LOCUS       sequence-213777-        8095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9, PCP-SoxS_R93A, J109 scRNA.b1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8095)
  AUTHORS   Dong C, Fontana J, Patel A, Carothers JM, Zalatan JG
  TITLE     Synthetic CRISPR-Cas gene activators for transcriptional 
            reprogramming in bacteria.
  JOURNAL   Nat Commun. 2018 Jun 27;9(1):2489. doi: 10.1038/s41467-018-04901-6.
  PUBMED    29950558
REFERENCE   2  (bases 1 to 8095)
  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: "2018-06-27"; volume: "9(1):2489. doi"; pages: " 
            10.1038/s41467-018-04901-6"
COMMENT     Sequence Label: pCD294.9
FEATURES             Location/Qualifiers
     source          1..8095
                     /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             join(555..581,582..584,585..3071,3072..3074,3075..4661)
                     /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"
     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)"
     rep_origin      complement(5824..6369)
                     /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(5904..5923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6477..6511
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     CDS             6643..6672
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6688..6705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6761..6778)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      6931..6977
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(7195..7289)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7310..7969)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7884..7903
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7970..8072)
                     /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 aaaaatggat aagaaatact caataggctt agctatcggc acaaatagcg
      601 tcggatgggc ggtgatcact gatgaatata aggttccgtc taaaaagttc aaggttctgg
      661 gaaatacaga ccgccacagt atcaaaaaaa atcttatagg ggctctttta tttgacagtg
      721 gagagacagc ggaagcgact cgtctcaaac ggacagctcg tagaaggtat acacgtcgga
      781 agaatcgtat ttgttatcta caggagattt tttcaaatga gatggcgaaa gtagatgata
      841 gtttctttca tcgacttgaa gagtcttttt tggtggaaga agacaagaag catgaacgtc
      901 atcctatttt tggaaatata gtagatgaag ttgcttatca tgagaaatat ccaactatct
      961 atcatctgcg aaaaaaattg gtagattcta ctgataaagc ggatttgcgc ttaatctatt
     1021 tggccttagc gcatatgatt aagtttcgtg gtcatttttt gattgaggga gatttaaatc
     1081 ctgataatag tgatgtggac aaactattta tccagttggt acaaacctac aatcaattat
     1141 ttgaagaaaa ccctattaac gcaagtggag tagatgctaa agcgattctt tctgcacgat
     1201 tgagtaaatc aagacgatta gaaaatctca ttgctcagct ccccggtgag aagaaaaatg
     1261 gcttatttgg gaatctcatt gctttgtcat tgggtttgac ccctaatttt aaatcaaatt
     1321 ttgatttggc agaagatgct aaattacagc tttcaaaaga tacttacgat gatgatttag
     1381 ataatttatt ggcgcaaatt ggagatcaat atgctgattt gtttttggca gctaagaatt
     1441 tatcagatgc tattttactt tcagatatcc taagagtaaa tactgaaata actaaggctc
     1501 ccctatcagc ttcaatgatt aaacgctacg atgaacatca tcaagacttg actcttttaa
     1561 aagctttagt tcgacaacaa cttccagaaa agtataaaga aatctttttt gatcaatcaa
     1621 aaaacggata tgcaggttat attgatgggg gagctagcca agaagaattt tataaattta
     1681 tcaaaccaat tttagaaaaa atggatggta ctgaggaatt attggtgaaa ctaaatcgtg
     1741 aagatttgct gcgcaagcaa cggacctttg acaacggctc tattccccat caaattcact
     1801 tgggtgagct gcatgctatt ttgagaagac aagaagactt ttatccattt ttaaaagaca
     1861 atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc ttattatgtt ggtccattgg
     1921 cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca attaccccat
     1981 ggaattttga agaagttgtc gataaaggtg cttcagctca atcatttatt gaacgcatga
     2041 caaactttga taaaaatctt ccaaatgaaa aagtactacc aaaacatagt ttgctttatg
     2101 agtattttac ggtttataac gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa
     2161 aaccagcatt tctttcaggt gaacagaaga aagccattgt tgatttactc ttcaaaacaa
     2221 atcgaaaagt aaccgttaag caattaaaag aagattattt caaaaaaata gaatgttttg
     2281 atagtgttga aatttcagga gttgaagata gatttaatgc ttcattaggt acctaccatg
     2341 atttgctaaa aattattaaa gataaagatt ttttggataa tgaagaaaat gaagatatct
     2401 tagaggatat tgttttaaca ttgaccttat ttgaagatag ggagatgatt gaggaaagac
     2461 ttaaaacata tgctcacctc tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt
     2521 atactggttg gggacgtttg tctcgaaaat tgattaatgg tattagggat aagcaatctg
     2581 gcaaaacaat attagatttt ttgaaatcag atggttttgc caatcgcaat tttatgcagc
     2641 tgatccatga tgatagtttg acatttaaag aagacattca aaaagcacaa gtgtctggac
     2701 aaggcgatag tttacatgaa catattgcaa atttagctgg tagccctgct attaaaaaag
     2761 gtattttaca gactgtaaaa gttgttgatg aattggtcaa agtaatgggg cggcataagc
     2821 cagaaaatat cgttattgaa atggcacgtg aaaatcagac aactcaaaag ggccagaaaa
     2881 attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga agtcagattc
     2941 ttaaagagca tcctgttgaa aatactcaat tgcaaaatga aaagctctat ctctattatc
     3001 tccaaaatgg aagagacatg tatgtggacc aagaattaga tattaatcgt ttaagtgatt
     3061 atgatgtcga tgccattgtt ccacaaagtt tccttaaaga cgattcaata gacaataagg
     3121 tcttaacgcg ttctgataaa aatcgtggta aatcggataa cgttccaagt gaagaagtag
     3181 tcaaaaagat gaaaaactat tggagacaac ttctaaacgc caagttaatc actcaacgta
     3241 agtttgataa tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat aaagctggtt
     3301 ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa gcatgtggca caaattttgg
