LOCUS       sequence-486136-        7638 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Constitutive expression of dCas9 and PspF-lambda-N22.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7638)
  AUTHORS   Kiattisewee C, Karanjia AV, Cardiff RAL, Olander KE, Leejareon P, 
            Alvi SS, Carothers JM, Zalatan JG
  TITLE     Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene
            Activation Reveals Antagonistic Effects.
  JOURNAL   ACS Synth Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358.
  PUBMED    40693287
REFERENCE   2  (bases 1 to 7638)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358."
COMMENT     Sequence Label: pCK071
FEATURES             Location/Qualifiers
     source          1..7638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      3925..3996
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4012..4039
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4092..4126
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             4136..4147
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(5185..5202)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5319..5864)
                     /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(5399..5418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5978..6072)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6093..6752)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6667..6686
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6753..6855)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(7059..7137)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(7433..7459,7460..7462,7463..7638,1..2311,2312..2314,
                     2315..3901)
                     /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"
ORIGIN
        1 tagaaggtat acacgtcgga agaatcgtat ttgttatcta caggagattt tttcaaatga
       61 gatggcgaaa gtagatgata gtttctttca tcgacttgaa gagtcttttt tggtggaaga
      121 agacaagaag catgaacgtc atcctatttt tggaaatata gtagatgaag ttgcttatca
      181 tgagaaatat ccaactatct atcatctgcg aaaaaaattg gtagattcta ctgataaagc
      241 ggatttgcgc ttaatctatt tggccttagc gcatatgatt aagtttcgtg gtcatttttt
      301 gattgaggga gatttaaatc ctgataatag tgatgtggac aaactattta tccagttggt
      361 acaaacctac aatcaattat ttgaagaaaa ccctattaac gcaagtggag tagatgctaa
      421 agcgattctt tctgcacgat tgagtaaatc aagacgatta gaaaatctca ttgctcagct
      481 ccccggtgag aagaaaaatg gcttatttgg gaatctcatt gctttgtcat tgggtttgac
      541 ccctaatttt aaatcaaatt ttgatttggc agaagatgct aaattacagc tttcaaaaga
      601 tacttacgat gatgatttag ataatttatt ggcgcaaatt ggagatcaat atgctgattt
      661 gtttttggca gctaagaatt tatcagatgc tattttactt tcagatatcc taagagtaaa
      721 tactgaaata actaaggctc ccctatcagc ttcaatgatt aaacgctacg atgaacatca
      781 tcaagacttg actcttttaa aagctttagt tcgacaacaa cttccagaaa agtataaaga
      841 aatctttttt gatcaatcaa aaaacggata tgcaggttat attgatgggg gagctagcca
      901 agaagaattt tataaattta tcaaaccaat tttagaaaaa atggatggta ctgaggaatt
      961 attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg acaacggctc
     1021 tattccccat caaattcact tgggtgagct gcatgctatt ttgagaagac aagaagactt
     1081 ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc
     1141 ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc
     1201 tgaagaaaca attaccccat ggaattttga agaagttgtc gataaaggtg cttcagctca
     1261 atcatttatt gaacgcatga caaactttga taaaaatctt ccaaatgaaa aagtactacc
     1321 aaaacatagt ttgctttatg agtattttac ggtttataac gaattgacaa aggtcaaata
     1381 tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga aagccattgt
     1441 tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag aagattattt
     1501 caaaaaaata gaatgttttg atagtgttga aatttcagga gttgaagata gatttaatgc
