LOCUS       sequence-381515-        7368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dCas9-NG 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: pCK668
FEATURES             Location/Qualifiers
     source          1..7368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(220..298)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     terminator      4724..4795
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4811..4838
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4891..4925
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             4935..4946
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(5714..5731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5848..6393)
                     /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(5928..5947)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6507..6601)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6622..7281)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7196..7215
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(7282..7368,1..16))
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 tcttacgtgc cgatcaacgt ctcattttcg ccagatatcg acgtcttaag ttacgaaatc
       61 atcctgtgga gcttagtagg tttagcaaga tggcagcgcc taaatgtaga atgataaaag
      121 gattaagaga ttaatttccc taaaaatgat aaaacaagcg ttttgaaagc gcttgttttt
      181 ttggtttgca gtcagagtag aatagaagta tcaaaaaaag caccgactcg gtgccacttt
      241 ttcaagttga taacggacta gccttatttt aacttgctat gctgttttga atggttccaa
      301 caagattatt ttataacttt tataacaaat aatcaaggag aaattcaaag aaatttatca
      361 gccataaaac aatacttaat actatagaat gataacaaaa taaactactt tttaaaagaa
      421 ttttgtgtta taatctattt attattaagt attgggtaat attttttgaa gagatatttt
      481 gaaaaagaaa aattaaagca tattaaacta atttcggagg tcattaaaac tattattgaa
      541 atcatcaaac tcattatgga tttaatttaa actttttatt ttaggaggca aaaatggata
      601 agaaatactc aataggctta gctatcggca caaatagcgt cggatgggcg gtgatcactg
      661 atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta
      721 tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc
      781 gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tgttatctac
      841 aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag
      901 agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag
      961 tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg
     1021 tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta
     1081 agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca
     1141 aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg
     1201 caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag
     1261 aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg
     1321 ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta
     1381 aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg
     1441 gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt
     1501 cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta
     1561 aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac
     1621 ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat gcaggttata
     1681 ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa
     1741 tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac
     1801 ggacctttga caacggctct attccccatc aaattcactt gggtgagctg catgctattt
     1861 tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa
     1921 tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat agtcgttttg
     1981 catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg
     2041 ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat aaaaatcttc
     2101 caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg
     2161 aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg
     2221 aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc
     2281 aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa atttcaggag
     2341 ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa attattaaag
     2401 ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat
     2461 tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct
     2521 ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggacgtttgt
     2581 ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata ttagattttt
     2641 tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat gatagtttga
     2701 catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac
     2761 atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag
     2821 ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa
     2881 tggcacgtga gaaccaaacc acccagaagg gacagaagaa cagtagggaa aggatgaaga
     2941 ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac ccagttgaaa
     3001 acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc agggacatgt
     3061 acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggac gctatcgtgc
     3121 cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga tccgataaaa
     3181 acagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg aaaaattatt
     3241 ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat ctgactaagg
     3301 ctgaacgagg tggcctgtct gagttggata aagccggttt catcaaaagg cagcttgttg
     3361 agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg aacaccaagt
     3421 acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag tctaagctgg
     3481 tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac aattaccacc
     3541 atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa aaatatccca
     3601 agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg aaaatgatcg
     3661 caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac agcaatatta
     3721 tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag cgaccactta
     3781 tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat ttcgcgacag
     3841 tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa gtacagaccg
     3901 gaggtttctc caaggaaagt atccgtccga aaaggaacag cgacaagctg atcgcacgca
     3961 aaaaagattg ggaccccaag aaatacggcg gattcgtgtc tcctacagtc gcttacagtg
     4021 tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc gtcaaggaac
     4081 tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc gactttctgg
     4141 aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc aagtactctc
     4201 tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgcgtttt ctgcagaaag
     4261 gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc agccactatg
     4321 aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg gaacaacaca
     4381 aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga gtgatcctcg
     4441 ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat aagcccatca
     4501 gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc gcgcctcgtg
     4561 ccttcaagta cttcgacact accatagaca gaaaggtgta ccgctctaca aaggaggtcc
     4621 tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga atcgacctct
     4681 ctcagctcgg tggagactaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc
     4741 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     4801 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatacctaga gcgcccggaa
     4861 gagagtcaat tcagggtggt gaatctgcag tttacggcta gctcagccct aggtattatg
     4921 ctagcgaatt cattaaagag gagaaaggta ccatggggcc cgcttctaac tttactcagt
     4981 tcgttctcgt cgacaatggc ggaactggcg acgtgactgt cgccccaagc aacttcgcta
     5041 acgggatcgc tgaatggatc agctctaact cgcgttcaca ggcttacaaa gtaacctgta
     5101 gcgttcgtca gagctctgcg cagaatcgca aatacaccat caaagtcgag gtgcctaaag
     5161 gcgcctggcg ttcgtactta aatatggaac taaccattcc aattttcgcc acgaattccg
     5221 actgcgagct tattgttaag gcaatgcaag gtctcctaaa agatggaaac ccgattccct
     5281 cagcaatcgc agcaaactcc ggcatctacg gtggcggagg tagcatgtcc catcagaaaa
     5341 ttattcagga tcttatcgca tggattgacg agcatattga ccagccgctt aacattgatg
     5401 tagtcgcaaa aaaatcaggc tattcaaagt ggtacttgca acgaatgttc cgcacggtga
     5461 cgcatcagac gcttggcgat tacattcgcc aacgccgcct gttactggcc gccgttgagt
     5521 tgcgcaccac cgagcgtccg atttttgata tcgcaatgga cctgggttat gtctcgcagc
     5581 agaccttctc ccgcgttttc gcgcggcagt ttgatcgcac tcccgcggat tatcgccacc
     5641 gcctgtaagc ggccgccacg caaaaaaccc cgcttcggcg gggttttttc gccctaggga
     5701 tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg
     5761 aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg
     5821 aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca
     5881 cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc
     5941 gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt
     6001 cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg
     6061 cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct
     6121 tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag
     6181 cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa
     6241 actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag
     6301 ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag
     6361 caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa
     6421 tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata
     6481 cgatataagt tgttactagt gcttggattc tcaccaataa aaaacgcccg gcggcaaccg
     6541 agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt
     6601 ccaagcgagc tcgatatcaa attacgcccc gccctgccac tcatcgcagt actgttgtaa
     6661 ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc
     6721 cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc
     6781 gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt
     6841 ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc
     6901 gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc
     6961 actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac
     7021 actatcccat atcaccagct caccgtcttt cattgccata cgaaattccg gatgagcatt
     7081 catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac
     7141 ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac
     7201 tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata
     7261 tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa
     7321 aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacc
//
