LOCUS       sequence-418867-        6963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc096 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 6963)
  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 6963)
  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: pBC069
FEATURES             Location/Qualifiers
     source          1..6963
                     /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  2967..3132
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3175..3620
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3621..3707,3708..5042)
                     /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      5125..5219
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5340..5359)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5351..6166
                     /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            6333..6441
                     /note="incP origin of transfer"
     rep_origin      6452..6840
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6871..6957)
                     /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 ctccccggca agtacaactg
     2701 agcgagttct cgaatctagc attaatgatg actagacagt tcggcctcct gctggcgatc
     2761 attaatgcaa ctgtgtccta tatagctacg ttaggtgttc attaatgtaa ccgcaaggct
     2821 ggaagtgaaa cgtcagaccc aggggatgct ggttgtcatt gcccctcctt cctctaagtg
     2881 ataaggttac ttcctctaaa tgaccaagca gggtatctaa tcctgttaac gctcgctcag
     2941 agaattcgca tgcggagtcg agggagaacc agataagtga aatctagttc caaactattt
     3001 tgtcattttt aattttcgta ttagcttacg acgctacacc cagttcccat ctattttgtc
     3061 actcttccct aaataatcct taaaaactcc atttccaccc ctcccagttc ccaactattt
     3121 tgtccgccca cacgctgagc gaattccacg cttgtggaat tcctcgaggg agttcacata
     3181 tacctgccgt tcactattat ttagtgaaat gagatattat gatattttct gaattgtgat
     3241 taaaaaggca actttatgcc catgcaacag aaactataaa aaatacagag aatgaaaaga
     3301 aacagataga ttttttagtt ctttaggccc gtagtctgca aatcctttta tgattttcta
     3361 tcaaacaaaa gaggaaaata gaccagttgc aatccaaacg agagtctaat agaatgaggt
     3421 cgaaaagtaa atcgcgcggg tttgttactg ataaagcagg caagacctaa aatgtgtaaa
     3481 gggcaaagtg tatactttgg cgtcacccct tacatatttt aggtcttttt ttattgtgcg
     3541 taactaactt gccatcttca aacaggaggg ctggaagaag cagaccgcta acacagtaca
     3601 taaaaaagga gacatgaacg atgaacatca aaaagtttgc aaaacaagca acagtattaa
     3661 cctttactac cgcactgctg gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc
     3721 aaaagccata taaggaaaca tacggcattt cccatattac acgccatgat atgctgcaaa
     3781 tccctgaaca gcaaaaaaat gaaaaatatc aagttcctga attcgattcg tccacaatta
     3841 aaaatatctc ttctgcaaaa ggcctggacg tttgggacag ctggccatta caaaacgctg
     3901 acggcactgt cgcaaactat cacggctacc acatcgtctt tgcattagcc ggagatccta
     3961 aaaatgcgga tgacacatcg atttacatgt tctatcaaaa agtcggcgaa acttctattg
     4021 acagctggaa aaacgctggc cgcgtcttta aagacagcga caaattcgat gcaaatgatt
     4081 ctatcctaaa agaccaaaca caagaatggt caggttcagc cacatttaca tctgacggaa
     4141 aaatccgttt attctacact gatttctccg gtaaacatta cggcaaacaa acactgacaa
     4201 ctgcacaagt taacgtatca gcatcagaca gctctttgaa catcaacggt gtagaggatt
     4261 ataaatcaat ctttgacggt gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg
     4321 aaggcaacta cagctcaggc gacaaccata cgctgagaga tcctcactac gtagaagata