     1561 ttcattaggt acctaccatg atttgctaaa aattattaaa gataaagatt ttttggataa
     1621 tgaagaaaat gaagatatct tagaggatat tgttttaaca ttgaccttat ttgaagatag
     1681 ggagatgatt gaggaaagac ttaaaacata tgctcacctc tttgatgata aggtgatgaa
     1741 acagcttaaa cgtcgccgtt atactggttg gggacgtttg tctcgaaaat tgattaatgg
     1801 tattagggat aagcaatctg gcaaaacaat attagatttt ttgaaatcag atggttttgc
     1861 caatcgcaat tttatgcagc tgatccatga tgatagtttg acatttaaag aagacattca
     1921 aaaagcacaa gtgtctggac aaggcgatag tttacatgaa catattgcaa atttagctgg
     1981 tagccctgct attaaaaaag gtattttaca gactgtaaaa gttgttgatg aattggtcaa
     2041 agtaatgggg cggcataagc cagaaaatat cgttattgaa atggcacgtg aaaatcagac
     2101 aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa
     2161 agaattagga agtcagattc ttaaagagca tcctgttgaa aatactcaat tgcaaaatga
     2221 aaagctctat ctctattatc tccaaaatgg aagagacatg tatgtggacc aagaattaga
     2281 tattaatcgt ttaagtgatt atgatgtcga tgccattgtt ccacaaagtt tccttaaaga
     2341 cgattcaata gacaataagg tcttaacgcg ttctgataaa aatcgtggta aatcggataa
     2401 cgttccaagt gaagaagtag tcaaaaagat gaaaaactat tggagacaac ttctaaacgc
     2461 caagttaatc actcaacgta agtttgataa tttaacgaaa gctgaacgtg gaggtttgag
     2521 tgaacttgat aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa
     2581 gcatgtggca caaattttgg atagtcgcat gaatactaaa tacgatgaaa atgataaact
     2641 tattcgagag gttaaagtga ttaccttaaa atctaaatta gtttctgact tccgaaaaga
     2701 tttccaattc tataaagtac gtgagattaa caattaccat catgcccatg atgcgtatct
     2761 aaatgccgtc gttggaactg ctttgattaa gaaatatcca aaacttgaat cggagtttgt
     2821 ctatggtgat tataaagttt atgatgttcg taaaatgatt gctaagtctg agcaagaaat
     2881 aggcaaagca accgcaaaat atttctttta ctctaatatc atgaacttct tcaaaacaga
     2941 aattacactt gcaaatggag agattcgcaa acgccctcta atcgaaacta atggggaaac
     3001 tggagaaatt gtctgggata aagggcgaga ttttgccaca gtgcgcaaag tattgtccat
     3061 gccccaagtc aatattgtca agaaaacaga agtacagaca ggcggattct ccaaggagtc
     3121 aattttacca aaaagaaatt cggacaagct tattgctcgt aaaaaagact gggatccaaa
     3181 aaaatatggt ggttttgata gtccaacggt agcttattca gtcctagtgg ttgctaaggt
     3241 ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag ttactaggga tcacaattat
     3301 ggaaagaagt tcctttgaaa aaaatccgat tgacttttta gaagctaaag gatataagga
     3361 agttaaaaaa gacttaatca ttaaactacc taaatatagt ctttttgagt tagaaaacgg
     3421 tcgtaaacgg atgctggcta gtgccggaga attacaaaaa ggaaatgagc tggctctgcc
     3481 aagcaaatat gtgaattttt tatatttagc tagtcattat gaaaagttga agggtagtcc
     3541 agaagataac gaacaaaaac aattgtttgt ggagcagcat aagcattatt tagatgagat
     3601 tattgagcaa atcagtgaat tttctaagcg tgttatttta gcagatgcca atttagataa
     3661 agttcttagt gcatataaca aacatagaga caaaccaata cgtgaacaag cagaaaatat
     3721 tattcattta tttacgttga cgaatcttgg agctcccgct gcttttaaat attttgatac
     3781 aacaattgat cgtaaacgat atacgtctac aaaagaagtt ttagatgcca ctcttatcca
     3841 tcaatccatc actggtcttt atgaaacacg cattgatttg agtcagctag gaggtgacta
     3901 actcgagtaa ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     3961 tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct
     4021 tcgggtgggc ctttctgcgt ttatacctag agcgcccgga agagagtcaa ttcagggtgg
     4081 tgaatctgca gtttacggct agctcagccc taggtattat gctagcgaat tcattaaaga
     4141 ggagaaaggt accatggggc ccatggcaga atacaaagat aatttacttg gtgaggcgaa
     4201 cagctttctc gaagtgctgg aacaggtttc gcatctcgca ccgctggaca aaccggtgct
     4261 catcatcggc gaacgcggca ccggtaaaga gctgattgcc agccgcctgc attatctctc
     4321 ctcccgttgg caagggccgt ttatttccct taactgcgcg gcgttaaatg aaaatctgct
     4381 ggattccgaa ctgtttggtc acgaagcggg ggcgtttacc ggtgcgcaaa aacgtcatcc
     4441 agggagattt gaacgtgccg acggcggtac gctatttctt gatgaactcg ctacggcacc
     4501 gatgatggtg caggagaaat tattgcgcgt gattgagtac ggtgaactgg agcgcgttgg