     3361 atagtcgcat gaatactaaa tacgatgaaa atgataaact tattcgagag gttaaagtga
     3421 ttaccttaaa atctaaatta gtttctgact tccgaaaaga tttccaattc tataaagtac
     3481 gtgagattaa caattaccat catgcccatg atgcgtatct aaatgccgtc gttggaactg
     3541 ctttgattaa gaaatatcca aaacttgaat cggagtttgt ctatggtgat tataaagttt
     3601 atgatgttcg taaaatgatt gctaagtctg agcaagaaat aggcaaagca accgcaaaat
     3661 atttctttta ctctaatatc atgaacttct tcaaaacaga aattacactt gcaaatggag
     3721 agattcgcaa acgccctcta atcgaaacta atggggaaac tggagaaatt gtctgggata
     3781 aagggcgaga ttttgccaca gtgcgcaaag tattgtccat gccccaagtc aatattgtca
     3841 agaaaacaga agtacagaca ggcggattct ccaaggagtc aattttacca aaaagaaatt
     3901 cggacaagct tattgctcgt aaaaaagact gggatccaaa aaaatatggt ggttttgata
     3961 gtccaacggt agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga
     4021 agttaaaatc cgttaaagag ttactaggga tcacaattat ggaaagaagt tcctttgaaa
     4081 aaaatccgat tgacttttta gaagctaaag gatataagga agttaaaaaa gacttaatca
     4141 ttaaactacc taaatatagt ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta
     4201 gtgccggaga attacaaaaa ggaaatgagc tggctctgcc aagcaaatat gtgaattttt
     4261 tatatttagc tagtcattat gaaaagttga agggtagtcc agaagataac gaacaaaaac
     4321 aattgtttgt ggagcagcat aagcattatt tagatgagat tattgagcaa atcagtgaat
     4381 tttctaagcg tgttatttta gcagatgcca atttagataa agttcttagt gcatataaca
     4441 aacatagaga caaaccaata cgtgaacaag cagaaaatat tattcattta tttacgttga
     4501 cgaatcttgg agctcccgct gcttttaaat attttgatac aacaattgat cgtaaacgat
     4561 atacgtctac aaaagaagtt ttagatgcca ctcttatcca tcaatccatc actggtcttt
     4621 atgaaacacg cattgatttg agtcagctag gaggtgacta 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 cctccaaaac catcgttctt tcggtcggcg aggctactcg cactctgact gagatccagt
     4981 ccaccgcaga ccgtcagatc ttcgaagaga aggtcgggcc tctggtgggt cggctgcgcc
     5041 tcacggcttc gctccgtcaa aacggagcca agaccgcgta tcgcgtcaac ctaaaactgg
     5101 atcaggcgga cgtcgttgat tccggacttc cgaaagtgcg ctacactcag gtatggtcgc
     5161 acgacgtgac aatcgttgcg aatagcaccg aggcctcgcg caaatcgttg tacgatttga
     5221 ccaagtccct cgtcgcgacc tcgcaggtcg aagatcttgt cgtcaacctt gtgccgctgg
     5281 gccgtggtgg cggaggtagc atgtcccatc agaaaattat tcaggatctt atcgcatgga
     5341 ttgacgagca tattgaccag ccgcttaaca ttgatgtagt cgcaaaaaaa tcaggctatt
     5401 caaagtggta cttgcaacga atgttccgca cggtgacgca tcagacgctt ggcgattaca
     5461 ttcgccaacg ccgcctgtta ctggccgccg ttgagttgcg caccaccgag cgtccgattt
     5521 ttgatatcgc aatggacctg ggttatgtct cgcagcagac cttctcccgc gttttcgcgc
     5581 ggcagtttga tcgcactccc agcgattatc gccaccgcct gtaagcggcc gccacgcaaa
     5641 aaaccccgct tcggcggggt tttttcgccc tagggatata ttccgcttcc tcgctcactg
     5701 actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag
     5761 atttcctgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc
     5821 gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg
     5881 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt
     5941 gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt
     6001 gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta
     6061 tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6121 ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag
     6181 gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac
     6241 tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa
     6301 aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa
     6361 cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt
     6421 atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgtt actagattga
     6481 cagctagctc agtcctaggt ataatactag taggtatcct gcggtgtcct ggttttagag
     6541 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag taacataagg
     6601 agtttatatg gaaaccctta tgtttttttg aagcttgggc ccgaacaaaa actcatctca
     6661 gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgagt ttaaacggtc
     6721 tccagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag
     6781 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
     6841 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
     6901 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     6961 tgggcctttc gttttatctg ttgtttgtcg gtgaactgga tccttactcg agtctagact
     7021 gcaggcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     7081 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     7141 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaactagtgc ttggattctc
     7201 accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga
     7261 ggtcattact ggatctatca acaggagtcc aagcgagctc gatatcaaat tacgccccgc
     7321 cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat
     7381 cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat
     7441 aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat
     7501 caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc
     7561 ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta
     7621 gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct
     7681 catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca
     7741 ttgccatacg aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg
     7801 gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa
     7861 cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat
     7921 gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct
     7981 tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat
     8041 ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccag atatc
//
