LOCUS       sequence-218612-        8166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9, MCP-TetD, J107 scRNA.b1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8166)
  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 8166)
  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: pCD296.7
FEATURES             Location/Qualifiers
     source          1..8166
                     /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(5902..6447)
                     /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(5982..6001)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6555..6589
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        6673..6691
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             6714..6743
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6759..6776
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6832..6849)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      7002..7048
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(7266..7360)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7381..8040)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7955..7974
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(8041..8143)
                     /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 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 gtagcatgta tattgaacag cattctcgct atcaaaataa agctaataac atccaattaa
     5341 gatatgatga taagcagttt catacaacgg ttatcaaaga tgttctatta tggattgaac
     5401 ataatttaga tcagtcttta ctgcttgatg atgtggcgaa taaagcgggt tataccaagt
     5461 ggtattttca gcggctgttc aaaaaagtaa caggggtcac actggctagc tatattcgtg
     5521 ctcgtcgttt gacgaaagcg gctgttgagt tgaggttgac gaaaaaaact atccttgaga
     5581 tcgcattaaa atatcaattt gattcccaac aatcttttac acgtcgattt aagtacattt
     5641 ttaaggttac accaagttat tatcggcgta ataaattatg ggaattggag gcaatgcact
     5701 aagcggccgc cacgcaaaaa accccgcttc ggcggggttt tttcgcccta gggatatatt
     5761 ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg
     5821 cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga
     5881 gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat
     5941 ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6001 ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc
     6061 gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc
     6121 gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg
     6181 gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca
     6241 ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa
     6301 aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta
     6361 gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag
     6421 attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc
     6481 tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat
     6541 aagttgttac tagattgaca gctagctcag tcctaggtat aatactagtc ggtgtcctgc
     6601 ggttaccaag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     6661 tgaaaaagtg gcacatgagg atcacccatg tgcttttttt gaagcttggg cccgaacaaa
     6721 aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat catcattgag
     6781 tttaaacggt ctccagcttg gctgttttgg cggatgagag aagattttca gcctgataca
     6841 gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc
     6901 ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag
     6961 tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc
     7021 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaactgg atccttactc
     7081 gagtctagac tgcaggcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     7141 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     7201 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaactagtg
     7261 cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa caaatccaga
     7321 tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct cgatatcaaa
     7381 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
     7441 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
     7501 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
     7561 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
     7621 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
     7681 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
     7741 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
     7801 accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg caagaatgtg
     7861 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
     7921 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
     7981 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
     8041 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
     8101 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
     8161 gatatc
//
