LOCUS       sequence-418904-        7165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc155 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 7165)
  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 7165)
  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: pBC128
FEATURES             Location/Qualifiers
     source          1..7165
                     /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  3169..3334
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3377..3822
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3823..3909,3910..5244)
                     /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      5327..5421
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5542..5561)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5553..6368
                     /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            6535..6643
                     /note="incP origin of transfer"
     rep_origin      6654..7042
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7073..7159)
                     /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 atcccaatcc tcgccgaggc tgatggcaag
     2941 tacaactgag cgagttctcg aatctagcat aatgcctaca accgtcggct gacgtcaagc
     3001 gtttcgcatt accgcaaggc tggaagtgaa acgtcagacc catggggatg ctggttgtca
     3061 ttgcccctcc ttcctctaag tgataaggtt acttcctcta aatgaccaag cagggtatct
     3121 aatcctgtta acgctcgctc agagaattcg catgcggagt cgagggagaa ccagataagt
     3181 gaaatctagt tccaaactat tttgtcattt ttaattttcg tattagctta cgacgctaca
     3241 cccagttccc atctattttg tcactcttcc ctaaataatc cttaaaaact ccatttccac
     3301 ccctcccagt tcccaactat tttgtccgcc cacacgctga gcgaattcca cgcttgtgga
     3361 attcctcgag ggagttcaca tatacctgcc gttcactatt atttagtgaa atgagatatt
     3421 atgatatttt ctgaattgtg attaaaaagg caactttatg cccatgcaac agaaactata
     3481 aaaaatacag agaatgaaaa gaaacagata gattttttag ttctttaggc ccgtagtctg
     3541 caaatccttt tatgattttc tatcaaacaa aagaggaaaa tagaccagtt gcaatccaaa
     3601 cgagagtcta atagaatgag gtcgaaaagt aaatcgcgcg ggtttgttac tgataaagca
     3661 ggcaagacct aaaatgtgta aagggcaaag tgtatacttt ggcgtcaccc cttacatatt
     3721 ttaggtcttt ttttattgtg cgtaactaac ttgccatctt caaacaggag ggctggaaga
     3781 agcagaccgc taacacagta cataaaaaag gagacatgaa cgatgaacat caaaaagttt
     3841 gcaaaacaag caacagtatt aacctttact accgcactgc tggcaggagg cgcaactcaa
     3901 gcgtttgcga aagaaacgaa ccaaaagcca tataaggaaa catacggcat ttcccatatt
     3961 acacgccatg atatgctgca aatccctgaa cagcaaaaaa atgaaaaata tcaagttcct
     4021 gaattcgatt cgtccacaat taaaaatatc tcttctgcaa aaggcctgga cgtttgggac
     4081 agctggccat tacaaaacgc tgacggcact gtcgcaaact atcacggcta ccacatcgtc
     4141 tttgcattag ccggagatcc taaaaatgcg gatgacacat cgatttacat gttctatcaa
     4201 aaagtcggcg aaacttctat tgacagctgg aaaaacgctg gccgcgtctt taaagacagc
     4261 gacaaattcg atgcaaatga ttctatccta aaagaccaaa cacaagaatg gtcaggttca
     4321 gccacattta catctgacgg aaaaatccgt ttattctaca ctgatttctc cggtaaacat
     4381 tacggcaaac aaacactgac aactgcacaa gttaacgtat cagcatcaga cagctctttg
     4441 aacatcaacg gtgtagagga ttataaatca atctttgacg gtgacggaaa aacgtatcaa
     4501 aatgtacagc agttcatcga tgaaggcaac tacagctcag gcgacaacca tacgctgaga
