LOCUS       sequence-419093-        6929 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc107 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 6929)
  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 6929)
  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: pBC080
FEATURES             Location/Qualifiers
     source          1..6929
                     /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  2933..3098
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3141..3586
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3587..3673,3674..5008)
                     /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      5091..5185
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5306..5325)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5317..6132
                     /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            6299..6407
                     /note="incP origin of transfer"
     rep_origin      6418..6806
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6837..6923)
                     /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 ccaatggcag
     2701 cctcggcgag gagtgggata tatctccgca taatgatcgc cagcaggagg ccgaactgtc
     2761 tagtcatcat ttggcgcagt aagctctact cgccagcgtt aactccaggg gatgctggtt
     2821 gtcattgccc ctccttcctc taagtgataa ggttacttcc tctaaatgac caagcagggt
     2881 atctaatcct gttaacgctc gctcagagaa ttcgcatgcg gagtcgaggg agaaccagat
     2941 aagtgaaatc tagttccaaa ctattttgtc atttttaatt ttcgtattag cttacgacgc
     3001 tacacccagt tcccatctat tttgtcactc ttccctaaat aatccttaaa aactccattt
     3061 ccacccctcc cagttcccaa ctattttgtc cgcccacacg ctgagcgaat tccacgcttg
     3121 tggaattcct cgagggagtt cacatatacc tgccgttcac tattatttag tgaaatgaga
     3181 tattatgata ttttctgaat tgtgattaaa aaggcaactt tatgcccatg caacagaaac
     3241 tataaaaaat acagagaatg aaaagaaaca gatagatttt ttagttcttt aggcccgtag
     3301 tctgcaaatc cttttatgat tttctatcaa acaaaagagg aaaatagacc agttgcaatc
     3361 caaacgagag tctaatagaa tgaggtcgaa aagtaaatcg cgcgggtttg ttactgataa
     3421 agcaggcaag acctaaaatg tgtaaagggc aaagtgtata ctttggcgtc accccttaca
     3481 tattttaggt ctttttttat tgtgcgtaac taacttgcca tcttcaaaca ggagggctgg
     3541 aagaagcaga ccgctaacac agtacataaa aaaggagaca tgaacgatga acatcaaaaa
     3601 gtttgcaaaa caagcaacag tattaacctt tactaccgca ctgctggcag gaggcgcaac
     3661 tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag gaaacatacg gcatttccca
     3721 tattacacgc catgatatgc tgcaaatccc tgaacagcaa aaaaatgaaa aatatcaagt
     3781 tcctgaattc gattcgtcca caattaaaaa tatctcttct gcaaaaggcc tggacgtttg
     3841 ggacagctgg ccattacaaa acgctgacgg cactgtcgca aactatcacg gctaccacat
     3901 cgtctttgca ttagccggag atcctaaaaa tgcggatgac acatcgattt acatgttcta
     3961 tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac gctggccgcg tctttaaaga
     4021 cagcgacaaa ttcgatgcaa atgattctat cctaaaagac caaacacaag aatggtcagg
     4081 ttcagccaca tttacatctg acggaaaaat ccgtttattc tacactgatt tctccggtaa
     4141 acattacggc aaacaaacac tgacaactgc acaagttaac gtatcagcat cagacagctc
     4201 tttgaacatc aacggtgtag aggattataa atcaatcttt gacggtgacg gaaaaacgta
     4261 tcaaaatgta cagcagttca tcgatgaagg caactacagc tcaggcgaca accatacgct
     4321 gagagatcct cactacgtag aagataaagg ccacaaatac ttagtatttg aagcaaacac
