LOCUS       sequence-417284-        7158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc161 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 7158)
  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 7158)
  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: pBC134
FEATURES             Location/Qualifiers
     source          1..7158
                     /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  3162..3327
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3370..3815
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3816..3902,3903..5237)
                     /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      5320..5414
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5535..5554)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5546..6361
                     /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            6528..6636
                     /note="incP origin of transfer"
     rep_origin      6647..7035
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7066..7152)
                     /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 ctccccctag acagttcggc ctcctgctgg cgatcataat gcaactgtgt cctatatagc
     2941 tacgttaggt gttcattaat gggtccttgg ttgtccctca ttaggtgatc cgacattaat
     3001 gtaaccgcaa ggctggaagt gaaacgtcag acccatgggg atgctggttg tcattgcccc
     3061 tccttcctct aagtgataag gttacttcct ctaaatgacc aagcagggta tctaatcctg
     3121 ttaacgctcg ctcagagaat tcgcatgcgg agtcgaggga gaaccagata agtgaaatct
     3181 agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct acacccagtt
     3241 cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc cacccctccc
     3301 agttcccaac tattttgtcc gcccacacgc tgagcgaatt ccacgcttgt ggaattcctc
     3361 gagggagttc acatatacct gccgttcact attatttagt gaaatgagat attatgatat
     3421 tttctgaatt gtgattaaaa aggcaacttt atgcccatgc aacagaaact ataaaaaata
     3481 cagagaatga aaagaaacag atagattttt tagttcttta ggcccgtagt ctgcaaatcc
     3541 ttttatgatt ttctatcaaa caaaagagga aaatagacca gttgcaatcc aaacgagagt
     3601 ctaatagaat gaggtcgaaa agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga
     3661 cctaaaatgt gtaaagggca aagtgtatac tttggcgtca ccccttacat attttaggtc
     3721 tttttttatt gtgcgtaact aacttgccat cttcaaacag gagggctgga agaagcagac
     3781 cgctaacaca gtacataaaa aaggagacat gaacgatgaa catcaaaaag tttgcaaaac
     3841 aagcaacagt attaaccttt actaccgcac tgctggcagg aggcgcaact caagcgtttg
     3901 cgaaagaaac gaaccaaaag ccatataagg aaacatacgg catttcccat attacacgcc
     3961 atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa atatcaagtt cctgaattcg
     4021 attcgtccac aattaaaaat atctcttctg caaaaggcct ggacgtttgg gacagctggc
     4081 cattacaaaa cgctgacggc actgtcgcaa actatcacgg ctaccacatc gtctttgcat
     4141 tagccggaga tcctaaaaat gcggatgaca catcgattta catgttctat caaaaagtcg
     4201 gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat
     4261 tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga atggtcaggt tcagccacat
     4321 ttacatctga cggaaaaatc cgtttattct acactgattt ctccggtaaa cattacggca
     4381 aacaaacact gacaactgca caagttaacg tatcagcatc agacagctct ttgaacatca
     4441 acggtgtaga ggattataaa tcaatctttg acggtgacgg aaaaacgtat caaaatgtac
     4501 agcagttcat cgatgaaggc aactacagct caggcgacaa ccatacgctg agagatcctc
