LOCUS       sequence-381518-        7368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSpRY and MCP-SoxS(R93A/S101A) on p15A-CmR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7368)
  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 7368)
  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: pCK341
FEATURES             Location/Qualifiers
     source          1..7368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(12..106)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(127..786)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     701..720
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(787..889)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(1093..1171)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(1467..1493,1494..1496,1497..3983,3984..3986,
                     3987..5573)
                     /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      5597..5668
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5684..5711
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        5764..5798
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             5808..5819
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(6587..6604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6721..7266)
                     /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(6801..6820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa
       61 tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag cgagctcgat
      121 atcaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
      181 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
      241 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat
      301 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
      361 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
      421 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
      481 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
      541 cagctcaccg tctttcattg ccatacgaaa ttccggatga gcattcatca ggcgggcaag
      601 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
      661 cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
      721 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
      781 ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag
      841 tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt
      901 tcgccagata tcgacgtctt aagttacgaa atcatcctgt ggagcttagt aggtttagca
      961 agatggcagc gcctaaatgt agaatgataa aaggattaag agattaattt ccctaaaaat
     1021 gataaaacaa gcgttttgaa agcgcttgtt tttttggttt gcagtcagag tagaatagaa
     1081 gtatcaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
     1141 tttaacttgc tatgctgttt tgaatggttc caacaagatt attttataac ttttataaca
     1201 aataatcaag gagaaattca aagaaattta tcagccataa aacaatactt aatactatag
     1261 aatgataaca aaataaacta ctttttaaaa gaattttgtg ttataatcta tttattatta
     1321 agtattgggt aatatttttt gaagagatat tttgaaaaag aaaaattaaa gcatattaaa
     1381 ctaatttcgg aggtcattaa aactattatt gaaatcatca aactcattat ggatttaatt
     1441 taaacttttt attttaggag gcaaaaatgg ataagaaata ctcaataggc ttagctatcg
     1501 gcacaaatag cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt
     1561 tcaaggttct gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt
     1621 tatttgacag tggagagaca gcggaacgta ctcgtctcaa acggacagct cgtagaaggt
     1681 atacacgtcg gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga
     1741 aagtagatga tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga
     1801 agcatgaacg tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat
     1861 atccaactat ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc
     1921 gcttaatcta tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg
     1981 gagatttaaa tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct
     2041 acaatcaatt atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc
     2101 tttctgcacg attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg
     2161 agaagaaaaa tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt
     2221 ttaaatcaaa ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg
     2281 atgatgattt agataattta ttggcgcaaa ttggagatca atatgctgat ttgtttttgg
     2341 cagctaagaa tttatcagat gctattttac tttcagatat cctaagagta aatactgaaa
     2401 taactaaggc tcccctatca gcttcaatga ttaaacgcta cgatgaacat catcaagact
     2461 tgactctttt aaaagcttta gttcgacaac aacttccaga aaagtataaa gaaatctttt
     2521 ttgatcaatc aaaaaacgga tatgcaggtt atattgatgg gggagctagc caagaagaat
     2581 tttataaatt tatcaaacca attttagaaa aaatggatgg tactgaggaa ttattggtga
     2641 aactaaatcg tgaagatttg ctgcgcaagc aacggacctt tgacaacggc tctattcccc
     2701 atcaaattca cttgggtgag ctgcatgcta ttttgagaag acaagaagac ttttatccat
     2761 ttttaaaaga caatcgtgag aagattgaaa aaatcttgac ttttcgaatt ccttattatg
     2821 ttggtccatt ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag tctgaagaaa
     2881 caattacccc atggaatttt gaagaagttg tcgataaagg tgcttcagct caatcattta
     2941 ttgaacgcat gacaaacttt gataaaaatc ttccaaatga aaaagtacta ccaaaacata
     3001 gtttgcttta tgagtatttt acggtttata acgaattgac aaaggtcaaa tatgttactg
     3061 aaggaatgcg aaaaccagca tttctttcag gtgaacagaa gaaagccatt gttgatttac
     3121 tcttcaaaac aaatcgaaaa gtaaccgtta agcaattaaa agaagattat ttcaaaaaaa
