LOCUS       sequence-418870-        6961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc099 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 6961)
  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 6961)
  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: pBC072
FEATURES             Location/Qualifiers
     source          1..6961
                     /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  2965..3130
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3173..3618
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3619..3705,3706..5040)
                     /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      5123..5217
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5338..5357)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5349..6164
                     /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            6331..6439
                     /note="incP origin of transfer"
     rep_origin      6450..6838
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6869..6955)
                     /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 agttaacgct
     2701 ggcgagtaga gcttactgcg ccaaatgact aactgatcct attcacgtgt tcctttacac
     2761 attaatgcta gattcgagaa ctcgctcagt tgtacttgcc aaatgcaact gtgtcctata
     2821 tagctacgtt aggtgttcag gggatgctgg ttgtcattgc ccctccttcc tctaagtgat
     2881 aaggttactt cctctaaatg accaagcagg gtatctaatc ctgttaacgc tcgctcagag
     2941 aattcgcatg cggagtcgag ggagaaccag ataagtgaaa tctagttcca aactattttg
     3001 tcatttttaa ttttcgtatt agcttacgac gctacaccca gttcccatct attttgtcac
     3061 tcttccctaa ataatcctta aaaactccat ttccacccct cccagttccc aactattttg
     3121 tccgcccaca cgctgagcga attccacgct tgtggaattc ctcgagggag ttcacatata
     3181 cctgccgttc actattattt agtgaaatga gatattatga tattttctga attgtgatta
     3241 aaaaggcaac tttatgccca tgcaacagaa actataaaaa atacagagaa tgaaaagaaa
     3301 cagatagatt ttttagttct ttaggcccgt agtctgcaaa tccttttatg attttctatc
     3361 aaacaaaaga ggaaaataga ccagttgcaa tccaaacgag agtctaatag aatgaggtcg
     3421 aaaagtaaat cgcgcgggtt tgttactgat aaagcaggca agacctaaaa tgtgtaaagg
     3481 gcaaagtgta tactttggcg tcacccctta catattttag gtcttttttt attgtgcgta
     3541 actaacttgc catcttcaaa caggagggct ggaagaagca gaccgctaac acagtacata
     3601 aaaaaggaga catgaacgat gaacatcaaa aagtttgcaa aacaagcaac agtattaacc
     3661 tttactaccg cactgctggc aggaggcgca actcaagcgt ttgcgaaaga aacgaaccaa
     3721 aagccatata aggaaacata cggcatttcc catattacac gccatgatat gctgcaaatc
     3781 cctgaacagc aaaaaaatga aaaatatcaa gttcctgaat tcgattcgtc cacaattaaa
     3841 aatatctctt ctgcaaaagg cctggacgtt tgggacagct ggccattaca aaacgctgac
     3901 ggcactgtcg caaactatca cggctaccac atcgtctttg cattagccgg agatcctaaa
     3961 aatgcggatg acacatcgat ttacatgttc tatcaaaaag tcggcgaaac ttctattgac
     4021 agctggaaaa acgctggccg cgtctttaaa gacagcgaca aattcgatgc aaatgattct
     4081 atcctaaaag accaaacaca agaatggtca ggttcagcca catttacatc tgacggaaaa
     4141 atccgtttat tctacactga tttctccggt aaacattacg gcaaacaaac actgacaact
     4201 gcacaagtta acgtatcagc atcagacagc tctttgaaca tcaacggtgt agaggattat
     4261 aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg tacagcagtt catcgatgaa
     4321 ggcaactaca gctcaggcga caaccatacg ctgagagatc ctcactacgt agaagataaa
