LOCUS       sequence-240457-        8462 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive dCas9 and GFP expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8462)
  AUTHORS   Zhang J, Poh CL
  TITLE     Regulating exopolysaccharide gene wcaF allows control of Escherichia
            coli biofilm formation.
  JOURNAL   Sci Rep. 2018 Sep 3;8(1):13127. doi: 10.1038/s41598-018-31161-7.
  PUBMED    30177768
REFERENCE   2  (bases 1 to 8462)
  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: "Sci Rep."; 
            date: "2018-09-3"; volume: "8(1):13127. doi"; pages: " 
            10.1038/s41598-018-31161-7"
COMMENT     Sequence Label: pD2-dcas9-J23101-GFP
FEATURES             Location/Qualifiers
     source          1..8462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1534..1545
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1552..2265
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1582..1610)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2205..2225
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2305..2376
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2392..2419
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2512..2606)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2627..3286)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3201..3220
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3287..3389)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     RBS             3739..3750
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(3757..3783,3784..3786,3787..6273,6274..6276,
                     6277..7863)
                     /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      7887..7958
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      7974..8001
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(8029..8046)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(8162..8462,1..245))
                     /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(8242..8261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt
       61 tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg
      121 ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac
      181 cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat
      241 ctcaagaaga tcatcttatt aatcagataa aatatttcta gatttcagtg tttacagcta
      301 gctcagtcct agggaaggtg cgaacaagtc cctgatatga gatcatgttt gtcatctgga
      361 gccatagaac agggttcatc atgagtcatc aacttacctt cgccgacagt gaattcagca
      421 gtaagcgccg tcagaccaga aaagagattt tcttgtcccg catggagcag attctgccat
      481 ggcaaaacat ggtggaagtc atcgagccgt tttaccccaa ggctggtaat ggccggcgac
      541 cttatccgct ggaaaccatg ctacgcattc actgcatgca gcattggtac aacctgagcg
      601 atggcgcgat ggaagatgct ctgtacgaaa tcgcctccat gcgtctgttt gcccggttat
      661 ccctggatag cgccttgccg gaccgcacca ccatcatgaa tttccgccac ctgctggagc
      721 agcatcaact ggcccgccaa ttgttcaaga ccatcaatcg ctggctggcc gaagcaggcg
      781 tcatgatgac tcaaggcacc ttggtcgatg ccaccatcat tgaggcaccc agctcgacca
      841 agaacaaaga gcagcaacgc gatccggaga tgcatcagac caagaaaggc aatcagtggc
      901 actttggcat gaaggcccac attggtgtcg atgccaagag tggcctgacc cacagcctgg
      961 tcaccaccgc ggccaacgag catgacctca atcagctggg taatctgctg catggagagg
     1021 agcaatttgt ctcagccgat gccggctacc aaggggcgcc acagcgcgag gagctggccg
     1081 aggtggatgt ggactggctg atcgccgagc gccccggcaa ggtaagaacc ttgaaacagc
     1141 atccacgcaa gaacaaaacg gccatcaaca tcgaatacat gaaagccagc atccgggcca
     1201 gggtggagca cccatttcgc atcatcaagc gacagttcgg cttcgtgaaa gccagataca
     1261 aggggttgct gaaaaacgat aaccaactgg cgatgttatt cacgctggcc aacctgtttc
     1321 gggcggacca aatgatacgt cagtgggaga gatctcacta aaaactgggg ataacgcctt
     1381 aaatggcgaa gaaacggtct aaataggctg attcaaggca tttacgggag aaaaaatcgg
     1441 ctcaaacatg aagaaatgaa atgactgagt cagccgagaa gaatttcccc gcttattcgc
     1501 accttcccta ggtattatgc tagctgtcta gagaaagagg agaaatacta gatgcgtaaa
     1561 ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat
     1621 gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc
     1681 cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact
     1741 ttcggttatg gtgttcaatg ctttgcgaga tacccagatc atatgaaaca gcatgacttt