     4561 gatcctcact acgtagaaga taaaggccac aaatacttag tatttgaagc aaacactgga
     4621 actgaagatg gctaccaagg cgaagaatct ttatttaaca aagcatacta tggcaaaagc
     4681 acatcattct tccgtcaaga aagtcaaaaa cttctgcaaa gcgataaaaa acgcacggct
     4741 gagttagcaa acggcgctct cggtatgatt gagctaaacg atgattacac actgaaaaaa
     4801 gtgatgaaac cgctgattgc atctaacaca gtaacagatg aaattgaacg cgcgaacgtc
     4861 tttaaaatga acggcaaatg gtacctgttc actgactccc gcggatcaaa aatgacgatt
     4921 gacggcatta cgtctaacga tatttacatg cttggttatg tttctaattc tttaactggc
     4981 ccatacaagc cgctgaacaa aactggcctt gtgttaaaaa tggatcttga tcctaacgat
     5041 gtaaccttta cttactcaca cttcgctgta cctcaagcga aaggaaacaa tgtcgtgatt
     5101 acaagctata tgacaaacag aggattctac gcagacaaac aatcaacgtt tgcgccaagc
     5161 ttcctgctga acatcaaagg caagaaaaca tctgttgtca aagacagcat ccttgaacaa
     5221 ggacaattaa cagttaacaa ataaattcgc tgagcgagga tgcacatgtg accgagggaa
     5281 gcttgcggcc gcgtcgtgac tgggaaaacc ctggcgacta gtcttggact cctgttgata
     5341 gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg
     5401 gcgtttttta ttggtgagaa tccaggggtc cccaataatt acgatttaaa tttgtgtctc
     5461 aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt
     5521 ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcagcgt gaaacgagct
     5581 gtagccgtcc gcgtctgaac agcaacatgg atgcggatct gtatggctat aaatgggcgc
     5641 gtgataacgt gggtcagagc ggcgcgacca tttatcgtct gtatggcaaa ccggatgcgc
     5701 cggaactgtt tctgaaacat ggcaaaggca gcgtggcgaa cgatgtgacc gatgaaatgg
     5761 tgcgtctgaa ctggctgacc gaatttatgc cgctgccgac cattaaacat tttattcgca
     5821 ccccggatga tgcgtggctg ctgaccaccg cgattccggg caaaaccgcg tttcaggtgc
     5881 tggaagaata tccggatagc ggcgaaaaca ttgtggatgc gctggccgtg tttctgcgtc
     5941 gtctgcatag cattccggtg tgcaactgcc cgtttaacag cgatcgtgtg tttcgtctgg
     6001 cccaggcgca gagccgtatg aacaacggcc tggtggatgc gagcgatttt gatgatgaac
     6061 gtaacggctg gccggtggaa caggtgtgga aagaaatgca taaactgctg ccgtttagcc
     6121 cggatagcgt ggtgacccac ggcgatttta gcctggataa cctgattttc gatgaaggca
     6181 aactgattgg ctgcattgat gtgggccgtg tgggcattgc ggatcgttat caggatctgg
     6241 ccattctgtg gaactgcctg ggcgaattta gcccgagcct gcaaaaacgt ctgtttcaga
     6301 aatatggcat tgataatccg gatatgaaca aactgcaatt tcatctgatg ctggatgaat
     6361 ttttctaata attaattgga ccgcggtccg cgcgttgtcc ttttccgctg cataaccctg
     6421 cttcggggtc attatagcga ttttttcggt atatccatcc tttttcgcac gatatacagg
     6481 attttgccaa agggttcgtg tagactttcc ttggtgtatc caacggcgtc agccgggcag
     6541 gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc cttattcgca
     6601 cctggcggtg ctcaacggga atcctgctct gcgaggctgg ccgtaggccg gccgatctga
     6661 agatcagcag ttcaacctgt tgatagtacg tactaagctc tcatgtttca cgtactaagc
     6721 tctcatgttt aacgtactaa gctctcatgt ttaacgaact aaaccctcat ggctaacgta
     6781 ctaagctctc atggctaacg tactaagctc tcatgtttca cgtactaagc tctcatgttt
     6841 gaacaataaa attaatataa atcagcaact taaatagcct ctaaggtttt aagttttata
     6901 agaaaaaaaa gaatatataa ggcttttaaa gcctttaagg tttaacggtt gtggacaaca
     6961 agccagggat gtaacgcact gagaagccct tagagcctct caaagcaatt ttgagtgaca
     7021 caggaacact taacggctga catggggcgc gcccagctgt ctagggcggc ggatttgtcc
     7081 tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact
     7141 gagcctttcg ttttatttga tgcct
//
