LOCUS       sequence-353903-        6520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6520)
  AUTHORS   Tickman BI, Burbano DA, Chavali VP, Kiattisewee C, Fontana J, 
            Khakimzhan A, Noireaux V, Zalatan JG, Carothers JM
  TITLE     Multi-layer CRISPRa/i circuits for dynamic genetic programs in 
            cell-free and bacterial systems.
  JOURNAL   Cell Syst. 2021 Nov 17. pii: S2405-4712(21)00419-1. doi: 
            10.1016/j.cels.2021.10.008.
  PUBMED    34800362
REFERENCE   2  (bases 1 to 6520)
  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: "Cell Syst. 
            2021 Nov 17. pii: S2405-4712(21)00419-1. doi: 
            10.1016/j.cels.2021.10.008."
COMMENT     Sequence Label: pCD017
FEATURES             Location/Qualifiers
     source          1..6520
                     /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"
     primer_bind     complement(4827..4844)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4961..5506)
                     /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(5041..5060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5620..5714)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5735..6394)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6309..6328
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6395..6497)
                     /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 ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga
     4861 ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag
     4921 atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc
     4981 ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat
     5041 aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc
     5101 ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc
     5161 agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc
     5221 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa
     5281 gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc
     5341 cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc
     5401 tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt
     5461 ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat
     5521 taatcagata aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     5581 agtcagcccc atacgatata agttgttact agtgcttgga ttctcaccaa taaaaaacgc
     5641 ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc
     5701 tatcaacagg agtccaagcg agctcgatat caaattacgc cccgccctgc cactcatcgc
     5761 agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat
     5821 gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg
     5881 tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac
     5941 tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg
     6001 ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat
     6061 cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt
     6121 aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc atacgaaatt
     6181 ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct
     6241 tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg
     6301 tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat
     6361 caacggtggt atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc
     6421 tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac
     6481 ctcttacgtg ccgatcaacg tctcattttc gccagatatc
//
