LOCUS       sequence-418934-        7195 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc156 through Tn7 
            bacterial insertion, Kanamycin selectable marker and tagRFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7195)
  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 7195)
  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: pBC129
FEATURES             Location/Qualifiers
     source          1..7195
                     /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      2683..2777
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3199..3364
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3407..3852
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3853..3939,3940..5274)
                     /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      5357..5451
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5572..5591)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5583..6398
                     /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            6565..6673
                     /note="incP origin of transfer"
     rep_origin      6684..7072
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7103..7189)
                     /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 ggaatttttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
     1921 cggataacaa ttttcacaca ggaaacagct aaatggtgag taaaggtgaa gagttgatta
     1981 aagagaacat gcatatgaag ttatacatgg agggaactgt caataatcac cactttaagt
     2041 gtacatcaga gggtgaggga aagccatacg agggaaccca aactatgaga atcaaagtag
     2101 tggagggagg tcctcttcca tttgcttttg atatactagc aacaagtttc atgtatggtt
     2161 ccaggacctt cattaaccat actcagggaa tccctgactt ctttaaacag tcttttcctg
     2221 aaggttttac atgggagagg gttaccactt acgaggacgg tggagtcttg acagcaaccc
     2281 aggacacttc attacaagat ggatgcctaa tatacaatgt gaaaattagg ggtgtgaatt
     2341 tccctagtaa cggaccagtt atgcagaaga aaacactagg ttgggaagct aatactgaaa
     2401 tgttgtaccc tgccgacgga ggtttagaag gtagatccga catggccctt aagctagtcg
     2461 gaggtggaca cttgatctgt aactttaaaa ccacatatag gtctaagaag ccagcaaaga
     2521 atctaaaaat gcctggtgtt tactatgtgg accatagact agaaaggata aaagaagcag
     2581 acaaagaaac ttacgtggag caacatgagg tcgccgtcgc taggtattgc gacttacctt
     2641 ccaagctagg tcacaaattg aacgcttaat tagctgagct tggactcctg ttgatagatc
     2701 cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt
     2761 tttttattgg tgagaatctc ccgctttacg aattcccatg gggagcatgc ggagttacgt
     2821 catccccacc ttccgctgcg ttcttcatcg atgcccgctt agtgatatgc ttaaaactcc
     2881 aagcacagcc ttctccccat gcggagatat atcccactcc tcgccgaggc tgccattatg
     2941 ggtccttggt tgtccctcat taggtgatcc gacactcctc gccgaggctg ccattatgac
     3001 taactgatcc tattcacgtg ttcctttaca cattaatgct agattcgaga actcgctcag
     3061 ttgtacttgc catggggatg ctggttgtca ttgcccctcc ttcctctaag tgataaggtt
     3121 acttcctcta aatgaccaag cagggtatct aatcctgtta acgctcgctc agagaattcg
     3181 catgcggagt cgagggagaa ccagataagt gaaatctagt tccaaactat tttgtcattt
     3241 ttaattttcg tattagctta cgacgctaca cccagttccc atctattttg tcactcttcc
     3301 ctaaataatc cttaaaaact ccatttccac ccctcccagt tcccaactat tttgtccgcc
     3361 cacacgctga gcgaattcca cgcttgtgga attcctcgag ggagttcaca tatacctgcc
     3421 gttcactatt atttagtgaa atgagatatt atgatatttt ctgaattgtg attaaaaagg
     3481 caactttatg cccatgcaac agaaactata aaaaatacag agaatgaaaa gaaacagata
     3541 gattttttag ttctttaggc ccgtagtctg caaatccttt tatgattttc tatcaaacaa
     3601 aagaggaaaa tagaccagtt gcaatccaaa cgagagtcta atagaatgag gtcgaaaagt
     3661 aaatcgcgcg ggtttgttac tgataaagca ggcaagacct aaaatgtgta aagggcaaag
     3721 tgtatacttt ggcgtcaccc cttacatatt ttaggtcttt ttttattgtg cgtaactaac
     3781 ttgccatctt caaacaggag ggctggaaga agcagaccgc taacacagta cataaaaaag
     3841 gagacatgaa cgatgaacat caaaaagttt gcaaaacaag caacagtatt aacctttact
     3901 accgcactgc tggcaggagg cgcaactcaa gcgtttgcga aagaaacgaa ccaaaagcca
     3961 tataaggaaa catacggcat ttcccatatt acacgccatg atatgctgca aatccctgaa
