LOCUS       sequence-308629-        8073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9, MCP-SoxS(R93A/S101A), J106 scRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8073)
  AUTHORS   Fontana J, Dong C, Kiattisewee C, Chavali VP, Tickman BI, Carothers 
            JM, Zalatan JG
  TITLE     Effective CRISPRa-mediated control of gene expression in bacteria 
            must overcome strict target site requirements.
  JOURNAL   Nat Commun. 2020 Apr 1;11(1):1618. doi: 10.1038/s41467-020-15454-y.
  PUBMED    32238808
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: "Nat 
            Commun."; date: "2020-04-1"; volume: "11(1):1618. doi"; pages: " 
            10.1038/s41467-020-15454-y"
COMMENT     Sequence Label: pCK005.6
FEATURES             Location/Qualifiers
     source          1..8073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(170..248)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(544..570,571..573,574..3060,3061..3063,3064..4650)
                     /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      4674..4745
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4761..4788
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4841..4875
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             4885..4896
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     rep_origin      complement(5798..6343)
                     /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(5878..5897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6451..6485
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        6569..6587
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             6610..6639
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6655..6672
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6728..6745)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      6898..6944
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(7162..7256)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7277..7936)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7851..7870
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7937..8039)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 ttacgaaatc atcctgtgga gcttagtagg tttagcaaga tggcagcgcc taaatgtaga
       61 atgataaaag gattaagaga ttaatttccc taaaaatgat aaaacaagcg ttttgaaagc
      121 gcttgttttt ttggtttgca gtcagagtag aatagaagta tcaaaaaaag caccgactcg
      181 gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat gctgttttga
      241 atggttccaa caagattatt ttataacttt tataacaaat aatcaaggag aaattcaaag
      301 aaatttatca gccataaaac aatacttaat actatagaat gataacaaaa taaactactt
      361 tttaaaagaa ttttgtgtta taatctattt attattaagt attgggtaat attttttgaa
      421 gagatatttt gaaaaagaaa aattaaagca tattaaacta atttcggagg tcattaaaac
      481 tattattgaa atcatcaaac tcattatgga tttaatttaa actttttatt ttaggaggca
      541 aaaatggata agaaatactc aataggctta gctatcggca caaatagcgt cggatgggcg
      601 gtgatcactg atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac
      661 cgccacagta tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg
      721 gaagcgactc gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt
      781 tgttatctac aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat
      841 cgacttgaag agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt
      901 ggaaatatag tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga
      961 aaaaaattgg tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg
     1021 catatgatta agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt
     1081 gatgtggaca aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac
     1141 cctattaacg caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca
     1201 agacgattag aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg
     1261 aatctcattg ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca
     1321 gaagatgcta aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg
     1381 gcgcaaattg gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct
     1441 attttacttt cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct
     1501 tcaatgatta aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt
     1561 cgacaacaac ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat
     1621 gcaggttata ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt
     1681 ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg
     1741 cgcaagcaac ggacctttga caacggctct attccccatc aaattcactt gggtgagctg
     1801 catgctattt tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag
     1861 attgaaaaaa tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat
     1921 agtcgttttg catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa
     1981 gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat
     2041 aaaaatcttc caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg
     2101 gtttataacg aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt
     2161 ctttcaggtg aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta
     2221 accgttaagc aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa
     2281 atttcaggag ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa
     2341 attattaaag ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt
     2401 gttttaacat tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat
     2461 gctcacctct ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg
     2521 ggacgtttgt ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata
     2581 ttagattttt tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat
     2641 gatagtttga catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt
     2701 ttacatgaac atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag
     2761 actgtaaaag ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc
     2821 gttattgaaa tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag
     2881 cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat
     2941 cctgttgaaa atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga
     3001 agagacatgt atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat
