LOCUS       sequence-418932-        7196 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc162 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 7196)
  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 7196)
  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: pBC135
FEATURES             Location/Qualifiers
     source          1..7196
                     /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  3200..3365
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3408..3853
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3854..3940,3941..5275)
                     /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      5358..5452
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5573..5592)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5584..6399
                     /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            6566..6674
                     /note="incP origin of transfer"
     rep_origin      6685..7073
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7104..7190)
                     /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 ggtctgacgt ttcacttcca gccttgcggt tacattaagt acaactgagc
     2941 gagttctcga atctagcatt aatgaacacc taacgtagct atataggaca cagttgcatt
     3001 ggtcctggtt gtccctcatt aggtgatccg acattaatgc ctacaaccgt cggctgacgt
     3061 caagcgtttc gcatggggat gctggttgtc attgcccctc cttcctctaa gtgataaggt
     3121 tacttcctct aaatgaccaa gcagggtatc taatcctgtt aacgctcgct cagagaattc
     3181 gcatgcggag tcgagggaga accagataag tgaaatctag ttccaaacta ttttgtcatt
     3241 tttaattttc gtattagctt acgacgctac acccagttcc catctatttt gtcactcttc
     3301 cctaaataat ccttaaaaac tccatttcca cccctcccag ttcccaacta ttttgtccgc
     3361 ccacacgctg agcgaattcc acgcttgtgg aattcctcga gggagttcac atatacctgc
     3421 cgttcactat tatttagtga aatgagatat tatgatattt tctgaattgt gattaaaaag
     3481 gcaactttat gcccatgcaa cagaaactat aaaaaataca gagaatgaaa agaaacagat
     3541 agatttttta gttctttagg cccgtagtct gcaaatcctt ttatgatttt ctatcaaaca
     3601 aaagaggaaa atagaccagt tgcaatccaa acgagagtct aatagaatga ggtcgaaaag
     3661 taaatcgcgc gggtttgtta ctgataaagc aggcaagacc taaaatgtgt aaagggcaaa
     3721 gtgtatactt tggcgtcacc ccttacatat tttaggtctt tttttattgt gcgtaactaa
     3781 cttgccatct tcaaacagga gggctggaag aagcagaccg ctaacacagt acataaaaaa
     3841 ggagacatga acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat taacctttac
     3901 taccgcactg ctggcaggag gcgcaactca agcgtttgcg aaagaaacga accaaaagcc
     3961 atataaggaa acatacggca tttcccatat tacacgccat gatatgctgc aaatccctga
     4021 acagcaaaaa aatgaaaaat atcaagttcc tgaattcgat tcgtccacaa ttaaaaatat
     4081 ctcttctgca aaaggcctgg acgtttggga cagctggcca ttacaaaacg ctgacggcac
     4141 tgtcgcaaac tatcacggct accacatcgt ctttgcatta gccggagatc ctaaaaatgc
     4201 ggatgacaca tcgatttaca tgttctatca aaaagtcggc gaaacttcta ttgacagctg
     4261 gaaaaacgct ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg attctatcct
     4321 aaaagaccaa acacaagaat ggtcaggttc agccacattt acatctgacg gaaaaatccg
     4381 tttattctac actgatttct ccggtaaaca ttacggcaaa caaacactga caactgcaca
     4441 agttaacgta tcagcatcag acagctcttt gaacatcaac ggtgtagagg attataaatc
     4501 aatctttgac ggtgacggaa aaacgtatca aaatgtacag cagttcatcg atgaaggcaa