     4561 actacgtaga agataaaggc cacaaatact tagtatttga agcaaacact ggaactgaag
     4621 atggctacca aggcgaagaa tctttattta acaaagcata ctatggcaaa agcacatcat
     4681 tcttccgtca agaaagtcaa aaacttctgc aaagcgataa aaaacgcacg gctgagttag
     4741 caaacggcgc tctcggtatg attgagctaa acgatgatta cacactgaaa aaagtgatga
     4801 aaccgctgat tgcatctaac acagtaacag atgaaattga acgcgcgaac gtctttaaaa
     4861 tgaacggcaa atggtacctg ttcactgact cccgcggatc aaaaatgacg attgacggca
     4921 ttacgtctaa cgatatttac atgcttggtt atgtttctaa ttctttaact ggcccataca
     4981 agccgctgaa caaaactggc cttgtgttaa aaatggatct tgatcctaac gatgtaacct
     5041 ttacttactc acacttcgct gtacctcaag cgaaaggaaa caatgtcgtg attacaagct
     5101 atatgacaaa cagaggattc tacgcagaca aacaatcaac gtttgcgcca agcttcctgc
     5161 tgaacatcaa aggcaagaaa acatctgttg tcaaagacag catccttgaa caaggacaat
     5221 taacagttaa caaataaatt cgctgagcga ggatgcacat gtgaccgagg gaagcttgcg
     5281 gccgcgtcgt gactgggaaa accctggcga ctagtcttgg actcctgttg atagatccag
     5341 taatgacctc agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt
     5401 ttattggtga gaatccaggg gtccccaata attacgattt aaatttgtgt ctcaaaatct
     5461 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     5521 cataaacagt aatacaaggg gtgttatgag ccatattcag cgtgaaacga gctgtagccg
     5581 tccgcgtctg aacagcaaca tggatgcgga tctgtatggc tataaatggg cgcgtgataa
     5641 cgtgggtcag agcggcgcga ccatttatcg tctgtatggc aaaccggatg cgccggaact
     5701 gtttctgaaa catggcaaag gcagcgtggc gaacgatgtg accgatgaaa tggtgcgtct
     5761 gaactggctg accgaattta tgccgctgcc gaccattaaa cattttattc gcaccccgga
     5821 tgatgcgtgg ctgctgacca ccgcgattcc gggcaaaacc gcgtttcagg tgctggaaga
     5881 atatccggat agcggcgaaa acattgtgga tgcgctggcc gtgtttctgc gtcgtctgca
     5941 tagcattccg gtgtgcaact gcccgtttaa cagcgatcgt gtgtttcgtc tggcccaggc
     6001 gcagagccgt atgaacaacg gcctggtgga tgcgagcgat tttgatgatg aacgtaacgg
     6061 ctggccggtg gaacaggtgt ggaaagaaat gcataaactg ctgccgttta gcccggatag
     6121 cgtggtgacc cacggcgatt ttagcctgga taacctgatt ttcgatgaag gcaaactgat
     6181 tggctgcatt gatgtgggcc gtgtgggcat tgcggatcgt tatcaggatc tggccattct
     6241 gtggaactgc ctgggcgaat ttagcccgag cctgcaaaaa cgtctgtttc agaaatatgg
     6301 cattgataat ccggatatga acaaactgca atttcatctg atgctggatg aatttttcta
     6361 ataattaatt ggaccgcggt ccgcgcgttg tccttttccg ctgcataacc ctgcttcggg
     6421 gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac aggattttgc
     6481 caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg caggataggt
     6541 gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc gcacctggcg
     6601 gtgctcaacg ggaatcctgc tctgcgaggc tggccgtagg ccggccgatc tgaagatcag
     6661 cagttcaacc tgttgatagt acgtactaag ctctcatgtt tcacgtacta agctctcatg
     6721 tttaacgtac taagctctca tgtttaacga actaaaccct catggctaac gtactaagct
     6781 ctcatggcta acgtactaag ctctcatgtt tcacgtacta agctctcatg tttgaacaat
     6841 aaaattaata taaatcagca acttaaatag cctctaaggt tttaagtttt ataagaaaaa
     6901 aaagaatata taaggctttt aaagccttta aggtttaacg gttgtggaca acaagccagg
     6961 gatgtaacgc actgagaagc ccttagagcc tctcaaagca attttgagtg acacaggaac
     7021 acttaacggc tgacatgggg cgcgcccagc tgtctagggc ggcggatttg tcctactcag
     7081 gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt
     7141 tcgttttatt tgatgcct
//
