LOCUS       sequence-359542-        7636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 KTK compatible plasmid. Contains dCas9 and lacZ dropout region
            with flanking D1.1 overhangs for insertion of gRNA expression 
            assembly.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7636)
  AUTHORS   Goosens VJ, Walker KT, Aragon SM, Singh A, Senthivel VR, Dekker L, 
            Caro-Astorga J, Buat MLA, Song W, Lee KY, Ellis T
  TITLE     Komagataeibacter Tool Kit (KTK): A Modular Cloning System for 
            Multigene Constructs and Programmed Protein Secretion from Cellulose
            Producing Bacteria.
  JOURNAL   ACS Synth Biol. 2021 Dec 17;10(12):3422-3434. doi: 
            10.1021/acssynbio.1c00358. Epub 2021 Nov 12.
  PUBMED    34767345
REFERENCE   2  (bases 1 to 7636)
  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"; doi: 
            "10.1021/acssynbio.1c00358"; journalName: "ACS Synth Biol"; date: 
            "2021-12-17- 17"; volume: "10"; issue: "12"; pages: "3422-3434"
COMMENT     Sequence Label: KTK_095
FEATURES             Location/Qualifiers
     source          1..7636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    25..46
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(61..66,67..84,85..91)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    99..115
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     104..126
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     123..139
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     123..139
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             135..458
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    152..208
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(209..226)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(209..225)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(218..240)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(434..453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     terminator      492..586
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        634..736
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             737..1396
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(803..822)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     oriT            1545..1653
                     /note="incP origin of transfer"
     CDS             complement(1664..2326)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      2327..3098
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(3213..3299)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        3312..3346
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     promoter        3312..3346
                     /label=BBa_J23118
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.56"
     CDS             join(3382..3408,3409..3411,3412..5898,5899..5901,
                     5902..7488)
                     /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      7512..7583
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gaccggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
       61 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      121 cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagg tcgactctag
      181 aggatccccg ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg
      241 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
      301 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
      361 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
      421 ggtgcactct cagtacaatc tgctctgatg ccgcataagg tctcaaacgc cctgggtctt
      481 cactagtctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt
      541 gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccag gggtccccaa
      601 taattacgat ttaaattggc gaaaatgaga cgttgatcgg cacgtaagag gttccaactt
      661 tcaccataat gaaataagat cactaccggg cgtatttttt gagttatcga gattttcagg
      721 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca
      781 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca
      841 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt
      901 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aatttcgtat
      961 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt
     1021 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
     1081 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
     1141 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag
     1201 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
     1261 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
     1321 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
     1381 gcagggcggg gcgtaatttg acttttgtcc ttttccgctg cataaccctg cttcggggtc
     1441 attatagcga ttttttcggt atatccatcc tttttcgcac gatatacagg attttgccaa
     1501 agggttcgtg tagactttcc ttggtgtatc caacggcgtc agccgggcag gataggtgaa
     1561 gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc cttattcgca cctggcggtg
     1621 ctcaacggga atcctgctct gcgaggctgg ccgtaggccg gccctaccgg cgcggcagcg
     1681 ttacccgtgt cggcggctcc aacggctcgc catcgtccag aaaacacggc tcatcgggca
     1741 tcggcaggcg ctgctgcccg cgccgttccc attcctccgt ttcggtcaag gctggcaggt
     1801 ctggttccat gcccggaatg ccgggctggc tgggcggctc ctcgccgggg ccggtcggta
     1861 gttgctgctc gcccggatac agggtcggga tgcggcgcag gtcgccatgc cccaacagcg
     1921 attcgtcctg gtcgtcgtga tcaaccacca cggcggcact gaacaccgac aggcgcaact
     1981 ggtcgcgggg ctggccccac gccacgcggt cattgaccac gtaggccgac acggtgccgg
     2041 ggccgttgag cttcacgacg gagatccagc gctcggccac caagtccttg actgcgtatt
     2101 ggaccgtccg caaagaacgt ccgatgagct tggaaagtgt tttctggctg accaccacgg
     2161 cgttctggtg gcccatctgc gccacgaggt gatgcagcag cattgccgcc gtgggtttcc
     2221 tcgcaataag cccggcccac gcctcatgcg ctttgcgttc cgtttgcacc cagtgaccgg
     2281 gcttgttctt ggcttgaatg ccgatttctc tggactgcgt ggccatgctt atctccatgc
     2341 ggtaggggtg ccgcacggtt gcggcaccat gcgcaatcag ctgcaacttt tcggcagcgc
     2401 gacaacaatt atgcgttgcg taaaagtggc agtcaattac agattttctt taacctacgc
     2461 aatgagctat tgcggggggt gccgcaatga gctgttgcgt accccccttt tttaagttgt
     2521 tgatttttaa gtctttcgca tttcgcccta tatctagttc tttggtgccc aaagaagggc
     2581 acccctgcgg ggttccccca cgccttcggc gcggctcccc ctccggcaaa aagtggcccc
     2641 tccggggctt gttgatcgac tgcgcggcct tcggccttgc ccaaggtggc gctgccccct
