LOCUS       sequence-419131-        6969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc108 through Tn7 
            bacterial insertion, Gentamicin selectable marker and GFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6969)
  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 6969)
  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: pBC081
FEATURES             Location/Qualifiers
     source          1..6969
                     /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)."
     promoter        join(711..716,717..733,734..739)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             928..1461
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     protein_bind    complement(1591..1638)
                     /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(1666..1671,1672..1687,1688..1694)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1702..1718
                     /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)."
     CDS             1741..2454
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSVMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1771..1799)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2394..2414
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2477..2571
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  2973..3138
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3181..3626
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3627..3713,3714..5048)
                     /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      5131..5225
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5346..5365)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5357..6172
                     /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            6339..6447
                     /note="incP origin of transfer"
     rep_origin      6458..6846
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6877..6963)
                     /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 aagatcccct gattcccttt gtcaacagca atggatcgaa ttgacataag
      721 cctgttcggt tcgtaaactg taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa
      781 ccttgaccga acgcagcggt ggtaacggcg cagtggcggt tttcatggct tgttatgact
      841 gtttttttgt acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc
      901 gatgtttgat gttatggagc agcaacgatg ttacgcagca gcaacgatgt tacgcagcag
      961 ggcagtcgcc ctaaaacaaa gttaggtggc tcaagtatgg gcatcattcg cacatgtagg
     1021 ctcggccctg accaagtcaa atccatgcgg gctgctcttg atcttttcgg tcgtgagttc
     1081 ggagacgtag ccacctactc ccaacatcag ccggactccg attacctcgg gaacttgctc
     1141 cgtagtaaga cattcatcgc gcttgctgcc ttcgaccaag aagcggttgt tggcgctctc
     1201 gcggcttacg ttctgcccaa gtttgagcag ccgcgtagtg agatctatat ctatgatctc
     1261 gcagtctccg gcgagcaccg gaggcagggc attgccaccg cgctcatcaa tctcctcaag
     1321 catgaggcca acgcgcttgg tgcttatgtg atctacgtgc aagcagatta cggtgacgat
     1381 cccgcagtgg ctctctatac aaagttgggc atacgggaag aagtgatgca ctttgatatc
     1441 gacccaagta ccgccaccta acaattcgtt caagccgaga tcggcttccc ggccgcggag
     1501 ttgttcggta aattgtcaca acgccgcggc caattcgatc tagaattcca ttgagtaagt
     1561 ttttaagcac atcagcttca aaagcgctct gaagttccta tactttctag agaataggaa
     1621 cttcggaata ggtacttccg ctactagaat tcgagcgggg aattcttgac aattaatcat
     1681 cggctcgtat aatgtgtgga attgtgagcg gataacaatt ttcacacagg aaacagctaa
     1741 atgcgtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
     1801 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga
     1861 aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt
     1921 gtcactactt tcggttatgg tgttcaatgc tttgcgagat acccagatca tatgaaacag
     1981 catgactttt tcaagagtgt catgcccgaa ggttatgtac aggaaagaac tatatttttc
     2041 aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt
     2101 aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa
     2161 ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga
     2221 atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac
     2281 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac
     2341 ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt
     2401 cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaatgagct
     2461 taattagctg agcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
     2521 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tctcccgctt
     2581 tacgaattcc catggggagc atgcggagtt acgtcatccc caccttccgc tgcgttcttc
     2641 atcgatgccc gcttagtgat atgcttaaaa ctccaagcac agccttctcc ccatggcaag
     2701 tacaactgag cgagttctcg aatctagcat taatgcgaaa cgcttgacgt cagccgacgg
     2761 ttgtaggcat ttgcgaaacg cttgacgtca gccgacggtt gtaggcataa tgtcggatca
     2821 cctaatgagg gacaaccaag gacccatggg gatgctggtt gtcattgccc ctccttcctc
     2881 taagtgataa ggttacttcc tctaaatgac caagcagggt atctaatcct gttaacgctc
     2941 gctcagagaa ttcgcatgcg gagtcgaggg agaaccagat aagtgaaatc tagttccaaa
     3001 ctattttgtc atttttaatt ttcgtattag cttacgacgc tacacccagt tcccatctat
