LOCUS       sequence-418869-        6958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc098 through Tn7 
            bacterial insertion, Gentamicin selectable marker and tagRFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6958)
  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 6958)
  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: pBC071
FEATURES             Location/Qualifiers
     source          1..6958
                     /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)."
     terminator      2471..2565
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  2962..3127
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3170..3615
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3616..3702,3703..5037)
                     /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      5120..5214
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5335..5354)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5346..6161
                     /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            6328..6436
                     /note="incP origin of transfer"
     rep_origin      6447..6835
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6866..6952)
                     /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 aatttttgac aattaatcat
     1681 cggctcgtat aatgtgtgga attgtgagcg gataacaatt ttcacacagg aaacagctaa
     1741 atggtgagta aaggtgaaga gttgattaaa gagaacatgc atatgaagtt atacatggag
     1801 ggaactgtca ataatcacca ctttaagtgt acatcagagg gtgagggaaa gccatacgag
     1861 ggaacccaaa ctatgagaat caaagtagtg gagggaggtc ctcttccatt tgcttttgat
     1921 atactagcaa caagtttcat gtatggttcc aggaccttca ttaaccatac tcagggaatc
     1981 cctgacttct ttaaacagtc ttttcctgaa ggttttacat gggagagggt taccacttac
     2041 gaggacggtg gagtcttgac agcaacccag gacacttcat tacaagatgg atgcctaata
     2101 tacaatgtga aaattagggg tgtgaatttc cctagtaacg gaccagttat gcagaagaaa
     2161 acactaggtt gggaagctaa tactgaaatg ttgtaccctg ccgacggagg tttagaaggt
     2221 agatccgaca tggcccttaa gctagtcgga ggtggacact tgatctgtaa ctttaaaacc
     2281 acatataggt ctaagaagcc agcaaagaat ctaaaaatgc ctggtgttta ctatgtggac
     2341 catagactag aaaggataaa agaagcagac aaagaaactt acgtggagca acatgaggtc
     2401 gccgtcgcta ggtattgcga cttaccttcc aagctaggtc acaaattgaa cgcttaatta
     2461 gctgagcttg gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg
     2521 ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatctccc gctttacgaa
     2581 ttcccatggg gagcatgcgg agttacgtca tccccacctt ccgctgcgtt cttcatcgat
     2641 gcccgcttag tgatatgctt aaaactccaa gcacagcctt ctccccatgg cagcctcggc
     2701 gaggagtggg atatatctcc gcattatgat gactagacag ttcggcctcc tgctggcgat
     2761 catttggcgc agtaagctct actcgccagc gttaactcca ttctacaacc gtcggctgac
     2821 gtcaagcgtt tcgcatgggg atgctggttg tcattgcccc tccttcctct aagtgataag
     2881 gttacttcct ctaaatgacc aagcagggta tctaatcctg ttaacgctcg ctcagagaat
     2941 tcgcatgcgg agtcgaggga gaaccagata agtgaaatct agttccaaac tattttgtca
     3001 tttttaattt tcgtattagc ttacgacgct acacccagtt cccatctatt ttgtcactct
     3061 tccctaaata atccttaaaa actccatttc cacccctccc agttcccaac tattttgtcc
     3121 gcccacacgc tgagcgaatt ccacgcttgt ggaattcctc gagggagttc acatatacct
     3181 gccgttcact attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa
     3241 aggcaacttt atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag
     3301 atagattttt tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa
     3361 caaaagagga aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa
     3421 agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca
     3481 aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact
     3541 aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa
     3601 aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt
     3661 actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag
     3721 ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct
     3781 gaacagcaaa aaaatgaaaa atatcaagtt cctgaattcg attcgtccac aattaaaaat
     3841 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc
     3901 actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat
     3961 gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc
     4021 tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc
     4081 ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc
     4141 cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca
     4201 caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa
     4261 tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc
     4321 aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc
     4381 cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa
     4441 tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa
     4501 aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg
     4561 attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac
     4621 acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg
     4681 ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac
     4741 atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc
     4801 cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct
     4861 gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc
     4921 tacgcagaca aacaatcaac gtttgcgcca agcttcctgc tgaacatcaa aggcaagaaa
     4981 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaatt
     5041 cgctgagcga ggatgcacat gtgaccgagg gaagcttgcg gccgcgtcgt gactgggaaa
     5101 accctggcga ctagtcttgg actcctgttg atagatccag taatgacctc agaactccat
     5161 ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaggg
     5221 gtccccaata attacgattt aaatttgtgt ctcaaaatct ctgatgttac attgcacaag
     5281 ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     5341 gtgttatgag ccatattcag cgtgaaacga gctgtagccg tccgcgtctg aacagcaaca
     5401 tggatgcgga tctgtatggc tataaatggg cgcgtgataa cgtgggtcag agcggcgcga
     5461 ccatttatcg tctgtatggc aaaccggatg cgccggaact gtttctgaaa catggcaaag
     5521 gcagcgtggc gaacgatgtg accgatgaaa tggtgcgtct gaactggctg accgaattta
     5581 tgccgctgcc gaccattaaa cattttattc gcaccccgga tgatgcgtgg ctgctgacca
     5641 ccgcgattcc gggcaaaacc gcgtttcagg tgctggaaga atatccggat agcggcgaaa
     5701 acattgtgga tgcgctggcc gtgtttctgc gtcgtctgca tagcattccg gtgtgcaact
     5761 gcccgtttaa cagcgatcgt gtgtttcgtc tggcccaggc gcagagccgt atgaacaacg
     5821 gcctggtgga tgcgagcgat tttgatgatg aacgtaacgg ctggccggtg gaacaggtgt
     5881 ggaaagaaat gcataaactg ctgccgttta gcccggatag cgtggtgacc cacggcgatt
     5941 ttagcctgga taacctgatt ttcgatgaag gcaaactgat tggctgcatt gatgtgggcc
     6001 gtgtgggcat tgcggatcgt tatcaggatc tggccattct gtggaactgc ctgggcgaat
     6061 ttagcccgag cctgcaaaaa cgtctgtttc agaaatatgg cattgataat ccggatatga
     6121 acaaactgca atttcatctg atgctggatg aatttttcta ataattaatt ggaccgcggt
     6181 ccgcgcgttg tccttttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc
     6241 ggtatatcca tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt
     6301 tccttggtgt atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag
     6361 cgggtgttcc ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc
     6421 tctgcgaggc tggccgtagg ccggccgatc tgaagatcag cagttcaacc tgttgatagt
     6481 acgtactaag ctctcatgtt tcacgtacta agctctcatg tttaacgtac taagctctca
     6541 tgtttaacga actaaaccct catggctaac gtactaagct ctcatggcta acgtactaag
     6601 ctctcatgtt tcacgtacta agctctcatg tttgaacaat aaaattaata taaatcagca
     6661 acttaaatag cctctaaggt tttaagtttt ataagaaaaa aaagaatata taaggctttt
     6721 aaagccttta aggtttaacg gttgtggaca acaagccagg gatgtaacgc actgagaagc
     6781 ccttagagcc tctcaaagca attttgagtg acacaggaac acttaacggc tgacatgggg
     6841 cgcgcccagc tgtctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac
     6901 aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcct
//
