LOCUS       sequence-418918-        7160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc159 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 7160)
  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 7160)
  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: pBC132
FEATURES             Location/Qualifiers
     source          1..7160
                     /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  3164..3329
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3372..3817
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3818..3904,3905..5239)
                     /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      5322..5416
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5537..5556)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5548..6363
                     /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            6530..6638
                     /note="incP origin of transfer"
     rep_origin      6649..7037
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7068..7154)
                     /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 ctccccatgc gaaacgcttg acgtcagccg acggttgtag gcataatgca actgtgtcct
     2941 atatagctac gttaggtgtt cattaatgtc ggatcaccta atgagggaca accaaggaca
     3001 atgctagatt cgagaactcg ctcagttgta cttgccatgg ggatgctggt tgtcattgcc
     3061 cctccttcct ctaagtgata aggttacttc ctctaaatga ccaagcaggg tatctaatcc
     3121 tgttaacgct cgctcagaga attcgcatgc ggagtcgagg gagaaccaga taagtgaaat
     3181 ctagttccaa actattttgt catttttaat tttcgtatta gcttacgacg ctacacccag
     3241 ttcccatcta ttttgtcact cttccctaaa taatccttaa aaactccatt tccacccctc
     3301 ccagttccca actattttgt ccgcccacac gctgagcgaa ttccacgctt gtggaattcc
     3361 tcgagggagt tcacatatac ctgccgttca ctattattta gtgaaatgag atattatgat
     3421 attttctgaa ttgtgattaa aaaggcaact ttatgcccat gcaacagaaa ctataaaaaa
     3481 tacagagaat gaaaagaaac agatagattt tttagttctt taggcccgta gtctgcaaat
     3541 ccttttatga ttttctatca aacaaaagag gaaaatagac cagttgcaat ccaaacgaga
     3601 gtctaataga atgaggtcga aaagtaaatc gcgcgggttt gttactgata aagcaggcaa
     3661 gacctaaaat gtgtaaaggg caaagtgtat actttggcgt caccccttac atattttagg
     3721 tcttttttta ttgtgcgtaa ctaacttgcc atcttcaaac aggagggctg gaagaagcag
     3781 accgctaaca cagtacataa aaaaggagac atgaacgatg aacatcaaaa agtttgcaaa
     3841 acaagcaaca gtattaacct ttactaccgc actgctggca ggaggcgcaa ctcaagcgtt
     3901 tgcgaaagaa acgaaccaaa agccatataa ggaaacatac ggcatttccc atattacacg
     3961 ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa aaatatcaag ttcctgaatt
     4021 cgattcgtcc acaattaaaa atatctcttc tgcaaaaggc ctggacgttt gggacagctg
     4081 gccattacaa aacgctgacg gcactgtcgc aaactatcac ggctaccaca tcgtctttgc
     4141 attagccgga gatcctaaaa atgcggatga cacatcgatt tacatgttct atcaaaaagt
     4201 cggcgaaact tctattgaca gctggaaaaa cgctggccgc gtctttaaag acagcgacaa
     4261 attcgatgca aatgattcta tcctaaaaga ccaaacacaa gaatggtcag gttcagccac
     4321 atttacatct gacggaaaaa tccgtttatt ctacactgat ttctccggta aacattacgg
     4381 caaacaaaca ctgacaactg cacaagttaa cgtatcagca tcagacagct ctttgaacat
     4441 caacggtgta gaggattata aatcaatctt tgacggtgac ggaaaaacgt atcaaaatgt
     4501 acagcagttc atcgatgaag gcaactacag ctcaggcgac aaccatacgc tgagagatcc