     2701 tggaaccccc gcactcgccg ccgtgaggct cggggggcag gcgggcgggc ttcgcccttc
     2761 gactgccccc actcgcatag gcttgggtcg ttccaggcgc gtcaaggcca agccgctgcg
     2821 cggtcgctgc gcgagccttg acccgccttc cacttggtgt ccaaccggca agcgaagcgc
     2881 gcaggccgca ggccggaggc ttttccccag agaaaattaa aaaaattgat ggggcaaggc
     2941 cgcaggccgc gcagttggag ccggtgggta tgtggtcgaa ggctgggtag ccggtgggca
     3001 atccctgtgg tcaagctcgt gggcaggcgc agcctgtcca tcagcttgtc cagcagggtt
     3061 gtccacgggc cgagcgaagc gagccagccg gtggccgctc gcggccatcg tccacatatc
     3121 cacgggctgg caagggagcg cagcgaccgc gcagggcgaa gcccggagag caagcccgta
     3181 gggggggcgc gcccagctgt ctagggcggc ggatttgtcc tactcaggag agcgttcacc
     3241 gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga
     3301 tgcctctcga gtttacggct agctcagtcc taggtattgt gctagcaaga attcaaaagc
     3361 tctacagagg agaaaggata tatggataag aaatactcaa taggcttagc tatcggcaca
     3421 aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa aaagttcaag
     3481 gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc tcttttattt
     3541 gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag aaggtataca
     3601 cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat ggcgaaagta
     3661 gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga caagaagcat
     3721 gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga gaaatatcca
     3781 actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga tttgcgctta
     3841 atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat tgagggagat
     3901 ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca aacctacaat
     3961 caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc gattctttct
     4021 gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc cggtgagaag
     4081 aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc taattttaaa
     4141 tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac ttacgatgat
     4201 gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt tttggcagct
     4261 aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac tgaaataact
     4321 aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca agacttgact
     4381 cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat cttttttgat
     4441 caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga agaattttat
     4501 aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt ggtgaaacta
     4561 aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat tccccatcaa
     4621 attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta tccattttta
     4681 aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta ttatgttggt
     4741 ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga agaaacaatt
     4801 accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc atttattgaa
     4861 cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa acatagtttg
     4921 ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt tactgaagga
     4981 atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga tttactcttc
     5041 aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa aaaaatagaa
     5101 tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc attaggtacc
     5161 taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga agaaaatgaa
     5221 gatatcttag aggatattgt tttaacattg accttatttg aagataggga gatgattgag
     5281 gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca gcttaaacgt
     5341 cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat tagggataag
     5401 caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa tcgcaatttt
     5461 atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa agcacaagtg
     5521 tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag ccctgctatt
     5581 aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt aatggggcgg
     5641 cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac tcaaaagggc
     5701 cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga attaggaagt
     5761 cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa gctctatctc
     5821 tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat taatcgttta
     5881 agtgattatg atgtcgatgc cattgttcca caaagtttcc ttaaagacga ttcaatagac
     5941 aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt tccaagtgaa
     6001 gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa gttaatcact
     6061 caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga acttgataaa
     6121 gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca tgtggcacaa
     6181 attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat tcgagaggtt
     6241 aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt ccaattctat
     6301 aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa tgccgtcgtt
     6361 ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta tggtgattat
     6421 aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg caaagcaacc
     6481 gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat tacacttgca
     6541 aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg agaaattgtc
     6601 tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc ccaagtcaat
     6661 attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat tttaccaaaa
     6721 agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa atatggtggt
     6781 tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga aaaagggaaa
     6841 tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga aagaagttcc
     6901 tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt taaaaaagac
     6961 ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg taaacggatg
     7021 ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag caaatatgtg
     7081 aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga agataacgaa
     7141 caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat tgagcaaatc
     7201 agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt tcttagtgca
     7261 tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat tcatttattt
     7321 acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac aattgatcgt
     7381 aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca atccatcact
     7441 ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgactaact cgagtaagga
     7501 tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     7561 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtttaagaa
     7621 gacggcgacg acctga
//
