LOCUS       sequence-420619-        6971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc109 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 6971)
  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 6971)
  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: pBC082
FEATURES             Location/Qualifiers
     source          1..6971
                     /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  2975..3140
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3183..3628
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3629..3715,3716..5050)
                     /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      5133..5227
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5348..5367)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5359..6174
                     /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            6341..6449
                     /note="incP origin of transfer"
     rep_origin      6460..6848
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6879..6965)
                     /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 ccatggcagc
     2701 ctcggcgagg agtgggatat atctccgcat tatgatgact agacagttcg gcctcctgct
     2761 ggcgatcatt aatgctagat tcgagaactc gctcagttgt acttgccatt aatgcgaaac
     2821 gcttgacgtc agccgacggt tgtaggcatg gggatgctgg ttgtcattgc ccctccttcc
     2881 tctaagtgat aaggttactt cctctaaatg accaagcagg gtatctaatc ctgttaacgc
     2941 tcgctcagag aattcgcatg cggagtcgag ggagaaccag ataagtgaaa tctagttcca
     3001 aactattttg tcatttttaa ttttcgtatt agcttacgac gctacaccca gttcccatct
     3061 attttgtcac tcttccctaa ataatcctta aaaactccat ttccacccct cccagttccc
     3121 aactattttg tccgcccaca cgctgagcga attccacgct tgtggaattc ctcgagggag
     3181 ttcacatata cctgccgttc actattattt agtgaaatga gatattatga tattttctga
     3241 attgtgatta aaaaggcaac tttatgccca tgcaacagaa actataaaaa atacagagaa
     3301 tgaaaagaaa cagatagatt ttttagttct ttaggcccgt agtctgcaaa tccttttatg
     3361 attttctatc aaacaaaaga ggaaaataga ccagttgcaa tccaaacgag agtctaatag
     3421 aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat aaagcaggca agacctaaaa
     3481 tgtgtaaagg gcaaagtgta tactttggcg tcacccctta catattttag gtcttttttt
     3541 attgtgcgta actaacttgc catcttcaaa caggagggct ggaagaagca gaccgctaac
     3601 acagtacata aaaaaggaga catgaacgat gaacatcaaa aagtttgcaa aacaagcaac
     3661 agtattaacc tttactaccg cactgctggc aggaggcgca actcaagcgt ttgcgaaaga
     3721 aacgaaccaa aagccatata aggaaacata cggcatttcc catattacac gccatgatat
     3781 gctgcaaatc cctgaacagc aaaaaaatga aaaatatcaa gttcctgaat tcgattcgtc
     3841 cacaattaaa aatatctctt ctgcaaaagg cctggacgtt tgggacagct ggccattaca
     3901 aaacgctgac ggcactgtcg caaactatca cggctaccac atcgtctttg cattagccgg
     3961 agatcctaaa aatgcggatg acacatcgat ttacatgttc tatcaaaaag tcggcgaaac
     4021 ttctattgac agctggaaaa acgctggccg cgtctttaaa gacagcgaca aattcgatgc
     4081 aaatgattct atcctaaaag accaaacaca agaatggtca ggttcagcca catttacatc
     4141 tgacggaaaa atccgtttat tctacactga tttctccggt aaacattacg gcaaacaaac
     4201 actgacaact gcacaagtta acgtatcagc atcagacagc tctttgaaca tcaacggtgt
     4261 agaggattat aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg tacagcagtt
     4321 catcgatgaa ggcaactaca gctcaggcga caaccatacg ctgagagatc ctcactacgt
     4381 agaagataaa ggccacaaat acttagtatt tgaagcaaac actggaactg aagatggcta
     4441 ccaaggcgaa gaatctttat ttaacaaagc atactatggc aaaagcacat cattcttccg
     4501 tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg
     4561 cgctctcggt atgattgagc taaacgatga ttacacactg aaaaaagtga tgaaaccgct
     4621 gattgcatct aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg
     4681 caaatggtac ctgttcactg actcccgcgg atcaaaaatg acgattgacg gcattacgtc
     4741 taacgatatt tacatgcttg gttatgtttc taattcttta actggcccat acaagccgct
     4801 gaacaaaact ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa cctttactta
     4861 ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa gctatatgac
     4921 aaacagagga ttctacgcag acaaacaatc aacgtttgcg ccaagcttcc tgctgaacat
     4981 caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac aattaacagt
     5041 taacaaataa attcgctgag cgaggatgca catgtgaccg agggaagctt gcggccgcgt
     5101 cgtgactggg aaaaccctgg cgactagtct tggactcctg ttgatagatc cagtaatgac
     5161 ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt tttttattgg
     5221 tgagaatcca ggggtcccca ataattacga tttaaatttg tgtctcaaaa tctctgatgt
     5281 tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac
     5341 agtaatacaa ggggtgttat gagccatatt cagcgtgaaa cgagctgtag ccgtccgcgt
     5401 ctgaacagca acatggatgc ggatctgtat ggctataaat gggcgcgtga taacgtgggt
     5461 cagagcggcg cgaccattta tcgtctgtat ggcaaaccgg atgcgccgga actgtttctg
     5521 aaacatggca aaggcagcgt ggcgaacgat gtgaccgatg aaatggtgcg tctgaactgg
     5581 ctgaccgaat ttatgccgct gccgaccatt aaacatttta ttcgcacccc ggatgatgcg
     5641 tggctgctga ccaccgcgat tccgggcaaa accgcgtttc aggtgctgga agaatatccg
     5701 gatagcggcg aaaacattgt ggatgcgctg gccgtgtttc tgcgtcgtct gcatagcatt
     5761 ccggtgtgca actgcccgtt taacagcgat cgtgtgtttc gtctggccca ggcgcagagc
     5821 cgtatgaaca acggcctggt ggatgcgagc gattttgatg atgaacgtaa cggctggccg
     5881 gtggaacagg tgtggaaaga aatgcataaa ctgctgccgt ttagcccgga tagcgtggtg
     5941 acccacggcg attttagcct ggataacctg attttcgatg aaggcaaact gattggctgc
     6001 attgatgtgg gccgtgtggg cattgcggat cgttatcagg atctggccat tctgtggaac
     6061 tgcctgggcg aatttagccc gagcctgcaa aaacgtctgt ttcagaaata tggcattgat
     6121 aatccggata tgaacaaact gcaatttcat ctgatgctgg atgaattttt ctaataatta
     6181 attggaccgc ggtccgcgcg ttgtcctttt ccgctgcata accctgcttc ggggtcatta
     6241 tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg
     6301 ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag
     6361 gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca
     6421 acgggaatcc tgctctgcga ggctggccgt aggccggccg atctgaagat cagcagttca
     6481 acctgttgat agtacgtact aagctctcat gtttcacgta ctaagctctc atgtttaacg
     6541 tactaagctc tcatgtttaa cgaactaaac cctcatggct aacgtactaa gctctcatgg
     6601 ctaacgtact aagctctcat gtttcacgta ctaagctctc atgtttgaac aataaaatta
     6661 atataaatca gcaacttaaa tagcctctaa ggttttaagt tttataagaa aaaaaagaat
     6721 atataaggct tttaaagcct ttaaggttta acggttgtgg acaacaagcc agggatgtaa
     6781 cgcactgaga agcccttaga gcctctcaaa gcaattttga gtgacacagg aacacttaac
     6841 ggctgacatg gggcgcgccc agctgtctag ggcggcggat ttgtcctact caggagagcg
     6901 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     6961 atttgatgcc t
//