     4561 cggcagccaa ccattgcagg tgaatgtgcg gttggtatgc gcgacgaatg ccgatctccc
     4621 ggcgatggtc aatgaaggca cttttcgcgc tgacctgctc gaccgactgg cttttgatgt
     4681 tgtacaactg ccaccactgc gcgagcgcga aagcgacata atgttgatgg cagaatactt
     4741 tgccatccag atgtgtcggg aaatcaagct gcctctgttc ccggggttta cggagcgcgc
     4801 cagagaaaca ttgctgaatt atcgttggcc gggaaatatt cgtgaattga aaaacgtggt
     4861 ggaacgttca gtgtatcgcc acggcaccag cgattatccg cttgatgaca tcattattga
     4921 tccctttaaa cggcgtccgc ctgaagacgc tatcgccgtt tcagaaacca cctcgcttcc
     4981 aacactgccg ctggatttac gtgagtttca gatgcagcag gaaaaagagt tgctgcaact
     5041 cagtttgcaa atgaatgcac gcacacgccg ccgcgaacgt cgcgcagaga aacaggctca
     5101 atggaaagca gcaaattaag cggccgccac gcaaaaaacc ccgcttcggc ggggtttttt
     5161 cgccctaggg atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact
     5221 gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat
     5281 acttaacagg gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct
     5341 gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa
     5401 agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg
     5461 tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag
     5521 ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga
     5581 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc
     5641 accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg
     5701 gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc
     5761 ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt
     5821 cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta
     5881 atcagataaa atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     5941 tcagccccat acgatataag ttgttactag tgcttggatt ctcaccaata aaaaacgccc
     6001 ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta
     6061 tcaacaggag tccaagcgag ctcgatatca aattacgccc cgccctgcca ctcatcgcag
     6121 tactgttgta attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga
     6181 acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg
     6241 aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc
     6301 acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc
     6361 aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg
     6421 tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa
     6481 caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat acgaaattcc
     6541 ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta
     6601 tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta
     6661 cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca
     6721 acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc
     6781 gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct
     6841 cttacgtgcc gatcaacgtc tcattttcgc cagatatcga cgtcttaagt tacgaaatca
     6901 tcctgtggag cttagtaggt ttagcaagat ggcagcgcct aaatgtagaa tgataaaagg
     6961 attaagagat taatttccct aaaaatgata aaacaagcgt tttgaaagcg cttgtttttt
     7021 tggtttgcag tcagagtaga atagaagtat caaaaaaagc accgactcgg tgccactttt
     7081 tcaagttgat aacggactag ccttatttta acttgctatg ctgttttgaa tggttccaac
     7141 aagattattt tataactttt ataacaaata atcaaggaga aattcaaaga aatttatcag
     7201 ccataaaaca atacttaata ctatagaatg ataacaaaat aaactacttt ttaaaagaat
     7261 tttgtgttat aatctattta ttattaagta ttgggtaata ttttttgaag agatattttg
     7321 aaaaagaaaa attaaagcat attaaactaa tttcggaggt cattaaaact attattgaaa
     7381 tcatcaaact cattatggat ttaatttaaa ctttttattt taggaggcaa aaatggataa
     7441 gaaatactca ataggcttag ctatcggcac aaatagcgtc ggatgggcgg tgatcactga
     7501 tgaatataag gttccgtcta aaaagttcaa ggttctggga aatacagacc gccacagtat
     7561 caaaaaaaat cttatagggg ctcttttatt tgacagtgga gagacagcgg aagcgactcg
     7621 tctcaaacgg acagctcg
//