     1801 ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac
     1861 gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc
     1921 gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac
     1981 aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtt
     2041 aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa
     2101 caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca
     2161 caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt
     2221 gtaacagctg ctgggattac acatggcatg gatgaactat acaaataata aaggatccaa
     2281 actcgagtaa ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     2341 tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct
     2401 tcgggtgggc ctttctgcgt ttatacctag ggatatatta gtgcaattta tctcttcaaa
     2461 tgtagcacct gaagtcagcc ccatacgata taagttgtta ctagtgcttg gattctcacc
     2521 aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt
     2581 cattactgga tctatcaaca ggagtccaag cgagctcgat atcaaattac gccccgccct
     2641 gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac
     2701 aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat
     2761 atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa
     2821 aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt
     2881 tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa
     2941 actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat
     3001 ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg
     3061 ccatacgaaa ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat
     3121 aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg
     3181 tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc
     3241 attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag
     3301 ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt
     3361 gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccagata tcgacgtctt
     3421 aagacccact ttcacattta agttgttttt ctaatccgca tatgatcaat tcaaggccga
     3481 ataagaaggc tggctctgca ccttggtgat caaataattc gatagcttgt cgtaataatg
     3541 gcggcatact atcagtagta ggtgtttccc tttcttcttt agcgacttga tgctcttgat
     3601 cttccaatac gcaacctaaa gtaaaatgcc ccacagcgct gagattttgg gctcgtcggg
     3661 gattcgccgg gtgctgcaaa accatctacg ctcaggactg ggcgagatga aaaactcgct
     3721 gaagaattca aaagatctaa agaggagaaa ggatctatgg ataagaaata ctcaataggc
     3781 ttagctatcg gcacaaatag cgtcggatgg gcggtgatca ctgatgaata taaggttccg
     3841 tctaaaaagt tcaaggttct gggaaataca gaccgccaca gtatcaaaaa aaatcttata
     3901 ggggctcttt tatttgacag tggagagaca gcggaagcga ctcgtctcaa acggacagct
     3961 cgtagaaggt atacacgtcg gaagaatcgt atttgttatc tacaggagat tttttcaaat
     4021 gagatggcga aagtagatga tagtttcttt catcgacttg aagagtcttt tttggtggaa
     4081 gaagacaaga agcatgaacg tcatcctatt tttggaaata tagtagatga agttgcttat
     4141 catgagaaat atccaactat ctatcatctg cgaaaaaaat tggtagattc tactgataaa
     4201 gcggatttgc gcttaatcta tttggcctta gcgcatatga ttaagtttcg tggtcatttt
     4261 ttgattgagg gagatttaaa tcctgataat agtgatgtgg acaaactatt tatccagttg
     4321 gtacaaacct acaatcaatt atttgaagaa aaccctatta acgcaagtgg agtagatgct
     4381 aaagcgattc tttctgcacg attgagtaaa tcaagacgat tagaaaatct cattgctcag
     4441 ctccccggtg agaagaaaaa tggcttattt gggaatctca ttgctttgtc attgggtttg
     4501 acccctaatt ttaaatcaaa ttttgatttg gcagaagatg ctaaattaca gctttcaaaa
     4561 gatacttacg atgatgattt agataattta ttggcgcaaa ttggagatca atatgctgat
     4621 ttgtttttgg cagctaagaa tttatcagat gctattttac tttcagatat cctaagagta
     4681 aatactgaaa taactaaggc tcccctatca gcttcaatga ttaaacgcta cgatgaacat
     4741 catcaagact tgactctttt aaaagcttta gttcgacaac aacttccaga aaagtataaa
     4801 gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt atattgatgg gggagctagc
     4861 caagaagaat tttataaatt tatcaaacca attttagaaa aaatggatgg tactgaggaa
     4921 ttattggtga aactaaatcg tgaagatttg ctgcgcaagc aacggacctt tgacaacggc
     4981 tctattcccc atcaaattca cttgggtgag ctgcatgcta ttttgagaag acaagaagac
     5041 ttttatccat ttttaaaaga caatcgtgag aagattgaaa aaatcttgac ttttcgaatt
     5101 ccttattatg ttggtccatt ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag
