LOCUS       sequence-418300-        7209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc154 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 7209)
  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 7209)
  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: pBC127
FEATURES             Location/Qualifiers
     source          1..7209
                     /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  3213..3378
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3421..3866
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3867..3953,3954..5288)
                     /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      5371..5465
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5586..5605)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5597..6412
                     /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            6579..6687
                     /note="incP origin of transfer"
     rep_origin      6698..7086
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7117..7203)
                     /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 ttctcccctg tacgaatctc ctactatggt cttcaaccgt gtcattatgg
     2941 gtccttggtt gtccctcatt aggtgatccg aattatgact agacagttcg gcctcctgct
     3001 ggcgatcatt aatgaacacc taacgtagct atataggaca cagttgcatt atggagttaa
     3061 cgctggcgag tagagcttac tgcgccaggg gatgctggtt gtcattgccc ctccttcctc
     3121 taagtgataa ggttacttcc tctaaatgac caagcagggt atctaatcct gttaacgctc
     3181 gctcagagaa ttcgcatgcg gagtcgaggg agaaccagat aagtgaaatc tagttccaaa
     3241 ctattttgtc atttttaatt ttcgtattag cttacgacgc tacacccagt tcccatctat
     3301 tttgtcactc ttccctaaat aatccttaaa aactccattt ccacccctcc cagttcccaa
     3361 ctattttgtc cgcccacacg ctgagcgaat tccacgcttg tggaattcct cgagggagtt
     3421 cacatatacc tgccgttcac tattatttag tgaaatgaga tattatgata ttttctgaat
     3481 tgtgattaaa aaggcaactt tatgcccatg caacagaaac tataaaaaat acagagaatg
     3541 aaaagaaaca gatagatttt ttagttcttt aggcccgtag tctgcaaatc cttttatgat
     3601 tttctatcaa acaaaagagg aaaatagacc agttgcaatc caaacgagag tctaatagaa
     3661 tgaggtcgaa aagtaaatcg cgcgggtttg ttactgataa agcaggcaag acctaaaatg
     3721 tgtaaagggc aaagtgtata ctttggcgtc accccttaca tattttaggt ctttttttat
     3781 tgtgcgtaac taacttgcca tcttcaaaca ggagggctgg aagaagcaga ccgctaacac
     3841 agtacataaa aaaggagaca tgaacgatga acatcaaaaa gtttgcaaaa caagcaacag
     3901 tattaacctt tactaccgca ctgctggcag gaggcgcaac tcaagcgttt gcgaaagaaa
     3961 cgaaccaaaa gccatataag gaaacatacg gcatttccca tattacacgc catgatatgc
     4021 tgcaaatccc tgaacagcaa aaaaatgaaa aatatcaagt tcctgaattc gattcgtcca
     4081 caattaaaaa tatctcttct gcaaaaggcc tggacgtttg ggacagctgg ccattacaaa
     4141 acgctgacgg cactgtcgca aactatcacg gctaccacat cgtctttgca ttagccggag
     4201 atcctaaaaa tgcggatgac acatcgattt acatgttcta tcaaaaagtc ggcgaaactt
     4261 ctattgacag ctggaaaaac gctggccgcg tctttaaaga cagcgacaaa ttcgatgcaa
     4321 atgattctat cctaaaagac caaacacaag aatggtcagg ttcagccaca tttacatctg
     4381 acggaaaaat ccgtttattc tacactgatt tctccggtaa acattacggc aaacaaacac
     4441 tgacaactgc acaagttaac gtatcagcat cagacagctc tttgaacatc aacggtgtag
     4501 aggattataa atcaatcttt gacggtgacg gaaaaacgta tcaaaatgta cagcagttca
     4561 tcgatgaagg caactacagc tcaggcgaca accatacgct gagagatcct cactacgtag
