LOCUS       sequence-418913-        7408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc145 through Tn7 
            bacterial insertion, Tetracyline selectable marker and GFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7408)
  AUTHORS   Ordon J, Thouin J, Nakano RT, Ma KW, Zhang P, Huettel B, 
            Garrido-Oter R, Schulze-Lefert P
  TITLE     Chromosomal barcodes for simultaneous tracking of near-isogenic 
            bacterial strains in plant microbiota.
  JOURNAL   Nat Microbiol. 2024 Apr;9(4):1117-1129. doi: 
            10.1038/s41564-024-01619-8. Epub 2024 Mar 19.
  PUBMED    38503974
REFERENCE   2  (bases 1 to 7408)
  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: 
            "10.1038/s41564-024-01619-8"; journalName: "Nat Microbiol"; date: 
            "2024-04"; volume: "9"; issue: "4"; pages: "1117-1129"
COMMENT     Sequence Label: pBC118
FEATURES             Location/Qualifiers
     source          1..7408
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      308..402
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      504..590
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    complement(623..670)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        join(689..694,695..711,712..717)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             765..1955
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(1117..1136)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    complement(2035..2082)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        join(2110..2115,2116..2131,2132..2138)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2146..2162
                     /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)."
     CDS             2185..2898
                     /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
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSVMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2215..2243)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2838..2858
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2921..3015
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3412..3577
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3620..4065
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4066..4152,4153..5487)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     terminator      5570..5664
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5785..5804)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5796..6611
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            6778..6886
                     /note="incP origin of transfer"
     rep_origin      6897..7285
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7316..7402)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaattaaag cggataacaa tttcacacag gaggccgcct aggccgcggc cgcgcgaatt
       61 tgccgaattc ggatccggag ggagtgtggg cggacaataa agtcttaaac tgaacaaaat
      121 agatctaaac tatgacaata aagtcttaaa ctagacagaa tagttgtaaa ctgaaatcag
      181 tccagttatg ctgtgaaaaa gcatactgga cttttgttat ggctaaagca aactcttcat
      241 tttctgaagt gcaaattgcc cgtcgtatta aagaggggcg tggcgctgca agaattcaag
      301 cttggaggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
      361 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaagcta gactgcgatg
      421 agtggcaggg cggggcgtaa tttcttaagg cagttattgg tgcccttaaa cgcctggggt
      481 aatgactctc tagcttgagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
      541 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgcccgccg
      601 ctgcaagaat tcaagcttgg aggaagttcc tatactttct agagaatagg aacttcggaa
      661 taggaacttc aatgaagaat tctcatgttt gacagcttat catcgataag ctttaatgcg
      721 gtagtttatc acagttaaat tgctaacgca gtcaggcacc gtgtatgaaa tctaacaatg
      781 cgctcatcgt catcctcggc accgtcaccc tggatgctgt aggcataggc ttggttatgc
      841 cggtactgcc gggcctcttg cgggatatcg tccattccga cagcatcgcc agtcactatg
      901 gcgtgctgct agcgctatat gcgttgatgc aatttctatg cgcacccgtt ctcggagcac
