     2641 tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc
     2701 agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg
     2761 gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc
     2821 ccgagaatat cgttatcgag atggcccgag agaaccaaac cacccagaag ggacagaaga
     2881 acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc
     2941 ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc
     3001 tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact
     3061 acgacgtgga cgctatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag
     3121 tgttgacaag atccgataaa aacagaggga agagtgataa cgtcccctca gaagaagttg
     3181 tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga
     3241 agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggtt
     3301 tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg
     3361 attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta
     3421 ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga
     3481 gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg
     3541 cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt
     3601 acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt
     3661 acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag
     3721 agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca
     3781 agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta
     3841 aaaagaccga agtacagacc ggaggtttct ccaaggaaag tatcctcccg aaaaggaaca
     3901 gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt
     3961 ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa
     4021 aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa
     4081 aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca
     4141 ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta
     4201 gtgcgggcgt gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct
     4261 tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc
     4321 agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat
     4381 tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata
     4441 agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga
     4501 ccaacttggg cgcgcctgca gccttcaagt acttcgacac taccatagac agaaagcggt
     4561 acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct
     4621 atgaaacaag aatcgacctc tctcagctcg gtggagacta actcgagtaa ggatctccag
     4681 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     4741 gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt
     4801 ttatacctag agcgcccgga agagagtcaa ttcagggtgg tgaatctgca gtttacggct
     4861 agctcagccc taggtattat gctagcgaat tcattaaaga ggagaaaggt accatggggc
     4921 ccgcttctaa ctttactcag ttcgttctcg tcgacaatgg cggaactggc gacgtgactg
     4981 tcgccccaag caacttcgct aacgggatcg ctgaatggat cagctctaac tcgcgttcac
     5041 aggcttacaa agtaacctgt agcgttcgtc agagctctgc gcagaatcgc aaatacacca
     5101 tcaaagtcga ggtgcctaaa ggcgcctggc gttcgtactt aaatatggaa ctaaccattc
     5161 caattttcgc cacgaattcc gactgcgagc ttattgttaa ggcaatgcaa ggtctcctaa
     5221 aagatggaaa cccgattccc tcagcaatcg cagcaaactc cggcatctac ggtggcggag
     5281 gtagcatgtc ccatcagaaa attattcagg atcttatcgc atggattgac gagcatattg
     5341 accagccgct taacattgat gtagtcgcaa aaaaatcagg ctattcaaag tggtacttgc
     5401 aacgaatgtt ccgcacggtg acgcatcaga cgcttggcga ttacattcgc caacgccgcc
     5461 tgttactggc cgccgttgag ttgcgcacca ccgagcgtcc gatttttgat atcgcaatgg
     5521 acctgggtta tgtctcgcag cagaccttct cccgcgtttt cgcgcggcag tttgatcgca
     5581 ctcccgcgga ttatcgccac cgcctgtaag cggccgccac gcaaaaaacc ccgcttcggc
     5641 ggggtttttt cgccctaggg atatattccg cttcctcgct cactgactcg ctacgctcgg
     5701 tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg
     5761 ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt tccataggct
     5821 ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac
     5881 aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc
     5941 tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc
     6001 tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg
     6061 ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac
     6121 atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag
     6181 tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc
     6241 cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag
     6301 gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat
     6361 catcttatta atcagataaa atatttctag atttcagtgc aatttatctc ttcaaatgta
     6421 gcacctgaag tcagccccat acgatataag ttgttactag tgcttggatt ctcaccaata
     6481 aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt
     6541 actggatcta tcaacaggag tccaagcgag ctcgatatca aattacgccc cgccctgcca
     6601 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacaaac
     6661 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt
     6721 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact
     6781 ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg
     6841 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg
     6901 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa
     6961 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat
     7021 acgaaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa
     7081 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg
     7141 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg
     7201 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc
     7261 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa
     7321 gttggaacct cttacgtgcc gatcaacgtc tcattttcgc cagatatc
//