     4381 ggccacaaat acttagtatt tgaagcaaac actggaactg aagatggcta ccaaggcgaa
     4441 gaatctttat ttaacaaagc atactatggc aaaagcacat cattcttccg tcaagaaagt
     4501 caaaaacttc tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg cgctctcggt
     4561 atgattgagc taaacgatga ttacacactg aaaaaagtga tgaaaccgct gattgcatct
     4621 aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg caaatggtac
     4681 ctgttcactg actcccgcgg atcaaaaatg acgattgacg gcattacgtc taacgatatt
     4741 tacatgcttg gttatgtttc taattcttta actggcccat acaagccgct gaacaaaact
     4801 ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa cctttactta ctcacacttc
     4861 gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa gctatatgac aaacagagga
     4921 ttctacgcag acaaacaatc aacgtttgcg ccaagcttcc tgctgaacat caaaggcaag
     4981 aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac aattaacagt taacaaataa
     5041 attcgctgag cgaggatgca catgtgaccg agggaagctt gcggccgcgt cgtgactggg
     5101 aaaaccctgg cgactagtct tggactcctg ttgatagatc cagtaatgac ctcagaactc
     5161 catctggatt tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca
     5221 ggggtcccca ataattacga tttaaatttg tgtctcaaaa tctctgatgt tacattgcac
     5281 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     5341 ggggtgttat gagccatatt cagcgtgaaa cgagctgtag ccgtccgcgt ctgaacagca
     5401 acatggatgc ggatctgtat ggctataaat gggcgcgtga taacgtgggt cagagcggcg
     5461 cgaccattta tcgtctgtat ggcaaaccgg atgcgccgga actgtttctg aaacatggca
     5521 aaggcagcgt ggcgaacgat gtgaccgatg aaatggtgcg tctgaactgg ctgaccgaat
     5581 ttatgccgct gccgaccatt aaacatttta ttcgcacccc ggatgatgcg tggctgctga
     5641 ccaccgcgat tccgggcaaa accgcgtttc aggtgctgga agaatatccg gatagcggcg
     5701 aaaacattgt ggatgcgctg gccgtgtttc tgcgtcgtct gcatagcatt ccggtgtgca
     5761 actgcccgtt taacagcgat cgtgtgtttc gtctggccca ggcgcagagc cgtatgaaca
     5821 acggcctggt ggatgcgagc gattttgatg atgaacgtaa cggctggccg gtggaacagg
     5881 tgtggaaaga aatgcataaa ctgctgccgt ttagcccgga tagcgtggtg acccacggcg
     5941 attttagcct ggataacctg attttcgatg aaggcaaact gattggctgc attgatgtgg
     6001 gccgtgtggg cattgcggat cgttatcagg atctggccat tctgtggaac tgcctgggcg
     6061 aatttagccc gagcctgcaa aaacgtctgt ttcagaaata tggcattgat aatccggata
     6121 tgaacaaact gcaatttcat ctgatgctgg atgaattttt ctaataatta attggaccgc
     6181 ggtccgcgcg ttgtcctttt ccgctgcata accctgcttc ggggtcatta tagcgatttt
     6241 ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga
     6301 ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc
     6361 gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca acgggaatcc
     6421 tgctctgcga ggctggccgt aggccggccg atctgaagat cagcagttca acctgttgat
     6481 agtacgtact aagctctcat gtttcacgta ctaagctctc atgtttaacg tactaagctc
     6541 tcatgtttaa cgaactaaac cctcatggct aacgtactaa gctctcatgg ctaacgtact
     6601 aagctctcat gtttcacgta ctaagctctc atgtttgaac aataaaatta atataaatca
     6661 gcaacttaaa tagcctctaa ggttttaagt tttataagaa aaaaaagaat atataaggct
     6721 tttaaagcct ttaaggttta acggttgtgg acaacaagcc agggatgtaa cgcactgaga
     6781 agcccttaga gcctctcaaa gcaattttga gtgacacagg aacacttaac ggctgacatg
     6841 gggcgcgccc agctgtctag ggcggcggat ttgtcctact caggagagcg ttcaccgaca
     6901 aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc
     6961 t
//
