LOCUS       sequence-418939-        7178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc152 through Tn7 
            bacterial insertion, Kanamycin selectable marker and GFP fluorescent
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7178)
  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 7178)
  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: pBC125
FEATURES             Location/Qualifiers
     source          1..7178
                     /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)."
     terminator      complement(681..933)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(953..1504)
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(1546..1725)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     protein_bind    complement(1803..1850)
                     /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(1878..1883,1884..1899,1900..1906)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1914..1930
                     /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             1953..2666
                     /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(1983..2011)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2606..2626
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2689..2783
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3182..3347
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3390..3835
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3836..3922,3923..5257)
                     /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      5340..5434
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5555..5574)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5566..6381
                     /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            6548..6656
                     /note="incP origin of transfer"
     rep_origin      6667..7055
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7086..7172)
                     /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 aatgccggtc gatctagtaa catagatgac accgcgcgcg ataatttatc
      721 ctagtttgcg cgctatattt tgttttctat cgcgtattaa atgtataatt gcgggactct
      781 aatcataaaa acccatctca taaataacgt catgcattac atgttaatta ttacatgctt
      841 aacgtaattc aacagaaatt atatgataat catcgcaaga ccggcaacag gattcaatct
      901 taagaaactt tattgccaaa tgtttgaacg atctgcttga ctctaggggt catcagattt
      961 cggtgacggg caggaccgga cggggcggca ccggcaggct gaagtccagc tgccagaaac
     1021 ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg cagcatgccg cggggggcat
     1081 atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg cagcccgatg acagcgacca
     1141 cgctcttgaa gccctgtgcc tccagggact tcagcaggtg ggtgtagagc gtggagccca
     1201 gtcccgtccg ctggtggcgg ggggagacgt acacggtcga ctcggccgtc cagtcgtagg
     1261 cgttgcgtgc cttccaggga cccgcgtagg cgatgccggc gacctcgccg tccacctcgg
     1321 cgacgagcca gggatagcgc tcccgcagac ggacgaggtc gtccgtccac tcctgcggtt
     1381 cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat gtagtggttg acgatggtgc
     1441 agaccgccgg catgtccgcc tcggtggcac ggcggatgtc ggccgggcgt cgttctgggc
     1501 tcatggtaga tcccctcgat cgagttgaga gtgaatatga gactctaatt ggataccgag
     1561 gggaatttat ggaacgtcag tggagcattt ttgacaagaa atatttgcta gctgatagtg
     1621 accttaggcg acttttgaac gcgcaataat ggtttctgac gtatgtgctt agctcattaa
     1681 actccagaaa cccgcggctc agtggctcct tcaacgttgc ggttctgtca gttccaaacg
     1741 taaaacggct tgtcccgcgt catcggcggg ggtcataacg tgactccctt aattctcagc
     1801 ttgaagttcc tatactttct agagaatagg aacttcggaa taggtacttc cgctactaga
     1861 attcgagcgg ggaattcttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
     1921 cggataacaa ttttcacaca ggaaacagct aaatgcgtaa aggagaagaa cttttcactg
     1981 gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca
     2041 gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta
     2101 ctggaaaact acctgttcca tggccaacac ttgtcactac tttcggttat ggtgttcaat
     2161 gctttgcgag atacccagat catatgaaac agcatgactt tttcaagagt gtcatgcccg
     2221 aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac aagacacgtg
     2281 ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt
     2341 ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac tcacacaatg
     2401 tatacatcat ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa attagacaca
     2461 acattgaaga tggaagcgtt caactagcag accattatca acaaaatact ccaattggcg
     2521 atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc ctttcgaaag
     2581 atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta
     2641 cacatggcat ggatgaacta tacaaatgag cttaattagc tgagcttgga ctcctgttga
     2701 tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc
     2761 gggcgttttt tattggtgag aatctcccgc tttacgaatt cccatgggga gcatgcggag
     2821 ttacgtcatc cccaccttcc gctgcgttct tcatcgatgc ccgcttagtg atatgcttaa
     2881 aactccaagc acagccttct ccccaatggg tctgacgttt cacttccagc cttgcggtta
     2941 cattaatgcg aaacgcttga cgtcagccga cggttgtagg cattgtcctt ggttgtccct
     3001 cattaggtga tccgacatta tgggtctgac gtttcacttc cagccttgcg gttacagggg
     3061 atgctggttg tcattgcccc tccttcctct aagtgataag gttacttcct ctaaatgacc
