LOCUS       sequence-358828-        3538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone for D2.2b cloning, contains LacZ dropout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3538)
  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 3538)
  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_016
FEATURES             Location/Qualifiers
     source          1..3538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      239..1010
                     /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(1125..1211)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    1269..1290
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1305..1310,1311..1328,1329..1335)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1343..1359
                     /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     1348..1370
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1367..1383
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1367..1383
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1379..1702
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1396..1452
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1453..1470)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1453..1469)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1462..1484)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1678..1697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     terminator      1736..1830
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1862..2653)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     oriT            2995..3103
                     /note="incP origin of transfer"
     CDS             complement(join(3114..3538,1..238))
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
ORIGIN
        1 tgactgcgta ttggaccgtc cgcaaagaac gtccgatgag cttggaaagt gttttctggc
       61 tgaccaccac ggcgttctgg tggcccatct gcgccacgag gtgatgcagc agcattgccg
      121 ccgtgggttt cctcgcaata agcccggccc acgcctcatg cgctttgcgt tccgtttgca
      181 cccagtgacc gggcttgttc ttggcttgaa tgccgatttc tctggactgc gtggccatgc
      241 ttatctccat gcggtagggg tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact
      301 tttcggcagc gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc
      361 tttaacctac gcaatgagct attgcggggg gtgccgcaat gagctgttgc gtacccccct
      421 tttttaagtt gttgattttt aagtctttcg catttcgccc tatatctagt tctttggtgc
      481 ccaaagaagg gcacccctgc ggggttcccc cacgccttcg gcgcggctcc ccctccggca
      541 aaaagtggcc cctccggggc ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg
      601 gcgctgcccc cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg
      661 gcttcgccct tcgactgccc ccactcgcat aggcttgggt cgttccaggc gcgtcaaggc
      721 caagccgctg cgcggtcgct gcgcgagcct tgacccgcct tccacttggt gtccaaccgg
      781 caagcgaagc gcgcaggccg caggccggag gcttttcccc agagaaaatt aaaaaaattg
      841 atggggcaag gccgcaggcc gcgcagttgg agccggtggg tatgtggtcg aaggctgggt
      901 agccggtggg caatccctgt ggtcaagctc gtgggcaggc gcagcctgtc catcagcttg
      961 tccagcaggg ttgtccacgg gccgagcgaa gcgagccagc cggtggccgc tcgcggccat
     1021 cgtccacata tccacgggct ggcaagggag cgcagcgacc gcgcagggcg aagcccggag
     1081 agcaagcccg tagggggggc gcgcccagct gtctagggcg gcggatttgt cctactcagg
     1141 agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga ctgagccttt
     1201 cgttttattt gatgccttta attaaggtct cgtatgcgac gggtcttcgg cagtgagcgc
     1261 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     1321 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     1381 gaccatgatt acgccaagct tgcatgcctg caggtcgact ctagaggatc cccgggtacc
     1441 gagctcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     1501 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     1561 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     1621 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac
     1681 aatctgctct gatgccgcat aagaagacgg ccgaaacgtg agaccactag tcttggactc
     1741 ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt
     1801 tgccgccggg cgttttttat tggtgagaat ccaggggtcc ccaataatta cgatttttgt
     1861 cttatttgcc gactaccttg gtgatctcgc ctttcacgta gtggacaaat tcttccaact
     1921 gatctgcgcg cgaggccaag cgatcttctt cttgtccaag ataagcctgt ctagcttcaa
     1981 gtatgacggg ctgatactgg gccggcaggc gctccattgc ccagtcggca gcgacatcct
     2041 tcggcgcgat tttgccggtt actgcgctgt accaaatgcg ggacaacgta agcactacat
     2101 ttcgctcatc gccagcccag tcgggcggcg agttccatag cgttaaggtt tcatttagcg
     2161 cctcaaatag atcctgttca ggaaccggat caaagagttc ctccgccgct ggacctacca
     2221 aggcaacgct atgttctctt gcttttgtca gcaagatagc cagatcaatg tcgatcgtgg
     2281 ctggctcgaa gatacctgca agaatgtcat tgcgctgcca ttctccaaat tgcagttcgc
     2341 gcttagctgg ataacgccac ggaatgatgt cgtcgtgcac aacaatggtg acttctacag
     2401 cgcggagaat ctcgctctct ccaggggaag ccgaagtttc caaaaggtcg ttgatcaaag
     2461 ctcgccgcgt tgtttcatca agccttacgg tcaccgtaac cagcaaatca atatcactgt
     2521 gtggcttcag gccgccatcc actgcggagc cgtacaaatg tacggccagc aacgtcggtt
     2581 cgagatggcg ctcgatgacg ccaactacct ctgatagttg agtcgatact tcggcgatca
     2641 ccgcttccct catgatgttt aactttgttt tagggcgact gccctgctgc gtaacatcgt
     2701 tgctgctcca taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc
     2761 gaggcataga ctgtacccca aaaaaacagt cataacaagc catgaaaacc gccactgcgc
     2821 cgttaccacc gctgcgttcg gtcaaggttc atttttgtcc ttttccgctg cataaccctg
     2881 cttcggggtc attatagcga ttttttcggt atatccatcc tttttcgcac gatatacagg
     2941 attttgccaa agggttcgtg tagactttcc ttggtgtatc caacggcgtc agccgggcag
     3001 gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc cttattcgca
     3061 cctggcggtg ctcaacggga atcctgctct gcgaggctgg ccgtaggccg gccctaccgg
     3121 cgcggcagcg ttacccgtgt cggcggctcc aacggctcgc catcgtccag aaaacacggc
     3181 tcatcgggca tcggcaggcg ctgctgcccg cgccgttccc attcctccgt ttcggtcaag
     3241 gctggcaggt ctggttccat gcccggaatg ccgggctggc tgggcggctc ctcgccgggg
     3301 ccggtcggta gttgctgctc gcccggatac agggtcggga tgcggcgcag gtcgccatgc
     3361 cccaacagcg attcgtcctg gtcgtcgtga tcaaccacca cggcggcact gaacaccgac
     3421 aggcgcaact ggtcgcgggg ctggccccac gccacgcggt cattgaccac gtaggccgac
     3481 acggtgccgg ggccgttgag cttcacgacg gagatccagc gctcggccac caagtcct
//