     5161 tctgaagaaa caattacccc atggaatttt gaagaagttg tcgataaagg tgcttcagct
     5221 caatcattta ttgaacgcat gacaaacttt gataaaaatc ttccaaatga aaaagtacta
     5281 ccaaaacata gtttgcttta tgagtatttt acggtttata acgaattgac aaaggtcaaa
     5341 tatgttactg aaggaatgcg aaaaccagca tttctttcag gtgaacagaa gaaagccatt
     5401 gttgatttac tcttcaaaac aaatcgaaaa gtaaccgtta agcaattaaa agaagattat
     5461 ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag gagttgaaga tagatttaat
     5521 gcttcattag gtacctacca tgatttgcta aaaattatta aagataaaga ttttttggat
     5581 aatgaagaaa atgaagatat cttagaggat attgttttaa cattgacctt atttgaagat
     5641 agggagatga ttgaggaaag acttaaaaca tatgctcacc tctttgatga taaggtgatg
     5701 aaacagctta aacgtcgccg ttatactggt tggggacgtt tgtctcgaaa attgattaat
     5761 ggtattaggg ataagcaatc tggcaaaaca atattagatt ttttgaaatc agatggtttt
     5821 gccaatcgca attttatgca gctgatccat gatgatagtt tgacatttaa agaagacatt
     5881 caaaaagcac aagtgtctgg acaaggcgat agtttacatg aacatattgc aaatttagct
     5941 ggtagccctg ctattaaaaa aggtatttta cagactgtaa aagttgttga tgaattggtc
     6001 aaagtaatgg ggcggcataa gccagaaaat atcgttattg aaatggcacg tgaaaatcag
     6061 acaactcaaa agggccagaa aaattcgcga gagcgtatga aacgaatcga agaaggtatc
     6121 aaagaattag gaagtcagat tcttaaagag catcctgttg aaaatactca attgcaaaat
     6181 gaaaagctct atctctatta tctccaaaat ggaagagaca tgtatgtgga ccaagaatta
     6241 gatattaatc gtttaagtga ttatgatgtc gatgccattg ttccacaaag tttccttaaa
     6301 gacgattcaa tagacaataa ggtcttaacg cgttctgata aaaatcgtgg taaatcggat
     6361 aacgttccaa gtgaagaagt agtcaaaaag atgaaaaact attggagaca acttctaaac
     6421 gccaagttaa tcactcaacg taagtttgat aatttaacga aagctgaacg tggaggtttg
     6481 agtgaacttg ataaagctgg ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact
     6541 aagcatgtgg cacaaatttt ggatagtcgc atgaatacta aatacgatga aaatgataaa
     6601 cttattcgag aggttaaagt gattacctta aaatctaaat tagtttctga cttccgaaaa
     6661 gatttccaat tctataaagt acgtgagatt aacaattacc atcatgccca tgatgcgtat
     6721 ctaaatgccg tcgttggaac tgctttgatt aagaaatatc caaaacttga atcggagttt
     6781 gtctatggtg attataaagt ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa
     6841 ataggcaaag caaccgcaaa atatttcttt tactctaata tcatgaactt cttcaaaaca
     6901 gaaattacac ttgcaaatgg agagattcgc aaacgccctc taatcgaaac taatggggaa
     6961 actggagaaa ttgtctggga taaagggcga gattttgcca cagtgcgcaa agtattgtcc
     7021 atgccccaag tcaatattgt caagaaaaca gaagtacaga caggcggatt ctccaaggag
     7081 tcaattttac caaaaagaaa ttcggacaag cttattgctc gtaaaaaaga ctgggatcca
     7141 aaaaaatatg gtggttttga tagtccaacg gtagcttatt cagtcctagt ggttgctaag
     7201 gtggaaaaag ggaaatcgaa gaagttaaaa tccgttaaag agttactagg gatcacaatt
     7261 atggaaagaa gttcctttga aaaaaatccg attgactttt tagaagctaa aggatataag
     7321 gaagttaaaa aagacttaat cattaaacta cctaaatata gtctttttga gttagaaaac
     7381 ggtcgtaaac ggatgctggc tagtgccgga gaattacaaa aaggaaatga gctggctctg
     7441 ccaagcaaat atgtgaattt tttatattta gctagtcatt atgaaaagtt gaagggtagt
     7501 ccagaagata acgaacaaaa acaattgttt gtggagcagc ataagcatta tttagatgag
     7561 attattgagc aaatcagtga attttctaag cgtgttattt tagcagatgc caatttagat
     7621 aaagttctta gtgcatataa caaacataga gacaaaccaa tacgtgaaca agcagaaaat
     7681 attattcatt tatttacgtt gacgaatctt ggagctcccg ctgcttttaa atattttgat
     7741 acaacaattg atcgtaaacg atatacgtct acaaaagaag ttttagatgc cactcttatc
     7801 catcaatcca tcactggtct ttatgaaaca cgcattgatt tgagtcagct aggaggtgac
     7861 taactcgagt aaggatctcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     7921 cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac
     7981 cttcgggtgg gcctttctgc gtttatacct agggatatat tccgcttcct cgctcactga
     8041 ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg gcttacgaac ggggcggaga
     8101 tttcctggaa gatgccagga agatacttaa cagggaagtg agagggccgc ggcaaagccg
     8161 ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt
     8221 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc
     8281 gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt
     8341 ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg
     8401 cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     8461 aa
//
