LOCUS       sequence-358850-        5579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  KTK compatible Intergration/suicide plasmid. Contains a LacZ dropout
            cassette flanked by regions homolgous to the arsenic resistance 
            cassete. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5579)
  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 5579)
  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_354
FEATURES             Location/Qualifiers
     source          1..5579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     453..472
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(691..795)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     863..881
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(919..941)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     protein_bind    2121..2142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2157..2162,2163..2180,2181..2187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2195..2211
                     /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     2200..2222
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2219..2235
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2219..2235
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2231..2554
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    2248..2304
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2305..2322)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2305..2321)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2314..2336)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      2588..2682
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     protein_bind    2701..2734
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      complement(2765..2799)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(2870..3529)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3444..3463
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    3635..3668
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     regulatory      4128..4137
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     rep_origin      complement(4650..5238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4730..4749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5409..5579,1..621,622..690))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
       61 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      121 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      181 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      241 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      301 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      361 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      421 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      481 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      541 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
      601 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
      661 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
      721 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      781 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
      841 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
      901 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
      961 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     1021 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatattcg
     1081 ccaatgatgg cgcggccctg ctgctgacac cgatcgtcat cgccatcctc acccagcttc
     1141 gcctgagccc cgccgcttcg ctcgccttta ttatcgcgac cggctttgtc gcggatacaa
     1201 ccagcttgcc gctggtaatt tccaatcttg tgaacatcgt cagcgccaat ttcttcgaca
     1261 ttccctttga ccgctacgcc gcgatcatgg tccccgtcga tctggccgcc cttggcgcga
     1321 cacttgccgt gctgtggctc tggtatcggc ggcaggtgcc ccagacctat cccgtcgccg
     1381 aactgccagc acccgccacc gccattcgtg accgccttgt gtttcgcgcg gcttttccgc
     1441 tgctggccct ggtgctggcg gcttatttcc tgaccgcgcc atttcatatt ccggtctgta
     1501 tcgtggcttg cctggctgcc attaccctgc tggcagttgc tggatatgga agggtcattc
     1561 ccatccgtcg ggtgctgcgc gacgccccgt ggcagatcgt gatcttctcg ctggggatgt
     1621 atctggtggt ctacggactg cgcaatgccg ggctgaccga tcatctggcg gcggcactgg
     1681 tctggcttgg ccatcagggt ccgttcaccg ccacgcttgg caccggcttt ctggcggcag
     1741 ccctgtcgtc ggttatgaac aacatgccca gcgttctgat cggcgcgctg gcgatccagc
     1801 aggcgcacga cattacgcct ttgacccgcg atctgatggt ttacgccaac gtcattggct
     1861 gtgacctcgg gccgaaattc acgccaatcg gcagtctcgc caccctgctg tggctgcatg
     1921 tgctggcatc caaaaaccat cgggtcactt gggggcaata catgacggtg ggtctggtga
     1981 tcacgccacc cgtcctgctg gtcacacttg ccgcactggc cttgtggctg cccctgatca
     2041 gccatccctg acaagaccga aggatctgac ggaacgaggt ctctgacccg acctgtcttc
     2101 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     2161 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     2221 ggaaacagct atgaccatga ttacgccaag cttgcatgcc tgcaggtcga ctctagagga
     2281 tccccgggta ccgagctcga attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     2341 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     2401 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     2461 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     2521 cactctcagt acaatctgct ctgatgccgc ataagaagac ggccgaaacg tgagaccact
     2581 agtcttggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
     2641 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaggggt ccccaataat
     2701 gaagttccta ttctctagaa agtataggaa cttcatctgc gctgatttag ctctgaccag
     2761 accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga
     2821 ggtcattact ggatctatca acaggagtcc aagcgagctc gatatcaaat tacgccccgc
     2881 cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat
     2941 cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat
     3001 aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat
     3061 caaaactggt gaaactcacc cagggattgg ctgaaacgaa aaacatattc tcaataaacc
     3121 ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta
     3181 gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct
     3241 catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca
     3301 ttgccatacg aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg
     3361 gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa
     3421 cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat
     3481 gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct
     3541 tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat
     3601 ggtgaaagtt ggaacctctt acgtgcccga tcaagaagtt cctattctct agaaagtata
     3661 ggaacttcca cgcggtgcgc ccgttcgtgc gcatgacggc tacgggcgtc gtctggcccg
     3721 gcggacagga agaagccatt gacgcgatca tctggtgtac aggcttccgt cccgcgctct
     3781 ctgcatttgc accactcgac gtgatcgagc cggacgggct gatccacgtg aacgggcagc
     3841 aggccatcag ggagccacgc ctgtggctgc tgggatatgg ctcttggtgc ggtcccgcat
     3901 ccgcaaccct cctgggggcg ggccgtgtcg cgcgatccat gatccggcaa ctcacggact
     3961 ggcttgcatc accgacacag gatgtccgct taccaaaacc ttgagcgact ctctttacga
     4021 ccgacatgac ggcgactgcc gcctgtctgc tcctacatca ctgagcagac aggcggacta
     4081 gggggggaag ccgaatgtct gcttcagaac tggcctgatc ataaactgcc accatgggag
     4141 cacgtatagt ccttgaattt tccatgagga tttttcagat tctgacacgt gaggatgagg
     4201 ccgcataact atttcgggta atcttccgtc aaatgcagaa tcgactggct gcgaaaaaag
     4261 tcaggagaac taattatcaa acggagaaag tgcttcaata attcggcatt ctgaaatatt
     4321 acctcccccg catgattcta tcatcgtaga aagttcatgg cgcaatgcga tgagatccgc
     4381 aattttgcgc tcaatttctg ccatatggtc acgagcaatt ctatcaactg tcttacaatc
     4441 ctgggttccc tgatccgtta gggataatag agctcttacc tggtctaatg aaaaacccag
     4501 atcccgagat cgacgaataa aagacagtct cgcgagtgcc tcatcatcat aagtgcgata
     4561 attgccatcg gtccgctgtg gcggagccac atgtgagcaa aaggccagca aaaggccagg
     4621 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4681 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4741 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4801 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4861 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4921 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4981 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5041 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5101 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5161 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5221 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5281 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5341 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5401 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5461 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5521 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatc
//
