LOCUS       sequence-419250-        7198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc160 through Tn7 
            bacterial insertion, Kanamycin selectable marker and tagBFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7198)
  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 7198)
  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: pBC133
FEATURES             Location/Qualifiers
     source          1..7198
                     /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)."
     terminator      2671..2765
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3202..3367
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3410..3855
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3856..3942,3943..5277)
                     /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      5360..5454
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5575..5594)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5586..6401
                     /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            6568..6676
                     /note="incP origin of transfer"
     rep_origin      6687..7075
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7106..7192)
                     /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 aaatgtctga attgattaaa gagaatatgc
     1981 atatgaagtt gtatatggaa ggtactgttg ataaccacca ttttaaatgt acatctgagg
     2041 gagaaggtaa gccttacgaa ggtactcaaa caatgcgtat taaagtagtt gagggaggtc
     2101 ctttaccatt cgcttttgac atattggcaa cttcattctt atatggatct aagacattta
     2161 ttaaccatac tcaaggtata ccagatttct ttaaacaatc atttcctgaa ggattcacat
     2221 gggagagagt aacaacttat gaagatggtg gagttttgac tgctactcag gatacatctt
     2281 tacaagacgg ttgtttgatt tataatgtta agattcgtgg agtaaacttc acttctaatg
     2341 gtccagttat gcagaagaaa acattgggat gggaggcatt cactgagaca ttataccctg
     2401 ctgatggtgg attggaaggt cgtaacgaca tggcattgaa attagtagga ggttcacact
     2461 tgatagctaa tattaagact acatatcgtt ctaagaaacc agctaaaaac ttgaaaatgc
     2521 ctggtgttta ctatgttgat tatcgtttag aaagaattaa ggaagcaaat aacgaaactt
     2581 acgttgagca acatgaagta gctgttgcac gttattgtga cttgccttct aagttaggac
     2641 acaaattgaa cgcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct
     2701 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     2761 agaatctccc gctttacgaa ttcccatggg gagcatgcgg agttacgtca tccccacctt
     2821 ccgctgcgtt cttcatcgat gcccgcttag tgatatgctt aaaactccaa gcacagcctt
     2881 ctccccaatg cggagatata tcccactcct cgccgaggct gccattaatg atcgccagca
     2941 ggaggccgaa ctgtctagtc atcataatgg caagtacaac tgagcgagtt ctcgaatcta
     3001 gcattacaac cgtcggctga cgtcaagcgt ttcgcattat gcaactgtgt cctatatagc
     3061 tacgttaggt gttcatgggg atgctggttg tcattgcccc tccttcctct aagtgataag
     3121 gttacttcct ctaaatgacc aagcagggta tctaatcctg ttaacgctcg ctcagagaat
     3181 tcgcatgcgg agtcgaggga gaaccagata agtgaaatct agttccaaac tattttgtca
     3241 tttttaattt tcgtattagc ttacgacgct acacccagtt cccatctatt ttgtcactct
     3301 tccctaaata atccttaaaa actccatttc cacccctccc agttcccaac tattttgtcc
     3361 gcccacacgc tgagcgaatt ccacgcttgt ggaattcctc gagggagttc acatatacct
     3421 gccgttcact attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa
     3481 aggcaacttt atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag
     3541 atagattttt tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa
     3601 caaaagagga aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa
     3661 agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca
     3721 aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact
     3781 aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa
     3841 aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt
     3901 actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag
     3961 ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct
     4021 gaacagcaaa aaaatgaaaa atatcaagtt cctgaattcg attcgtccac aattaaaaat
     4081 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc
     4141 actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat
     4201 gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc
     4261 tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc
     4321 ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc
     4381 cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca
     4441 caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa
     4501 tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc
     4561 aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc
     4621 cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa
     4681 tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa
     4741 aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg
     4801 attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac
     4861 acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg
     4921 ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac
     4981 atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc
     5041 cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct
     5101 gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc
     5161 tacgcagaca aacaatcaac gtttgcgcca agcttcctgc tgaacatcaa aggcaagaaa
     5221 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaatt
     5281 cgctgagcga ggatgcacat gtgaccgagg gaagcttgcg gccgcgtcgt gactgggaaa
     5341 accctggcga ctagtcttgg actcctgttg atagatccag taatgacctc agaactccat
     5401 ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaggg
     5461 gtccccaata attacgattt aaatttgtgt ctcaaaatct ctgatgttac attgcacaag
     5521 ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     5581 gtgttatgag ccatattcag cgtgaaacga gctgtagccg tccgcgtctg aacagcaaca
     5641 tggatgcgga tctgtatggc tataaatggg cgcgtgataa cgtgggtcag agcggcgcga
     5701 ccatttatcg tctgtatggc aaaccggatg cgccggaact gtttctgaaa catggcaaag
     5761 gcagcgtggc gaacgatgtg accgatgaaa tggtgcgtct gaactggctg accgaattta
     5821 tgccgctgcc gaccattaaa cattttattc gcaccccgga tgatgcgtgg ctgctgacca
     5881 ccgcgattcc gggcaaaacc gcgtttcagg tgctggaaga atatccggat agcggcgaaa
     5941 acattgtgga tgcgctggcc gtgtttctgc gtcgtctgca tagcattccg gtgtgcaact
     6001 gcccgtttaa cagcgatcgt gtgtttcgtc tggcccaggc gcagagccgt atgaacaacg
     6061 gcctggtgga tgcgagcgat tttgatgatg aacgtaacgg ctggccggtg gaacaggtgt
     6121 ggaaagaaat gcataaactg ctgccgttta gcccggatag cgtggtgacc cacggcgatt
     6181 ttagcctgga taacctgatt ttcgatgaag gcaaactgat tggctgcatt gatgtgggcc
     6241 gtgtgggcat tgcggatcgt tatcaggatc tggccattct gtggaactgc ctgggcgaat
     6301 ttagcccgag cctgcaaaaa cgtctgtttc agaaatatgg cattgataat ccggatatga
     6361 acaaactgca atttcatctg atgctggatg aatttttcta ataattaatt ggaccgcggt
     6421 ccgcgcgttg tccttttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc
     6481 ggtatatcca tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt
     6541 tccttggtgt atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag
     6601 cgggtgttcc ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc
     6661 tctgcgaggc tggccgtagg ccggccgatc tgaagatcag cagttcaacc tgttgatagt
     6721 acgtactaag ctctcatgtt tcacgtacta agctctcatg tttaacgtac taagctctca
     6781 tgtttaacga actaaaccct catggctaac gtactaagct ctcatggcta acgtactaag
     6841 ctctcatgtt tcacgtacta agctctcatg tttgaacaat aaaattaata taaatcagca
     6901 acttaaatag cctctaaggt tttaagtttt ataagaaaaa aaagaatata taaggctttt
     6961 aaagccttta aggtttaacg gttgtggaca acaagccagg gatgtaacgc actgagaagc
     7021 ccttagagcc tctcaaagca attttgagtg acacaggaac acttaacggc tgacatgggg
     7081 cgcgcccagc tgtctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac
     7141 aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcct
//
