LOCUS       sequence-381520-        8073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSpRY and MCP-SoxS(R93A/S101A) and scRNA J306 on p15A-CmR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8073)
  AUTHORS   Kiattisewee C, Karanjia AV, Legut M, Daniloski Z, Koplik SE, Nelson 
            J, Kleinstiver BP, Sanjana NE, Carothers JM, Zalatan JG
  TITLE     Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible
            dCas9 Variants.
  JOURNAL   ACS Synth Biol. 2022 Nov 15. doi: 10.1021/acssynbio.2c00405.
  PUBMED    36378874
REFERENCE   2  (bases 1 to 8073)
  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: "ACS Synth 
            Biol. 2022 Nov 15. doi: 10.1021/acssynbio.2c00405."
COMMENT     Sequence Label: pCK364.306
FEATURES             Location/Qualifiers
     source          1..8073
                     /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
                     NLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVL
                     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(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"
     promoter        6462..6496
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        6580..6598
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             6621..6650
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6666..6683
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6739..6756)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      6909..6955
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(7173..7267)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7288..7947)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7862..7881
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7948..8050)
                     /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 ggaacgtact 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 tgcgattgtt 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 aattcgtcca aaaagaaatt
     3901 cggacaagct tattgctcgt aaaaaagact gggatccaaa aaaatatggt ggttttctgt
     3961 ggccaacggt 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 gtgccaaaca gttacaaaaa 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 cgcgtcttgg agctccccgt gcttttaaat attttgatac aacaattgat ccgaaacagt
     4561 atcgttctac 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 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 attgacagct agctcagtcc
     6481 taggtataat actagtttgt gtccagaacg ctccgtgttt tagagctaga aatagcaagt
     6541 taaaataagg ctagtccgtt atcaacttga aaaagtggca catgaggatc acccatgtgc
     6601 tttttttgaa gcttgggccc gaacaaaaac tcatctcaga agaggatctg aatagcgccg
     6661 tcgaccatca tcatcatcat cattgagttt aaacggtctc cagcttggct gttttggcgg
     6721 atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     6781 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     6841 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg
     6901 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     6961 gtttgtcggt gaactggatc cttactcgag tctagactgc aggcttcctc gctcactgac
     7021 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     7081 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     7141 aaggccagga accgtaaaaa actagtgctt ggattctcac caataaaaaa cgcccggcgg
     7201 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     7261 aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg
     7321 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     7381 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     7441 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     7501 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     7561 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     7621 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     7681 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg
     7741 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     7801 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     7861 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     7921 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa
     7981 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac
     8041 gtgccgatca acgtctcatt ttcgccagat atc
//