     3121 aagcagggta tctaatcctg ttaacgctcg ctcagagaat tcgcatgcgg agtcgaggga
     3181 gaaccagata agtgaaatct agttccaaac tattttgtca tttttaattt tcgtattagc
     3241 ttacgacgct acacccagtt cccatctatt ttgtcactct tccctaaata atccttaaaa
     3301 actccatttc cacccctccc agttcccaac tattttgtcc gcccacacgc tgagcgaatt
     3361 ccacgcttgt ggaattcctc gagggagttc acatatacct gccgttcact attatttagt
     3421 gaaatgagat attatgatat tttctgaatt gtgattaaaa aggcaacttt atgcccatgc
     3481 aacagaaact ataaaaaata cagagaatga aaagaaacag atagattttt tagttcttta
     3541 ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga aaatagacca
     3601 gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc gcgggtttgt
     3661 tactgataaa gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac tttggcgtca
     3721 ccccttacat attttaggtc tttttttatt gtgcgtaact aacttgccat cttcaaacag
     3781 gagggctgga agaagcagac cgctaacaca gtacataaaa aaggagacat gaacgatgaa
     3841 catcaaaaag tttgcaaaac aagcaacagt attaaccttt actaccgcac tgctggcagg
     3901 aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag ccatataagg aaacatacgg
     3961 catttcccat attacacgcc atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa
     4021 atatcaagtt cctgaattcg attcgtccac aattaaaaat atctcttctg caaaaggcct
     4081 ggacgtttgg gacagctggc cattacaaaa cgctgacggc actgtcgcaa actatcacgg
     4141 ctaccacatc gtctttgcat tagccggaga tcctaaaaat gcggatgaca catcgattta
     4201 catgttctat caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt
     4261 ctttaaagac agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga
     4321 atggtcaggt tcagccacat ttacatctga cggaaaaatc cgtttattct acactgattt
     4381 ctccggtaaa cattacggca aacaaacact gacaactgca caagttaacg tatcagcatc
     4441 agacagctct ttgaacatca acggtgtaga ggattataaa tcaatctttg acggtgacgg
     4501 aaaaacgtat caaaatgtac agcagttcat cgatgaaggc aactacagct caggcgacaa
     4561 ccatacgctg agagatcctc actacgtaga agataaaggc cacaaatact tagtatttga
     4621 agcaaacact ggaactgaag atggctacca aggcgaagaa tctttattta acaaagcata
     4681 ctatggcaaa agcacatcat tcttccgtca agaaagtcaa aaacttctgc aaagcgataa
     4741 aaaacgcacg gctgagttag caaacggcgc tctcggtatg attgagctaa acgatgatta
     4801 cacactgaaa aaagtgatga aaccgctgat tgcatctaac acagtaacag atgaaattga
     4861 acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg ttcactgact cccgcggatc
     4921 aaaaatgacg attgacggca ttacgtctaa cgatatttac atgcttggtt atgtttctaa
     4981 ttctttaact ggcccataca agccgctgaa caaaactggc cttgtgttaa aaatggatct
     5041 tgatcctaac gatgtaacct ttacttactc acacttcgct gtacctcaag cgaaaggaaa
     5101 caatgtcgtg attacaagct atatgacaaa cagaggattc tacgcagaca aacaatcaac
     5161 gtttgcgcca agcttcctgc tgaacatcaa aggcaagaaa acatctgttg tcaaagacag
     5221 catccttgaa caaggacaat taacagttaa caaataaatt cgctgagcga ggatgcacat
     5281 gtgaccgagg gaagcttgcg gccgcgtcgt gactgggaaa accctggcga ctagtcttgg
     5341 actcctgttg atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct
     5401 cggttgccgc cgggcgtttt ttattggtga gaatccaggg gtccccaata attacgattt
     5461 aaatttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     5521 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcag
     5581 cgtgaaacga gctgtagccg tccgcgtctg aacagcaaca tggatgcgga tctgtatggc
     5641 tataaatggg cgcgtgataa cgtgggtcag agcggcgcga ccatttatcg tctgtatggc
     5701 aaaccggatg cgccggaact gtttctgaaa catggcaaag gcagcgtggc gaacgatgtg
     5761 accgatgaaa tggtgcgtct gaactggctg accgaattta tgccgctgcc gaccattaaa
     5821 cattttattc gcaccccgga tgatgcgtgg ctgctgacca ccgcgattcc gggcaaaacc
     5881 gcgtttcagg tgctggaaga atatccggat agcggcgaaa acattgtgga tgcgctggcc
     5941 gtgtttctgc gtcgtctgca tagcattccg gtgtgcaact gcccgtttaa cagcgatcgt
     6001 gtgtttcgtc tggcccaggc gcagagccgt atgaacaacg gcctggtgga tgcgagcgat
     6061 tttgatgatg aacgtaacgg ctggccggtg gaacaggtgt ggaaagaaat gcataaactg
     6121 ctgccgttta gcccggatag cgtggtgacc cacggcgatt ttagcctgga taacctgatt
     6181 ttcgatgaag gcaaactgat tggctgcatt gatgtgggcc gtgtgggcat tgcggatcgt
     6241 tatcaggatc tggccattct gtggaactgc ctgggcgaat ttagcccgag cctgcaaaaa
     6301 cgtctgtttc agaaatatgg cattgataat ccggatatga acaaactgca atttcatctg
     6361 atgctggatg aatttttcta ataattaatt ggaccgcggt ccgcgcgttg tccttttccg
     6421 ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca tcctttttcg
     6481 cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt atccaacggc
     6541 gtcagccggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg
     6601 tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc tggccgtagg
     6661 ccggccgatc tgaagatcag cagttcaacc tgttgatagt acgtactaag ctctcatgtt
     6721 tcacgtacta agctctcatg tttaacgtac taagctctca tgtttaacga actaaaccct
     6781 catggctaac gtactaagct ctcatggcta acgtactaag ctctcatgtt tcacgtacta
     6841 agctctcatg tttgaacaat aaaattaata taaatcagca acttaaatag cctctaaggt
     6901 tttaagtttt ataagaaaaa aaagaatata taaggctttt aaagccttta aggtttaacg
     6961 gttgtggaca acaagccagg gatgtaacgc actgagaagc ccttagagcc tctcaaagca
     7021 attttgagtg acacaggaac acttaacggc tgacatgggg cgcgcccagc tgtctagggc
     7081 ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc
     7141 cagtctttcg actgagcctt tcgttttatt tgatgcct
//