     4381 tggaactgaa gatggctacc aaggcgaaga atctttattt aacaaagcat actatggcaa
     4441 aagcacatca ttcttccgtc aagaaagtca aaaacttctg caaagcgata aaaaacgcac
     4501 ggctgagtta gcaaacggcg ctctcggtat gattgagcta aacgatgatt acacactgaa
     4561 aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca gatgaaattg aacgcgcgaa
     4621 cgtctttaaa atgaacggca aatggtacct gttcactgac tcccgcggat caaaaatgac
     4681 gattgacggc attacgtcta acgatattta catgcttggt tatgtttcta attctttaac
     4741 tggcccatac aagccgctga acaaaactgg ccttgtgtta aaaatggatc ttgatcctaa
     4801 cgatgtaacc tttacttact cacacttcgc tgtacctcaa gcgaaaggaa acaatgtcgt
     4861 gattacaagc tatatgacaa acagaggatt ctacgcagac aaacaatcaa cgtttgcgcc
     4921 aagcttcctg ctgaacatca aaggcaagaa aacatctgtt gtcaaagaca gcatccttga
     4981 acaaggacaa ttaacagtta acaaataaat tcgctgagcg aggatgcaca tgtgaccgag
     5041 ggaagcttgc ggccgcgtcg tgactgggaa aaccctggcg actagtcttg gactcctgtt
     5101 gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg
     5161 ccgggcgttt tttattggtg agaatccagg ggtccccaat aattacgatt taaatttgtg
     5221 tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa
     5281 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca gcgtgaaacg
     5341 agctgtagcc gtccgcgtct gaacagcaac atggatgcgg atctgtatgg ctataaatgg
     5401 gcgcgtgata acgtgggtca gagcggcgcg accatttatc gtctgtatgg caaaccggat
     5461 gcgccggaac tgtttctgaa acatggcaaa ggcagcgtgg cgaacgatgt gaccgatgaa
     5521 atggtgcgtc tgaactggct gaccgaattt atgccgctgc cgaccattaa acattttatt
     5581 cgcaccccgg atgatgcgtg gctgctgacc accgcgattc cgggcaaaac cgcgtttcag
     5641 gtgctggaag aatatccgga tagcggcgaa aacattgtgg atgcgctggc cgtgtttctg
     5701 cgtcgtctgc atagcattcc ggtgtgcaac tgcccgttta acagcgatcg tgtgtttcgt
     5761 ctggcccagg cgcagagccg tatgaacaac ggcctggtgg atgcgagcga ttttgatgat
     5821 gaacgtaacg gctggccggt ggaacaggtg tggaaagaaa tgcataaact gctgccgttt
     5881 agcccggata gcgtggtgac ccacggcgat tttagcctgg ataacctgat tttcgatgaa
     5941 ggcaaactga ttggctgcat tgatgtgggc cgtgtgggca ttgcggatcg ttatcaggat
     6001 ctggccattc tgtggaactg cctgggcgaa tttagcccga gcctgcaaaa acgtctgttt
     6061 cagaaatatg gcattgataa tccggatatg aacaaactgc aatttcatct gatgctggat
     6121 gaatttttct aataattaat tggaccgcgg tccgcgcgtt gtccttttcc gctgcataac
     6181 cctgcttcgg ggtcattata gcgatttttt cggtatatcc atcctttttc gcacgatata
     6241 caggattttg ccaaagggtt cgtgtagact ttccttggtg tatccaacgg cgtcagccgg
     6301 gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc cttcttcact gtcccttatt
     6361 cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg ctggccgtag gccggccgat
     6421 ctgaagatca gcagttcaac ctgttgatag tacgtactaa gctctcatgt ttcacgtact
     6481 aagctctcat gtttaacgta ctaagctctc atgtttaacg aactaaaccc tcatggctaa
     6541 cgtactaagc tctcatggct aacgtactaa gctctcatgt ttcacgtact aagctctcat
     6601 gtttgaacaa taaaattaat ataaatcagc aacttaaata gcctctaagg ttttaagttt
     6661 tataagaaaa aaaagaatat ataaggcttt taaagccttt aaggtttaac ggttgtggac
     6721 aacaagccag ggatgtaacg cactgagaag cccttagagc ctctcaaagc aattttgagt
     6781 gacacaggaa cacttaacgg ctgacatggg gcgcgcccag ctgtctaggg cggcggattt
     6841 gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc
     6901 gactgagcct ttcgttttat ttgatgcct
//
