LOCUS       sequence-418914-        7411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc144 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 7411)
  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 7411)
  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: pBC117
FEATURES             Location/Qualifiers
     source          1..7411
                     /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  3415..3580
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3623..4068
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4069..4155,4156..5490)
                     /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      5573..5667
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5788..5807)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5799..6614
                     /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            6781..6889
                     /note="incP origin of transfer"
     rep_origin      6900..7288
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7319..7405)
                     /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 ctccccatgc gaaacgcttg acgtcagccg acggttgtag gatgatgact
     3181 agacagttcg gcctcctgct ggcgatcatt atgtacgaat ctcctactat ggtcttcaac
     3241 cgtgtcatta atgatgacta gacagttcgg cctcctgctg gcgatcattg gggatgctgg
     3301 ttgtcattgc ccctccttcc tctaagtgat aaggttactt cctctaaatg accaagcagg
     3361 gtatctaatc ctgttaacgc tcgctcagag aattcgcatg cggagtcgag ggagaaccag
     3421 ataagtgaaa tctagttcca aactattttg tcatttttaa ttttcgtatt agcttacgac
     3481 gctacaccca gttcccatct attttgtcac tcttccctaa ataatcctta aaaactccat
     3541 ttccacccct cccagttccc aactattttg tccgcccaca cgctgagcga attccacgct
     3601 tgtggaattc ctcgagggag ttcacatata cctgccgttc actattattt agtgaaatga
     3661 gatattatga tattttctga attgtgatta aaaaggcaac tttatgccca tgcaacagaa
     3721 actataaaaa atacagagaa tgaaaagaaa cagatagatt ttttagttct ttaggcccgt
     3781 agtctgcaaa tccttttatg attttctatc aaacaaaaga ggaaaataga ccagttgcaa
     3841 tccaaacgag agtctaatag aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat
     3901 aaagcaggca agacctaaaa tgtgtaaagg gcaaagtgta tactttggcg tcacccctta
     3961 catattttag gtcttttttt attgtgcgta actaacttgc catcttcaaa caggagggct
     4021 ggaagaagca gaccgctaac acagtacata aaaaaggaga catgaacgat gaacatcaaa
     4081 aagtttgcaa aacaagcaac agtattaacc tttactaccg cactgctggc aggaggcgca
     4141 actcaagcgt ttgcgaaaga aacgaaccaa aagccatata aggaaacata cggcatttcc
     4201 catattacac gccatgatat gctgcaaatc cctgaacagc aaaaaaatga aaaatatcaa
     4261 gttcctgaat tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg cctggacgtt
     4321 tgggacagct ggccattaca aaacgctgac ggcactgtcg caaactatca cggctaccac
     4381 atcgtctttg cattagccgg agatcctaaa aatgcggatg acacatcgat ttacatgttc
     4441 tatcaaaaag tcggcgaaac ttctattgac agctggaaaa acgctggccg cgtctttaaa
     4501 gacagcgaca aattcgatgc aaatgattct atcctaaaag accaaacaca agaatggtca
     4561 ggttcagcca catttacatc tgacggaaaa atccgtttat tctacactga tttctccggt
     4621 aaacattacg gcaaacaaac actgacaact gcacaagtta acgtatcagc atcagacagc
     4681 tctttgaaca tcaacggtgt agaggattat aaatcaatct ttgacggtga cggaaaaacg
     4741 tatcaaaatg tacagcagtt catcgatgaa ggcaactaca gctcaggcga caaccatacg
     4801 ctgagagatc ctcactacgt agaagataaa ggccacaaat acttagtatt tgaagcaaac
     4861 actggaactg aagatggcta ccaaggcgaa gaatctttat ttaacaaagc atactatggc
     4921 aaaagcacat cattcttccg tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc
     4981 acggctgagt tagcaaacgg cgctctcggt atgattgagc taaacgatga ttacacactg
     5041 aaaaaagtga tgaaaccgct gattgcatct aacacagtaa cagatgaaat tgaacgcgcg
     5101 aacgtcttta aaatgaacgg caaatggtac ctgttcactg actcccgcgg atcaaaaatg
     5161 acgattgacg gcattacgtc taacgatatt tacatgcttg gttatgtttc taattcttta
     5221 actggcccat acaagccgct gaacaaaact ggccttgtgt taaaaatgga tcttgatcct
     5281 aacgatgtaa cctttactta ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc
     5341 gtgattacaa gctatatgac aaacagagga ttctacgcag acaaacaatc aacgtttgcg
     5401 ccaagcttcc tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt
     5461 gaacaaggac aattaacagt taacaaataa attcgctgag cgaggatgca catgtgaccg
     5521 agggaagctt gcggccgcgt cgtgactggg aaaaccctgg cgactagtct tggactcctg
     5581 ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc
     5641 cgccgggcgt tttttattgg tgagaatcca ggggtcccca ataattacga tttaaatttg
     5701 tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa
     5761 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt cagcgtgaaa
     5821 cgagctgtag ccgtccgcgt ctgaacagca acatggatgc ggatctgtat ggctataaat
     5881 gggcgcgtga taacgtgggt cagagcggcg cgaccattta tcgtctgtat ggcaaaccgg
     5941 atgcgccgga actgtttctg aaacatggca aaggcagcgt ggcgaacgat gtgaccgatg
     6001 aaatggtgcg tctgaactgg ctgaccgaat ttatgccgct gccgaccatt aaacatttta
     6061 ttcgcacccc ggatgatgcg tggctgctga ccaccgcgat tccgggcaaa accgcgtttc
     6121 aggtgctgga agaatatccg gatagcggcg aaaacattgt ggatgcgctg gccgtgtttc
     6181 tgcgtcgtct gcatagcatt ccggtgtgca actgcccgtt taacagcgat cgtgtgtttc
     6241 gtctggccca ggcgcagagc cgtatgaaca acggcctggt ggatgcgagc gattttgatg
     6301 atgaacgtaa cggctggccg gtggaacagg tgtggaaaga aatgcataaa ctgctgccgt
     6361 ttagcccgga tagcgtggtg acccacggcg attttagcct ggataacctg attttcgatg
     6421 aaggcaaact gattggctgc attgatgtgg gccgtgtggg cattgcggat cgttatcagg
     6481 atctggccat tctgtggaac tgcctgggcg aatttagccc gagcctgcaa aaacgtctgt
     6541 ttcagaaata tggcattgat aatccggata tgaacaaact gcaatttcat ctgatgctgg
     6601 atgaattttt ctaataatta attggaccgc ggtccgcgcg ttgtcctttt ccgctgcata
     6661 accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt tcgcacgata
     6721 tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc
     6781 gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta
     6841 ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgt aggccggccg
     6901 atctgaagat cagcagttca acctgttgat agtacgtact aagctctcat gtttcacgta
     6961 ctaagctctc atgtttaacg tactaagctc tcatgtttaa cgaactaaac cctcatggct
     7021 aacgtactaa gctctcatgg ctaacgtact aagctctcat gtttcacgta ctaagctctc
     7081 atgtttgaac aataaaatta atataaatca gcaacttaaa tagcctctaa ggttttaagt
     7141 tttataagaa aaaaaagaat atataaggct tttaaagcct ttaaggttta acggttgtgg
     7201 acaacaagcc agggatgtaa cgcactgaga agcccttaga gcctctcaaa gcaattttga
     7261 gtgacacagg aacacttaac ggctgacatg gggcgcgccc agctgtctag ggcggcggat
     7321 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     7381 tcgactgagc ctttcgtttt atttgatgcc t
//