     3061 tttgtcactc ttccctaaat aatccttaaa aactccattt ccacccctcc cagttcccaa
     3121 ctattttgtc cgcccacacg ctgagcgaat tccacgcttg tggaattcct cgagggagtt
     3181 cacatatacc tgccgttcac tattatttag tgaaatgaga tattatgata ttttctgaat
     3241 tgtgattaaa aaggcaactt tatgcccatg caacagaaac tataaaaaat acagagaatg
     3301 aaaagaaaca gatagatttt ttagttcttt aggcccgtag tctgcaaatc cttttatgat
     3361 tttctatcaa acaaaagagg aaaatagacc agttgcaatc caaacgagag tctaatagaa
     3421 tgaggtcgaa aagtaaatcg cgcgggtttg ttactgataa agcaggcaag acctaaaatg
     3481 tgtaaagggc aaagtgtata ctttggcgtc accccttaca tattttaggt ctttttttat
     3541 tgtgcgtaac taacttgcca tcttcaaaca ggagggctgg aagaagcaga ccgctaacac
     3601 agtacataaa aaaggagaca tgaacgatga acatcaaaaa gtttgcaaaa caagcaacag
     3661 tattaacctt tactaccgca ctgctggcag gaggcgcaac tcaagcgttt gcgaaagaaa
     3721 cgaaccaaaa gccatataag gaaacatacg gcatttccca tattacacgc catgatatgc
     3781 tgcaaatccc tgaacagcaa aaaaatgaaa aatatcaagt tcctgaattc gattcgtcca
     3841 caattaaaaa tatctcttct gcaaaaggcc tggacgtttg ggacagctgg ccattacaaa
     3901 acgctgacgg cactgtcgca aactatcacg gctaccacat cgtctttgca ttagccggag
     3961 atcctaaaaa tgcggatgac acatcgattt acatgttcta tcaaaaagtc ggcgaaactt
     4021 ctattgacag ctggaaaaac gctggccgcg tctttaaaga cagcgacaaa ttcgatgcaa
     4081 atgattctat cctaaaagac caaacacaag aatggtcagg ttcagccaca tttacatctg
     4141 acggaaaaat ccgtttattc tacactgatt tctccggtaa acattacggc aaacaaacac
     4201 tgacaactgc acaagttaac gtatcagcat cagacagctc tttgaacatc aacggtgtag
     4261 aggattataa atcaatcttt gacggtgacg gaaaaacgta tcaaaatgta cagcagttca
     4321 tcgatgaagg caactacagc tcaggcgaca accatacgct gagagatcct cactacgtag
     4381 aagataaagg ccacaaatac ttagtatttg aagcaaacac tggaactgaa gatggctacc
     4441 aaggcgaaga atctttattt aacaaagcat actatggcaa aagcacatca ttcttccgtc
     4501 aagaaagtca aaaacttctg caaagcgata aaaaacgcac ggctgagtta gcaaacggcg
     4561 ctctcggtat gattgagcta aacgatgatt acacactgaa aaaagtgatg aaaccgctga
     4621 ttgcatctaa cacagtaaca gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca
     4681 aatggtacct gttcactgac tcccgcggat caaaaatgac gattgacggc attacgtcta
     4741 acgatattta catgcttggt tatgtttcta attctttaac tggcccatac aagccgctga
     4801 acaaaactgg ccttgtgtta aaaatggatc ttgatcctaa cgatgtaacc tttacttact
     4861 cacacttcgc tgtacctcaa gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa
     4921 acagaggatt ctacgcagac aaacaatcaa cgtttgcgcc aagcttcctg ctgaacatca
     4981 aaggcaagaa aacatctgtt gtcaaagaca gcatccttga acaaggacaa ttaacagtta
     5041 acaaataaat tcgctgagcg aggatgcaca tgtgaccgag ggaagcttgc ggccgcgtcg
     5101 tgactgggaa aaccctggcg actagtcttg gactcctgtt gatagatcca gtaatgacct
     5161 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     5221 agaatccagg ggtccccaat aattacgatt taaatttgtg tctcaaaatc tctgatgtta
     5281 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     5341 taatacaagg ggtgttatga gccatattca gcgtgaaacg agctgtagcc gtccgcgtct
     5401 gaacagcaac atggatgcgg atctgtatgg ctataaatgg gcgcgtgata acgtgggtca
     5461 gagcggcgcg accatttatc gtctgtatgg caaaccggat gcgccggaac tgtttctgaa
     5521 acatggcaaa ggcagcgtgg cgaacgatgt gaccgatgaa atggtgcgtc tgaactggct
     5581 gaccgaattt atgccgctgc cgaccattaa acattttatt cgcaccccgg atgatgcgtg
     5641 gctgctgacc accgcgattc cgggcaaaac cgcgtttcag gtgctggaag aatatccgga
     5701 tagcggcgaa aacattgtgg atgcgctggc cgtgtttctg cgtcgtctgc atagcattcc
     5761 ggtgtgcaac tgcccgttta acagcgatcg tgtgtttcgt ctggcccagg cgcagagccg
     5821 tatgaacaac ggcctggtgg atgcgagcga ttttgatgat gaacgtaacg gctggccggt
     5881 ggaacaggtg tggaaagaaa tgcataaact gctgccgttt agcccggata gcgtggtgac
     5941 ccacggcgat tttagcctgg ataacctgat tttcgatgaa ggcaaactga ttggctgcat
     6001 tgatgtgggc cgtgtgggca ttgcggatcg ttatcaggat ctggccattc tgtggaactg
     6061 cctgggcgaa tttagcccga gcctgcaaaa acgtctgttt cagaaatatg gcattgataa
     6121 tccggatatg aacaaactgc aatttcatct gatgctggat gaatttttct aataattaat
     6181 tggaccgcgg tccgcgcgtt gtccttttcc gctgcataac cctgcttcgg ggtcattata
     6241 gcgatttttt cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt
     6301 cgtgtagact ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc
     6361 ccacccgcga gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac
     6421 gggaatcctg ctctgcgagg ctggccgtag gccggccgat ctgaagatca gcagttcaac
     6481 ctgttgatag tacgtactaa gctctcatgt ttcacgtact aagctctcat gtttaacgta
     6541 ctaagctctc atgtttaacg aactaaaccc tcatggctaa cgtactaagc tctcatggct
     6601 aacgtactaa gctctcatgt ttcacgtact aagctctcat gtttgaacaa taaaattaat
     6661 ataaatcagc aacttaaata gcctctaagg ttttaagttt tataagaaaa aaaagaatat
     6721 ataaggcttt taaagccttt aaggtttaac ggttgtggac aacaagccag ggatgtaacg
     6781 cactgagaag cccttagagc ctctcaaagc aattttgagt gacacaggaa cacttaacgg
     6841 ctgacatggg gcgcgcccag ctgtctaggg cggcggattt gtcctactca ggagagcgtt
     6901 caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat
     6961 ttgatgcct
//