     4561 tcactacgta gaagataaag gccacaaata cttagtattt gaagcaaaca ctggaactga
     4621 agatggctac caaggcgaag aatctttatt taacaaagca tactatggca aaagcacatc
     4681 attcttccgt caagaaagtc aaaaacttct gcaaagcgat aaaaaacgca cggctgagtt
     4741 agcaaacggc gctctcggta tgattgagct aaacgatgat tacacactga aaaaagtgat
     4801 gaaaccgctg attgcatcta acacagtaac agatgaaatt gaacgcgcga acgtctttaa
     4861 aatgaacggc aaatggtacc tgttcactga ctcccgcgga tcaaaaatga cgattgacgg
     4921 cattacgtct aacgatattt acatgcttgg ttatgtttct aattctttaa ctggcccata
     4981 caagccgctg aacaaaactg gccttgtgtt aaaaatggat cttgatccta acgatgtaac
     5041 ctttacttac tcacacttcg ctgtacctca agcgaaagga aacaatgtcg tgattacaag
     5101 ctatatgaca aacagaggat tctacgcaga caaacaatca acgtttgcgc caagcttcct
     5161 gctgaacatc aaaggcaaga aaacatctgt tgtcaaagac agcatccttg aacaaggaca
     5221 attaacagtt aacaaataaa ttcgctgagc gaggatgcac atgtgaccga gggaagcttg
     5281 cggccgcgtc gtgactggga aaaccctggc gactagtctt ggactcctgt tgatagatcc
     5341 agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt
     5401 ttttattggt gagaatccag gggtccccaa taattacgat ttaaatttgt gtctcaaaat
     5461 ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct
     5521 tacataaaca gtaatacaag gggtgttatg agccatattc agcgtgaaac gagctgtagc
     5581 cgtccgcgtc tgaacagcaa catggatgcg gatctgtatg gctataaatg ggcgcgtgat
     5641 aacgtgggtc agagcggcgc gaccatttat cgtctgtatg gcaaaccgga tgcgccggaa
     5701 ctgtttctga aacatggcaa aggcagcgtg gcgaacgatg tgaccgatga aatggtgcgt
     5761 ctgaactggc tgaccgaatt tatgccgctg ccgaccatta aacattttat tcgcaccccg
     5821 gatgatgcgt ggctgctgac caccgcgatt ccgggcaaaa ccgcgtttca ggtgctggaa
     5881 gaatatccgg atagcggcga aaacattgtg gatgcgctgg ccgtgtttct gcgtcgtctg
     5941 catagcattc cggtgtgcaa ctgcccgttt aacagcgatc gtgtgtttcg tctggcccag
     6001 gcgcagagcc gtatgaacaa cggcctggtg gatgcgagcg attttgatga tgaacgtaac
     6061 ggctggccgg tggaacaggt gtggaaagaa atgcataaac tgctgccgtt tagcccggat
     6121 agcgtggtga cccacggcga ttttagcctg gataacctga ttttcgatga aggcaaactg
     6181 attggctgca ttgatgtggg ccgtgtgggc attgcggatc gttatcagga tctggccatt
     6241 ctgtggaact gcctgggcga atttagcccg agcctgcaaa aacgtctgtt tcagaaatat
     6301 ggcattgata atccggatat gaacaaactg caatttcatc tgatgctgga tgaatttttc
     6361 taataattaa ttggaccgcg gtccgcgcgt tgtccttttc cgctgcataa ccctgcttcg
     6421 gggtcattat agcgattttt tcggtatatc catccttttt cgcacgatat acaggatttt
     6481 gccaaagggt tcgtgtagac tttccttggt gtatccaacg gcgtcagccg ggcaggatag
     6541 gtgaagtagg cccacccgcg agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg
     6601 cggtgctcaa cgggaatcct gctctgcgag gctggccgta ggccggccga tctgaagatc
     6661 agcagttcaa cctgttgata gtacgtacta agctctcatg tttcacgtac taagctctca
     6721 tgtttaacgt actaagctct catgtttaac gaactaaacc ctcatggcta acgtactaag
     6781 ctctcatggc taacgtacta agctctcatg tttcacgtac taagctctca tgtttgaaca
     6841 ataaaattaa tataaatcag caacttaaat agcctctaag gttttaagtt ttataagaaa
     6901 aaaaagaata tataaggctt ttaaagcctt taaggtttaa cggttgtgga caacaagcca
     6961 gggatgtaac gcactgagaa gcccttagag cctctcaaag caattttgag tgacacagga
     7021 acacttaacg gctgacatgg ggcgcgccca gctgtctagg gcggcggatt tgtcctactc
     7081 aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc
     7141 tttcgtttta tttgatgcct
//
