LOCUS       sequence-345705-        6184 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 for modular CRISPRa. SYNZIP-dCas9. Expresses dCas9 with a 
            SYNZIP18 domain fused to the N terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6184)
  AUTHORS   Villegas Kcam MC, Tsong AJ, Chappell J
  TITLE     Rational engineering of a modular bacterial CRISPR-Cas activation 
            platform with expanded target range.
  JOURNAL   Nucleic Acids Res. 2021 May 7;49(8):4793-4802. doi: 
            10.1093/nar/gkab211.
  PUBMED    33823546
REFERENCE   2  (bases 1 to 6184)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2021-05-7- 7"; volume: "49"; issue: "8";
            pages: "4793-4802"
COMMENT     Sequence Label: pJEC578
FEATURES             Location/Qualifiers
     source          1..6184
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      58..85
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(113..130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(247..792)
                     /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(327..346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(906..1000)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1021..1680)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1595..1614
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1681..1783)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        1813..1847
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     promoter        1813..1847
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     promoter        1813..1847
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     RBS             1863..1874
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(2028..2051,2052..2054,2055..4541,4542..4544,
                     4545..6131)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     terminator      join(6155..6184,1..42)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc
       61 tcaccttcgg gtgggccttt ctgcgtttat acctagggat atattccgct tcctcgctca
      121 ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg
      181 gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa
      241 gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca
      301 gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct
      361 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg
      421 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact
      481 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg
      541 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta
      601 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt
      661 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc
      721 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca
      781 aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa
      841 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gttactagtg
      901 cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa caaatccaga
      961 tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct cgatatcaaa
     1021 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
     1081 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
     1141 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
     1201 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
     1261 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
     1321 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
     1381 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
     1441 accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg caagaatgtg
     1501 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
     1561 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
     1621 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
     1681 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
     1741 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
     1801 gatatcgacg tctttacggc tagctcagtc ctaggtatta tgctagcgaa ttcaaaagat
     1861 ctaaagagga gaaaggatct atgtctatcg cggccaccct ggaaaacgac ttagcacgcc
     1921 ttgagaatga aaatgctcgt ctggagaaag atatcgcgaa tctcgagcgt gacctggcaa
     1981 aattagagcg tgaggaagct tatttcggtg gcggtggctc tggtggagat aagaaatact
     2041 caataggctt agctatcggc acaaatagcg tcggatgggc ggtgatcact gatgaatata
     2101 aggttccgtc taaaaagttc aaggttctgg gaaatacaga ccgccacagt atcaaaaaaa
     2161 atcttatagg ggctctttta tttgacagtg gagagacagc ggaagcgact cgtctcaaac
     2221 ggacagctcg tagaaggtat acacgtcgga agaatcgtat ttgttatcta caggagattt
     2281 tttcaaatga gatggcgaaa gtagatgata gtttctttca tcgacttgaa gagtcttttt
     2341 tggtggaaga agacaagaag catgaacgtc atcctatttt tggaaatata gtagatgaag
     2401 ttgcttatca tgagaaatat ccaactatct atcatctgcg aaaaaaattg gtagattcta
     2461 ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt aagtttcgtg
     2521 gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac aaactattta
     2581 tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac gcaagtggag
     2641 tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta gaaaatctca
     2701 ttgctcagct ccccggtgag aagaaaaatg gcttatttgg gaatctcatt gctttgtcat
     2761 tgggtttgac ccctaatttt aaatcaaatt ttgatttggc agaagatgct aaattacagc
     2821 tttcaaaaga tacttacgat gatgatttag ataatttatt ggcgcaaatt ggagatcaat
     2881 atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt tcagatatcc
     2941 taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt aaacgctacg
     3001 atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa cttccagaaa
     3061 agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat attgatgggg
     3121 gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa atggatggta
     3181 ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg
     3241 acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt ttgagaagac
     3301 aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt
     3361 ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga
     3421 ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc gataaaggtg
     3481 cttcagctca atcatttatt gaacgcatga caaactttga taaaaatctt ccaaatgaaa
     3541 aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac gaattgacaa
     3601 aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga
     3661 aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag
     3721 aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga gttgaagata
     3781 gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa gataaagatt
     3841 ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca ttgaccttat
     3901 ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc tttgatgata
     3961 aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggacgtttg tctcgaaaat
     4021 tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt ttgaaatcag
     4081 atggttttgc caatcgcaat tttatgcagc tgatccatga tgatagtttg acatttaaag
     4141 aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa catattgcaa
     4201 atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa gttgttgatg
     4261 aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa atggcacgtg
     4321 aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag
     4381 aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa aatactcaat
     4441 tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg tatgtggacc
     4501 aagaattaga tattaatcgt ttaagtgatt atgatgtcga tgccattgtt ccacaaagtt
     4561 tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa aatcgtggta
     4621 aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat tggagacaac
     4681 ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa gctgaacgtg
     4741 gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc
     4801 aaatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa tacgatgaaa
     4861 atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta gtttctgact
     4921 tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat catgcccatg
     4981 atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca aaacttgaat
     5041 cggagtttgt ctatggtgat tataaagttt atgatgttcg taaaatgatt gctaagtctg
     5101 agcaagaaat aggcaaagca accgcaaaat atttctttta ctctaatatc atgaacttct
     5161 tcaaaacaga aattacactt gcaaatggag agattcgcaa acgccctcta atcgaaacta
     5221 atggggaaac tggagaaatt gtctgggata aagggcgaga ttttgccaca gtgcgcaaag
     5281 tattgtccat gccccaagtc aatattgtca agaaaacaga agtacagaca ggcggattct
     5341 ccaaggagtc aattttacca aaaagaaatt cggacaagct tattgctcgt aaaaaagact
     5401 gggatccaaa aaaatatggt ggttttgata gtccaacggt agcttattca gtcctagtgg
     5461 ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag ttactaggga
     5521 tcacaattat ggaaagaagt tcctttgaaa aaaatccgat tgacttttta gaagctaaag
     5581 gatataagga agttaaaaaa gacttaatca ttaaactacc taaatatagt ctttttgagt
     5641 tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga attacaaaaa ggaaatgagc
     5701 tggctctgcc aagcaaatat gtgaattttt tatatttagc tagtcattat gaaaagttga
     5761 agggtagtcc agaagataac gaacaaaaac aattgtttgt ggagcagcat aagcattatt
     5821 tagatgagat tattgagcaa atcagtgaat tttctaagcg tgttatttta gcagatgcca
     5881 atttagataa agttcttagt gcatataaca aacatagaga caaaccaata cgtgaacaag
     5941 cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct gcttttaaat
     6001 attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt ttagatgcca
     6061 ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg agtcagctag
     6121 gaggtgacta actcgagtaa ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa
     6181 agac
//