      961 tgtccgaccg ctttggccgc cgcccagtcc tgctcgcttc gctacttgga gccactatcg
     1021 actacgcgat catggcgacc acacccgtcc tgtggatcct ctacgccgga cgcatcgtgg
     1081 ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     1141 gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg
     1201 caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg
     1261 cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata
     1321 agggagagcg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     1381 cgcggggcat gactatcgtc gccgcactta tgactgtttt ctttatcatg caactcgtag
     1441 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     1501 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     1561 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     1621 ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca
     1681 ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca
     1741 ggcaggtaga tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc
     1801 taacttcgat cattggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat
     1861 ggaacgggtt ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc
     1921 gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa
     1981 cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg gcttgaagtt
     2041 cctatacttt ctagagaata ggaacttcgg aataggtact tccgctacta gaattcgagc
     2101 ggggaattct tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac
     2161 aattttcaca caggaaacag ctaaatgcgt aaaggagaag aacttttcac tggagttgtc
     2221 ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag
     2281 ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa
     2341 ctacctgttc catggccaac acttgtcact actttcggtt atggtgttca atgctttgcg
     2401 agatacccag atcatatgaa acagcatgac tttttcaaga gtgtcatgcc cgaaggttat
     2461 gtacaggaaa gaactatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc
     2521 aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa
     2581 gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa tgtatacatc
     2641 atggcagaca aacaaaagaa tggaatcaaa gttaacttca aaattagaca caacattgaa
     2701 gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct
     2761 gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa agatcccaac
     2821 gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc
     2881 atggatgaac tatacaaatg agcttaatta gctgagcttg gactcctgtt gatagatcca
     2941 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     3001 tttattggtg agaatctccc gctttacgaa ttcccatggg gagcatgcgg agttacgtca
     3061 tccccacctt ccgctgcgtt cttcatcgat gcccgcttag tgatatgctt aaaactccaa
     3121 gcacagcctt ctccccaatg ctagattcga gaactcgctc agttgtactt gccataatgt
     3181 gtaaaggaac acgtgaatag gatcagttag tcattaatgc aactgtgtcc tatatagcta
     3241 cgttaggtgt tcatgattcg agaactcgct cagttgtact tgccatgggg atgctggttg
     3301 tcattgcccc tccttcctct aagtgataag gttacttcct ctaaatgacc aagcagggta
     3361 tctaatcctg ttaacgctcg ctcagagaat tcgcatgcgg agtcgaggga gaaccagata
     3421 agtgaaatct agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct
     3481 acacccagtt cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc
     3541 cacccctccc agttcccaac tattttgtcc gcccacacgc tgagcgaatt ccacgcttgt
     3601 ggaattcctc gagggagttc acatatacct gccgttcact attatttagt gaaatgagat
     3661 attatgatat tttctgaatt gtgattaaaa aggcaacttt atgcccatgc aacagaaact
     3721 ataaaaaata cagagaatga aaagaaacag atagattttt tagttcttta ggcccgtagt
     3781 ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga aaatagacca gttgcaatcc
     3841 aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc gcgggtttgt tactgataaa
     3901 gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac tttggcgtca ccccttacat
     3961 attttaggtc tttttttatt gtgcgtaact aacttgccat cttcaaacag gagggctgga
     4021 agaagcagac cgctaacaca gtacataaaa aaggagacat gaacgatgaa catcaaaaag
     4081 tttgcaaaac aagcaacagt attaaccttt actaccgcac tgctggcagg aggcgcaact
     4141 caagcgtttg cgaaagaaac gaaccaaaag ccatataagg aaacatacgg catttcccat
     4201 attacacgcc atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa atatcaagtt