     4561 ctacagctca ggcgacaacc atacgctgag agatcctcac tacgtagaag ataaaggcca
     4621 caaatactta gtatttgaag caaacactgg aactgaagat ggctaccaag gcgaagaatc
     4681 tttatttaac aaagcatact atggcaaaag cacatcattc ttccgtcaag aaagtcaaaa
     4741 acttctgcaa agcgataaaa aacgcacggc tgagttagca aacggcgctc tcggtatgat
     4801 tgagctaaac gatgattaca cactgaaaaa agtgatgaaa ccgctgattg catctaacac
     4861 agtaacagat gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat ggtacctgtt
     4921 cactgactcc cgcggatcaa aaatgacgat tgacggcatt acgtctaacg atatttacat
     4981 gcttggttat gtttctaatt ctttaactgg cccatacaag ccgctgaaca aaactggcct
     5041 tgtgttaaaa atggatcttg atcctaacga tgtaaccttt acttactcac acttcgctgt
     5101 acctcaagcg aaaggaaaca atgtcgtgat tacaagctat atgacaaaca gaggattcta
     5161 cgcagacaaa caatcaacgt ttgcgccaag cttcctgctg aacatcaaag gcaagaaaac
     5221 atctgttgtc aaagacagca tccttgaaca aggacaatta acagttaaca aataaattcg
     5281 ctgagcgagg atgcacatgt gaccgaggga agcttgcggc cgcgtcgtga ctgggaaaac
     5341 cctggcgact agtcttggac tcctgttgat agatccagta atgacctcag aactccatct
     5401 ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaggggt
     5461 ccccaataat tacgatttaa atttgtgtct caaaatctct gatgttacat tgcacaagat
     5521 aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     5581 gttatgagcc atattcagcg tgaaacgagc tgtagccgtc cgcgtctgaa cagcaacatg
     5641 gatgcggatc tgtatggcta taaatgggcg cgtgataacg tgggtcagag cggcgcgacc
     5701 atttatcgtc tgtatggcaa accggatgcg ccggaactgt ttctgaaaca tggcaaaggc
     5761 agcgtggcga acgatgtgac cgatgaaatg gtgcgtctga actggctgac cgaatttatg
     5821 ccgctgccga ccattaaaca ttttattcgc accccggatg atgcgtggct gctgaccacc
     5881 gcgattccgg gcaaaaccgc gtttcaggtg ctggaagaat atccggatag cggcgaaaac
     5941 attgtggatg cgctggccgt gtttctgcgt cgtctgcata gcattccggt gtgcaactgc
     6001 ccgtttaaca gcgatcgtgt gtttcgtctg gcccaggcgc agagccgtat gaacaacggc
     6061 ctggtggatg cgagcgattt tgatgatgaa cgtaacggct ggccggtgga acaggtgtgg
     6121 aaagaaatgc ataaactgct gccgtttagc ccggatagcg tggtgaccca cggcgatttt
     6181 agcctggata acctgatttt cgatgaaggc aaactgattg gctgcattga tgtgggccgt
     6241 gtgggcattg cggatcgtta tcaggatctg gccattctgt ggaactgcct gggcgaattt
     6301 agcccgagcc tgcaaaaacg tctgtttcag aaatatggca ttgataatcc ggatatgaac
     6361 aaactgcaat ttcatctgat gctggatgaa tttttctaat aattaattgg accgcggtcc
     6421 gcgcgttgtc cttttccgct gcataaccct gcttcggggt cattatagcg attttttcgg
     6481 tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc
     6541 cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg
     6601 ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc
     6661 tgcgaggctg gccgtaggcc ggccgatctg aagatcagca gttcaacctg ttgatagtac
     6721 gtactaagct ctcatgtttc acgtactaag ctctcatgtt taacgtacta agctctcatg
     6781 tttaacgaac taaaccctca tggctaacgt actaagctct catggctaac gtactaagct
     6841 ctcatgtttc acgtactaag ctctcatgtt tgaacaataa aattaatata aatcagcaac
     6901 ttaaatagcc tctaaggttt taagttttat aagaaaaaaa agaatatata aggcttttaa
     6961 agcctttaag gtttaacggt tgtggacaac aagccaggga tgtaacgcac tgagaagccc
     7021 ttagagcctc tcaaagcaat tttgagtgac acaggaacac ttaacggctg acatggggcg
     7081 cgcccagctg tctagggcgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
     7141 cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcct
//