     3061 gccattgttc cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt
     3121 tctgataaaa atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg
     3181 aaaaactatt ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat
     3241 ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc
     3301 caattggttg aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg
     3361 aatactaaat acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa
     3421 tctaaattag tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac
     3481 aattaccatc atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag
     3541 aaatatccaa aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt
     3601 aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac
     3661 tctaatatca tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa
     3721 cgccctctaa tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat
     3781 tttgccacag tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa
     3841 gtacagacag gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt
     3901 attgctcgta aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta
     3961 gcttattcag tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc
     4021 gttaaagagt tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt
     4081 gactttttag aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct
     4141 aaatatagtc tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa
     4201 ttacaaaaag gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct
     4261 agtcattatg aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg
     4321 gagcagcata agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt
     4381 gttattttag cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac
     4441 aaaccaatac gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga
     4501 gctcccgctg cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca
     4561 aaagaagttt tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc
     4621 attgatttga gtcagctagg aggtgactaa ctcgagtaag gatctccagg catcaaataa
     4681 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     4741 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatacctaga
     4801 gcgcccggaa gagagtcaat tcagggtggt gaatctgcag tttacggcta gctcagccct
     4861 aggtattatg ctagcgaatt cattaaagag gagaaaggta ccatggggcc cgcttctaac
     4921 tttactcagt tcgttctcgt cgacaatggc ggaactggcg acgtgactgt cgccccaagc
     4981 aacttcgcta acgggatcgc tgaatggatc agctctaact cgcgttcaca ggcttacaaa
     5041 gtaacctgta gcgttcgtca gagctctgcg cagaatcgca aatacaccat caaagtcgag
     5101 gtgcctaaag gcgcctggcg ttcgtactta aatatggaac taaccattcc aattttcgcc
     5161 acgaattccg actgcgagct tattgttaag gcaatgcaag gtctcctaaa agatggaaac
     5221 ccgattccct cagcaatcgc agcaaactcc ggcatctacg gtggcggagg tagcatgtcc
     5281 catcagaaaa ttattcagga tcttatcgca tggattgacg agcatattga ccagccgctt
     5341 aacattgatg tagtcgcaaa aaaatcaggc tattcaaagt ggtacttgca acgaatgttc
     5401 cgcacggtga cgcatcagac gcttggcgat tacattcgcc aacgccgcct gttactggcc
     5461 gccgttgagt tgcgcaccac cgagcgtccg atttttgata tcgcaatgga cctgggttat
     5521 gtctcgcagc agaccttctc ccgcgttttc gcgcggcagt ttgatcgcac tcccgcggat
     5581 tatcgccacc gcctgtaagc ggccgccacg caaaaaaccc cgcttcggcg gggttttttc
     5641 gccctaggga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg
     5701 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata
     5761 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg
     5821 acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa
     5881 gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt
     5941 ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt
     6001 tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac
     6061 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca
     6121 ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg
     6181 ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg
     6241 gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc
     6301 gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa
     6361 tcagataaaa tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt
     6421 cagccccata cgatataagt tgttactaga ttgacagcta gctcagtcct aggtataata
     6481 ctagtaggac gcctttggta accgcgtttt agagctagaa atagcaagtt aaaataaggc
     6541 tagtccgtta tcaacttgaa aaagtggcac atgaggatca cccatgtgct ttttttgaag
     6601 cttgggcccg aacaaaaact catctcagaa gaggatctga atagcgccgt cgaccatcat
     6661 catcatcatc attgagttta aacggtctcc agcttggctg ttttggcgga tgagagaaga
     6721 ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc
     6781 ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc
     6841 gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa
     6901 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     6961 aactggatcc ttactcgagt ctagactgca ggcttcctcg ctcactgact cgctgcgctc
     7021 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     7081 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     7141 ccgtaaaaaa ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt
     7201 tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag
     7261 cgagctcgat atcaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat
     7321 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
     7381 gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga
     7441 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
     7501 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
     7561 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc
     7621 agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
     7681 cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga gcattcatca
     7741 ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct
     7801 ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact
     7861 gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag
     7921 tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
     7981 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     8041 cgtctcattt tcgccagata tcgacgtctt aag
//