     4261 cctgaattcg attcgtccac aattaaaaat atctcttctg caaaaggcct ggacgtttgg
     4321 gacagctggc cattacaaaa cgctgacggc actgtcgcaa actatcacgg ctaccacatc
     4381 gtctttgcat tagccggaga tcctaaaaat gcggatgaca catcgattta catgttctat
     4441 caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt ctttaaagac
     4501 agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga atggtcaggt
     4561 tcagccacat ttacatctga cggaaaaatc cgtttattct acactgattt ctccggtaaa
     4621 cattacggca aacaaacact gacaactgca caagttaacg tatcagcatc agacagctct
     4681 ttgaacatca acggtgtaga ggattataaa tcaatctttg acggtgacgg aaaaacgtat
     4741 caaaatgtac agcagttcat cgatgaaggc aactacagct caggcgacaa ccatacgctg
     4801 agagatcctc actacgtaga agataaaggc cacaaatact tagtatttga agcaaacact
     4861 ggaactgaag atggctacca aggcgaagaa tctttattta acaaagcata ctatggcaaa
     4921 agcacatcat tcttccgtca agaaagtcaa aaacttctgc aaagcgataa aaaacgcacg
     4981 gctgagttag caaacggcgc tctcggtatg attgagctaa acgatgatta cacactgaaa
     5041 aaagtgatga aaccgctgat tgcatctaac acagtaacag atgaaattga acgcgcgaac
     5101 gtctttaaaa tgaacggcaa atggtacctg ttcactgact cccgcggatc aaaaatgacg
     5161 attgacggca ttacgtctaa cgatatttac atgcttggtt atgtttctaa ttctttaact
     5221 ggcccataca agccgctgaa caaaactggc cttgtgttaa aaatggatct tgatcctaac
     5281 gatgtaacct ttacttactc acacttcgct gtacctcaag cgaaaggaaa caatgtcgtg
     5341 attacaagct atatgacaaa cagaggattc tacgcagaca aacaatcaac gtttgcgcca
     5401 agcttcctgc tgaacatcaa aggcaagaaa acatctgttg tcaaagacag catccttgaa
     5461 caaggacaat taacagttaa caaataaatt cgctgagcga ggatgcacat gtgaccgagg
     5521 gaagcttgcg gccgcgtcgt gactgggaaa accctggcga ctagtcttgg actcctgttg
     5581 atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct cggttgccgc
     5641 cgggcgtttt ttattggtga gaatccaggg gtccccaata attacgattt aaatttgtgt
     5701 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
     5761 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcag cgtgaaacga
     5821 gctgtagccg tccgcgtctg aacagcaaca tggatgcgga tctgtatggc tataaatggg
     5881 cgcgtgataa cgtgggtcag agcggcgcga ccatttatcg tctgtatggc aaaccggatg
     5941 cgccggaact gtttctgaaa catggcaaag gcagcgtggc gaacgatgtg accgatgaaa
     6001 tggtgcgtct gaactggctg accgaattta tgccgctgcc gaccattaaa cattttattc
     6061 gcaccccgga tgatgcgtgg ctgctgacca ccgcgattcc gggcaaaacc gcgtttcagg
     6121 tgctggaaga atatccggat agcggcgaaa acattgtgga tgcgctggcc gtgtttctgc
     6181 gtcgtctgca tagcattccg gtgtgcaact gcccgtttaa cagcgatcgt gtgtttcgtc
     6241 tggcccaggc gcagagccgt atgaacaacg gcctggtgga tgcgagcgat tttgatgatg
     6301 aacgtaacgg ctggccggtg gaacaggtgt ggaaagaaat gcataaactg ctgccgttta
     6361 gcccggatag cgtggtgacc cacggcgatt ttagcctgga taacctgatt ttcgatgaag
     6421 gcaaactgat tggctgcatt gatgtgggcc gtgtgggcat tgcggatcgt tatcaggatc
     6481 tggccattct gtggaactgc ctgggcgaat ttagcccgag cctgcaaaaa cgtctgtttc
     6541 agaaatatgg cattgataat ccggatatga acaaactgca atttcatctg atgctggatg
     6601 aatttttcta ataattaatt ggaccgcggt ccgcgcgttg tccttttccg ctgcataacc
     6661 ctgcttcggg gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac
     6721 aggattttgc caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg
     6781 caggataggt gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc
     6841 gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc tggccgtagg ccggccgatc
     6901 tgaagatcag cagttcaacc tgttgatagt acgtactaag ctctcatgtt tcacgtacta
     6961 agctctcatg tttaacgtac taagctctca tgtttaacga actaaaccct catggctaac
     7021 gtactaagct ctcatggcta acgtactaag ctctcatgtt tcacgtacta agctctcatg
     7081 tttgaacaat aaaattaata taaatcagca acttaaatag cctctaaggt tttaagtttt
     7141 ataagaaaaa aaagaatata taaggctttt aaagccttta aggtttaacg gttgtggaca
     7201 acaagccagg gatgtaacgc actgagaagc ccttagagcc tctcaaagca attttgagtg
     7261 acacaggaac acttaacggc tgacatgggg cgcgcccagc tgtctagggc ggcggatttg
     7321 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg
     7381 actgagcctt tcgttttatt tgatgcct
//