     4621 aagataaagg ccacaaatac ttagtatttg aagcaaacac tggaactgaa gatggctacc
     4681 aaggcgaaga atctttattt aacaaagcat actatggcaa aagcacatca ttcttccgtc
     4741 aagaaagtca aaaacttctg caaagcgata aaaaacgcac ggctgagtta gcaaacggcg
     4801 ctctcggtat gattgagcta aacgatgatt acacactgaa aaaagtgatg aaaccgctga
     4861 ttgcatctaa cacagtaaca gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca
     4921 aatggtacct gttcactgac tcccgcggat caaaaatgac gattgacggc attacgtcta
     4981 acgatattta catgcttggt tatgtttcta attctttaac tggcccatac aagccgctga
     5041 acaaaactgg ccttgtgtta aaaatggatc ttgatcctaa cgatgtaacc tttacttact
     5101 cacacttcgc tgtacctcaa gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa
     5161 acagaggatt ctacgcagac aaacaatcaa cgtttgcgcc aagcttcctg ctgaacatca
     5221 aaggcaagaa aacatctgtt gtcaaagaca gcatccttga acaaggacaa ttaacagtta
     5281 acaaataaat tcgctgagcg aggatgcaca tgtgaccgag ggaagcttgc ggccgcgtcg
     5341 tgactgggaa aaccctggcg actagtcttg gactcctgtt gatagatcca gtaatgacct
     5401 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     5461 agaatccagg ggtccccaat aattacgatt taaatttgtg tctcaaaatc tctgatgtta
     5521 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     5581 taatacaagg ggtgttatga gccatattca gcgtgaaacg agctgtagcc gtccgcgtct
     5641 gaacagcaac atggatgcgg atctgtatgg ctataaatgg gcgcgtgata acgtgggtca
     5701 gagcggcgcg accatttatc gtctgtatgg caaaccggat gcgccggaac tgtttctgaa
     5761 acatggcaaa ggcagcgtgg cgaacgatgt gaccgatgaa atggtgcgtc tgaactggct
     5821 gaccgaattt atgccgctgc cgaccattaa acattttatt cgcaccccgg atgatgcgtg
     5881 gctgctgacc accgcgattc cgggcaaaac cgcgtttcag gtgctggaag aatatccgga
     5941 tagcggcgaa aacattgtgg atgcgctggc cgtgtttctg cgtcgtctgc atagcattcc
     6001 ggtgtgcaac tgcccgttta acagcgatcg tgtgtttcgt ctggcccagg cgcagagccg
     6061 tatgaacaac ggcctggtgg atgcgagcga ttttgatgat gaacgtaacg gctggccggt
     6121 ggaacaggtg tggaaagaaa tgcataaact gctgccgttt agcccggata gcgtggtgac
     6181 ccacggcgat tttagcctgg ataacctgat tttcgatgaa ggcaaactga ttggctgcat
     6241 tgatgtgggc cgtgtgggca ttgcggatcg ttatcaggat ctggccattc tgtggaactg
     6301 cctgggcgaa tttagcccga gcctgcaaaa acgtctgttt cagaaatatg gcattgataa
     6361 tccggatatg aacaaactgc aatttcatct gatgctggat gaatttttct aataattaat
     6421 tggaccgcgg tccgcgcgtt gtccttttcc gctgcataac cctgcttcgg ggtcattata
     6481 gcgatttttt cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt
     6541 cgtgtagact ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc
     6601 ccacccgcga gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac
     6661 gggaatcctg ctctgcgagg ctggccgtag gccggccgat ctgaagatca gcagttcaac
     6721 ctgttgatag tacgtactaa gctctcatgt ttcacgtact aagctctcat gtttaacgta
     6781 ctaagctctc atgtttaacg aactaaaccc tcatggctaa cgtactaagc tctcatggct
     6841 aacgtactaa gctctcatgt ttcacgtact aagctctcat gtttgaacaa taaaattaat
     6901 ataaatcagc aacttaaata gcctctaagg ttttaagttt tataagaaaa aaaagaatat
     6961 ataaggcttt taaagccttt aaggtttaac ggttgtggac aacaagccag ggatgtaacg
     7021 cactgagaag cccttagagc ctctcaaagc aattttgagt gacacaggaa cacttaacgg
     7081 ctgacatggg gcgcgcccag ctgtctaggg cggcggattt gtcctactca ggagagcgtt
     7141 caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat
     7201 ttgatgcct
//