     3181 tagaatgttt tgatagtgtt gaaatttcag gagttgaaga tagatttaat gcttcattag
     3241 gtacctacca tgatttgcta aaaattatta aagataaaga ttttttggat aatgaagaaa
     3301 atgaagatat cttagaggat attgttttaa cattgacctt atttgaagat agggagatga
     3361 ttgaggaaag acttaaaaca tatgctcacc tctttgatga taaggtgatg aaacagctta
     3421 aacgtcgccg ttatactggt tggggacgtt tgtctcgaaa attgattaat ggtattaggg
     3481 ataagcaatc tggcaaaaca atattagatt ttttgaaatc agatggtttt gccaatcgca
     3541 attttatgca gctgatccat gatgatagtt tgacatttaa agaagacatt caaaaagcac
     3601 aagtgtctgg acaaggcgat agtttacatg aacatattgc aaatttagct ggtagccctg
     3661 ctattaaaaa aggtatttta cagactgtaa aagttgttga tgaattggtc aaagtaatgg
     3721 ggcggcataa gccagaaaat atcgttattg aaatggcacg tgaaaatcag acaactcaaa
     3781 agggccagaa aaattcgcga gagcgtatga aacgaatcga agaaggtatc aaagaattag
     3841 gaagtcagat tcttaaagag catcctgttg aaaatactca attgcaaaat gaaaagctct
     3901 atctctatta tctccaaaat ggaagagaca tgtatgtgga ccaagaatta gatattaatc
     3961 gtttaagtga ttatgatgtc gatgcgattg ttccacaaag tttccttaaa gacgattcaa
     4021 tagacaataa ggtcttaacg cgttctgata aaaatcgtgg taaatcggat aacgttccaa
     4081 gtgaagaagt agtcaaaaag atgaaaaact attggagaca acttctaaac gccaagttaa
     4141 tcactcaacg taagtttgat aatttaacga aagctgaacg tggaggtttg agtgaacttg
     4201 ataaagctgg ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact aagcatgtgg
     4261 cacaaatttt ggatagtcgc atgaatacta aatacgatga aaatgataaa cttattcgag
     4321 aggttaaagt gattacctta aaatctaaat tagtttctga cttccgaaaa gatttccaat
     4381 tctataaagt acgtgagatt aacaattacc atcatgccca tgatgcgtat ctaaatgccg
     4441 tcgttggaac tgctttgatt aagaaatatc caaaacttga atcggagttt gtctatggtg
     4501 attataaagt ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa ataggcaaag
     4561 caaccgcaaa atatttcttt tactctaata tcatgaactt cttcaaaaca gaaattacac
     4621 ttgcaaatgg agagattcgc aaacgccctc taatcgaaac taatggggaa actggagaaa
     4681 ttgtctggga taaagggcga gattttgcca cagtgcgcaa agtattgtcc atgccccaag
     4741 tcaatattgt caagaaaaca gaagtacaga caggcggatt ctccaaggag tcaattcgtc
     4801 caaaaagaaa ttcggacaag cttattgctc gtaaaaaaga ctgggatcca aaaaaatatg
     4861 gtggttttct gtggccaacg gtagcttatt cagtcctagt ggttgctaag gtggaaaaag
     4921 ggaaatcgaa gaagttaaaa tccgttaaag agttactagg gatcacaatt atggaaagaa
     4981 gttcctttga aaaaaatccg attgactttt tagaagctaa aggatataag gaagttaaaa
     5041 aagacttaat cattaaacta cctaaatata gtctttttga gttagaaaac ggtcgtaaac
     5101 ggatgctggc tagtgccaaa cagttacaaa aaggaaatga gctggctctg ccaagcaaat
     5161 atgtgaattt tttatattta gctagtcatt atgaaaagtt gaagggtagt ccagaagata
     5221 acgaacaaaa acaattgttt gtggagcagc ataagcatta tttagatgag attattgagc
     5281 aaatcagtga attttctaag cgtgttattt tagcagatgc caatttagat aaagttctta
     5341 gtgcatataa caaacataga gacaaaccaa tacgtgaaca agcagaaaat attattcatt
     5401 tatttacgtt gacgcgtctt ggagctcccc gtgcttttaa atattttgat acaacaattg
     5461 atccgaaaca gtatcgttct acaaaagaag ttttagatgc cactcttatc catcaatcca
     5521 tcactggtct ttatgaaaca cgcattgatt tgagtcagct aggaggtgac taactcgagt
     5581 aaggatctcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     5641 tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg
     5701 gcctttctgc gtttatacct agagcgcccg gaagagagtc aattcagggt ggtgaatctg
     5761 cagtttacgg ctagctcagc cctaggtatt atgctagcga attcattaaa gaggagaaag
     5821 gtaccatggg gcccgcttct aactttactc agttcgttct cgtcgacaat ggcggaactg
     5881 gcgacgtgac tgtcgcccca agcaacttcg ctaacgggat cgctgaatgg atcagctcta
     5941 actcgcgttc acaggcttac aaagtaacct gtagcgttcg tcagagctct gcgcagaatc
     6001 gcaaatacac catcaaagtc gaggtgccta aaggcgcctg gcgttcgtac ttaaatatgg
     6061 aactaaccat tccaattttc gccacgaatt ccgactgcga gcttattgtt aaggcaatgc
     6121 aaggtctcct aaaagatgga aacccgattc cctcagcaat cgcagcaaac tccggcatct
     6181 acggtggcgg aggtagcatg tcccatcaga aaattattca ggatcttatc gcatggattg
     6241 acgagcatat tgaccagccg cttaacattg atgtagtcgc aaaaaaatca ggctattcaa
     6301 agtggtactt gcaacgaatg ttccgcacgg tgacgcatca gacgcttggc gattacattc
     6361 gccaacgccg cctgttactg gccgccgttg agttgcgcac caccgagcgt ccgatttttg
     6421 atatcgcaat ggacctgggt tatgtctcgc agcagacctt ctcccgcgtt ttcgcgcggc
     6481 agtttgatcg cactcccgcg gattatcgcc accgcctgta agcggccgcc acgcaaaaaa
     6541 ccccgcttcg gcggggtttt ttcgccctag ggatatattc cgcttcctcg ctcactgact
     6601 cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt
     6661 tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt
     6721 tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg
     6781 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg
     6841 ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc
     6901 tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc
     6961 acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     7021 acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag
     7081 ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc
     7141 gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa
     7201 ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga
     7261 tctcaagaag atcatcttat taatcagata aaatatttct agatttcagt gcaatttatc
     7321 tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtta
//