     4021 cagcaaaaaa atgaaaaata tcaagttcct gaattcgatt cgtccacaat taaaaatatc
     4081 tcttctgcaa aaggcctgga cgtttgggac agctggccat tacaaaacgc tgacggcact
     4141 gtcgcaaact atcacggcta ccacatcgtc tttgcattag ccggagatcc taaaaatgcg
     4201 gatgacacat cgatttacat gttctatcaa aaagtcggcg aaacttctat tgacagctgg
     4261 aaaaacgctg gccgcgtctt taaagacagc gacaaattcg atgcaaatga ttctatccta
     4321 aaagaccaaa cacaagaatg gtcaggttca gccacattta catctgacgg aaaaatccgt
     4381 ttattctaca ctgatttctc cggtaaacat tacggcaaac aaacactgac aactgcacaa
     4441 gttaacgtat cagcatcaga cagctctttg aacatcaacg gtgtagagga ttataaatca
     4501 atctttgacg gtgacggaaa aacgtatcaa aatgtacagc agttcatcga tgaaggcaac
     4561 tacagctcag gcgacaacca tacgctgaga gatcctcact acgtagaaga taaaggccac
     4621 aaatacttag tatttgaagc aaacactgga actgaagatg gctaccaagg cgaagaatct
     4681 ttatttaaca aagcatacta tggcaaaagc acatcattct tccgtcaaga aagtcaaaaa
     4741 cttctgcaaa gcgataaaaa acgcacggct gagttagcaa acggcgctct cggtatgatt
     4801 gagctaaacg atgattacac actgaaaaaa gtgatgaaac cgctgattgc atctaacaca
     4861 gtaacagatg aaattgaacg cgcgaacgtc tttaaaatga acggcaaatg gtacctgttc
     4921 actgactccc gcggatcaaa aatgacgatt gacggcatta cgtctaacga tatttacatg
     4981 cttggttatg tttctaattc tttaactggc ccatacaagc cgctgaacaa aactggcctt
     5041 gtgttaaaaa tggatcttga tcctaacgat gtaaccttta cttactcaca cttcgctgta
     5101 cctcaagcga aaggaaacaa tgtcgtgatt acaagctata tgacaaacag aggattctac
     5161 gcagacaaac aatcaacgtt tgcgccaagc ttcctgctga acatcaaagg caagaaaaca
     5221 tctgttgtca aagacagcat ccttgaacaa ggacaattaa cagttaacaa ataaattcgc
     5281 tgagcgagga tgcacatgtg accgagggaa gcttgcggcc gcgtcgtgac tgggaaaacc
     5341 ctggcgacta gtcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
     5401 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaggggtc
     5461 cccaataatt acgatttaaa tttgtgtctc aaaatctctg atgttacatt gcacaagata
     5521 aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
     5581 ttatgagcca tattcagcgt gaaacgagct gtagccgtcc gcgtctgaac agcaacatgg
     5641 atgcggatct gtatggctat aaatgggcgc gtgataacgt gggtcagagc ggcgcgacca
     5701 tttatcgtct gtatggcaaa ccggatgcgc cggaactgtt tctgaaacat ggcaaaggca
     5761 gcgtggcgaa cgatgtgacc gatgaaatgg tgcgtctgaa ctggctgacc gaatttatgc
     5821 cgctgccgac cattaaacat tttattcgca ccccggatga tgcgtggctg ctgaccaccg
     5881 cgattccggg caaaaccgcg tttcaggtgc tggaagaata tccggatagc ggcgaaaaca
     5941 ttgtggatgc gctggccgtg tttctgcgtc gtctgcatag cattccggtg tgcaactgcc
     6001 cgtttaacag cgatcgtgtg tttcgtctgg cccaggcgca gagccgtatg aacaacggcc
     6061 tggtggatgc gagcgatttt gatgatgaac gtaacggctg gccggtggaa caggtgtgga
     6121 aagaaatgca taaactgctg ccgtttagcc cggatagcgt ggtgacccac ggcgatttta
     6181 gcctggataa cctgattttc gatgaaggca aactgattgg ctgcattgat gtgggccgtg
     6241 tgggcattgc ggatcgttat caggatctgg ccattctgtg gaactgcctg ggcgaattta
     6301 gcccgagcct gcaaaaacgt ctgtttcaga aatatggcat tgataatccg gatatgaaca
     6361 aactgcaatt tcatctgatg ctggatgaat ttttctaata attaattgga ccgcggtccg
     6421 cgcgttgtcc ttttccgctg cataaccctg cttcggggtc attatagcga ttttttcggt
     6481 atatccatcc tttttcgcac gatatacagg attttgccaa agggttcgtg tagactttcc
     6541 ttggtgtatc caacggcgtc agccgggcag gataggtgaa gtaggcccac ccgcgagcgg
     6601 gtgttccttc ttcactgtcc cttattcgca cctggcggtg ctcaacggga atcctgctct
     6661 gcgaggctgg ccgtaggccg gccgatctga agatcagcag ttcaacctgt tgatagtacg
     6721 tactaagctc tcatgtttca cgtactaagc tctcatgttt aacgtactaa gctctcatgt
     6781 ttaacgaact aaaccctcat ggctaacgta ctaagctctc atggctaacg tactaagctc
     6841 tcatgtttca cgtactaagc tctcatgttt gaacaataaa attaatataa atcagcaact
     6901 taaatagcct ctaaggtttt aagttttata agaaaaaaaa gaatatataa ggcttttaaa
     6961 gcctttaagg tttaacggtt gtggacaaca agccagggat gtaacgcact gagaagccct
     7021 tagagcctct caaagcaatt ttgagtgaca caggaacact taacggctga catggggcgc
     7081 gcccagctgt ctagggcggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac
     7141 agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcct
//