     4381 aaggccacaa atacttagta tttgaagcaa acactggaac tgaagatggc taccaaggcg
     4441 aagaatcttt atttaacaaa gcatactatg gcaaaagcac atcattcttc cgtcaagaaa
     4501 gtcaaaaact tctgcaaagc gataaaaaac gcacggctga gttagcaaac ggcgctctcg
     4561 gtatgattga gctaaacgat gattacacac tgaaaaaagt gatgaaaccg ctgattgcat
     4621 ctaacacagt aacagatgaa attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt
     4681 acctgttcac tgactcccgc ggatcaaaaa tgacgattga cggcattacg tctaacgata
     4741 tttacatgct tggttatgtt tctaattctt taactggccc atacaagccg ctgaacaaaa
     4801 ctggccttgt gttaaaaatg gatcttgatc ctaacgatgt aacctttact tactcacact
     4861 tcgctgtacc tcaagcgaaa ggaaacaatg tcgtgattac aagctatatg acaaacagag
     4921 gattctacgc agacaaacaa tcaacgtttg cgccaagctt cctgctgaac atcaaaggca
     4981 agaaaacatc tgttgtcaaa gacagcatcc ttgaacaagg acaattaaca gttaacaaat
     5041 aaattcgctg agcgaggatg cacatgtgac cgagggaagc ttgcggccgc gtcgtgactg
     5101 ggaaaaccct ggcgactagt cttggactcc tgttgataga tccagtaatg acctcagaac
     5161 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc
     5221 caggggtccc caataattac gatttaaatt tgtgtctcaa aatctctgat gttacattgc
     5281 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     5341 aaggggtgtt atgagccata ttcagcgtga aacgagctgt agccgtccgc gtctgaacag
     5401 caacatggat gcggatctgt atggctataa atgggcgcgt gataacgtgg gtcagagcgg
     5461 cgcgaccatt tatcgtctgt atggcaaacc ggatgcgccg gaactgtttc tgaaacatgg
     5521 caaaggcagc gtggcgaacg atgtgaccga tgaaatggtg cgtctgaact ggctgaccga
     5581 atttatgccg ctgccgacca ttaaacattt tattcgcacc ccggatgatg cgtggctgct
     5641 gaccaccgcg attccgggca aaaccgcgtt tcaggtgctg gaagaatatc cggatagcgg
     5701 cgaaaacatt gtggatgcgc tggccgtgtt tctgcgtcgt ctgcatagca ttccggtgtg
     5761 caactgcccg tttaacagcg atcgtgtgtt tcgtctggcc caggcgcaga gccgtatgaa
     5821 caacggcctg gtggatgcga gcgattttga tgatgaacgt aacggctggc cggtggaaca
     5881 ggtgtggaaa gaaatgcata aactgctgcc gtttagcccg gatagcgtgg tgacccacgg
     5941 cgattttagc ctggataacc tgattttcga tgaaggcaaa ctgattggct gcattgatgt
     6001 gggccgtgtg ggcattgcgg atcgttatca ggatctggcc attctgtgga actgcctggg
     6061 cgaatttagc ccgagcctgc aaaaacgtct gtttcagaaa tatggcattg ataatccgga
     6121 tatgaacaaa ctgcaatttc atctgatgct ggatgaattt ttctaataat taattggacc
     6181 gcggtccgcg cgttgtcctt ttccgctgca taaccctgct tcggggtcat tatagcgatt
     6241 ttttcggtat atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta
     6301 gactttcctt ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc
     6361 gcgagcgggt gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat
     6421 cctgctctgc gaggctggcc gtaggccggc cgatctgaag atcagcagtt caacctgttg
     6481 atagtacgta ctaagctctc atgtttcacg tactaagctc tcatgtttaa cgtactaagc
     6541 tctcatgttt aacgaactaa accctcatgg ctaacgtact aagctctcat ggctaacgta
     6601 ctaagctctc atgtttcacg tactaagctc tcatgtttga acaataaaat taatataaat
     6661 cagcaactta aatagcctct aaggttttaa gttttataag aaaaaaaaga atatataagg
     6721 cttttaaagc ctttaaggtt taacggttgt ggacaacaag ccagggatgt aacgcactga
     6781 gaagccctta gagcctctca aagcaatttt gagtgacaca ggaacactta acggctgaca
     6841 tggggcgcgc ccagctgtct agggcggcgg atttgtccta ctcaggagag cgttcaccga
     6901 caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg
     6961 cct
//
