LOCUS       sequence-418897-        7373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc142 through Tn7 
            bacterial insertion, Tetracyline selectable marker and far-red-FP 
            fluorescent marker mPlum.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7373)
  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 7373)
  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: pBC115
FEATURES             Location/Qualifiers
     source          1..7373
                     /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(689..694,695..711,712..717)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             765..1955
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(1117..1136)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    complement(2035..2082)
                     /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(2110..2115,2116..2131,2132..2138)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2146..2162
                     /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             2185..2868
                     /codon_start=1
                     /product="far-red monomeric derivative of DsRed fluorescent
                     protein (Wang et al., 2004)"
                     /label=mPlum
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKEHMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     RLKVTKGGPLPFAWDILSPQIMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGEFIYKVKVRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEMK
                     MRLRLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRH
                     STGAA"
     primer_bind     2780..2803
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      2882..2976
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3377..3542
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3585..4030
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4031..4117,4118..5452)
                     /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      5535..5629
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5750..5769)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5761..6576
                     /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            6743..6851
                     /note="incP origin of transfer"
     rep_origin      6862..7250
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7281..7367)
                     /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 aatgaagaat tctcatgttt gacagcttat catcgataag ctttaatgcg
      721 gtagtttatc acagttaaat tgctaacgca gtcaggcacc gtgtatgaaa tctaacaatg
      781 cgctcatcgt catcctcggc accgtcaccc tggatgctgt aggcataggc ttggttatgc
      841 cggtactgcc gggcctcttg cgggatatcg tccattccga cagcatcgcc agtcactatg
      901 gcgtgctgct agcgctatat gcgttgatgc aatttctatg cgcacccgtt ctcggagcac
      961 tgtccgaccg ctttggccgc cgcccagtcc tgctcgcttc gctacttgga gccactatcg
     1021 actacgcgat catggcgacc acacccgtcc tgtggatcct ctacgccgga cgcatcgtgg
     1081 ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     1141 gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg
     1201 caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg
     1261 cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata
     1321 agggagagcg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     1381 cgcggggcat gactatcgtc gccgcactta tgactgtttt ctttatcatg caactcgtag
     1441 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     1501 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     1561 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     1621 ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca
     1681 ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca
     1741 ggcaggtaga tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc
     1801 taacttcgat cattggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat
     1861 ggaacgggtt ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc
     1921 gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa
     1981 cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg gcttgaagtt
     2041 cctatacttt ctagagaata ggaacttcgg aataggtact tccgctacta gaattcgagc
     2101 ggggaattct tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac
     2161 aattttcaca caggaaacag ctaaatggtg agcaagggcg aggaggtcat caaggagttc
     2221 atgcgcttca aggagcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     2281 gagggcgagg gccgccccta cgagggcacc cagaccgcca ggctgaaggt gaccaagggt
     2341 ggccccctgc ccttcgcctg ggacatcctg tcccctcaga tcatgtacgg ctccaaggcc
     2401 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     2461 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     2521 tccctgcagg acggcgagtt catctacaag gtgaaggtgc gcggcaccaa cttcccctcc
     2581 gacggccccg taatgcagaa aaagaccatg ggctgggagg cctcctccga gcggatgtac
     2641 cccgaggacg gcgccctgaa gggcgagatg aagatgaggc tgaggctgaa ggacggcggc
     2701 cactacgacg ccgaggtcaa gaccacctac atggccaaga agcccgtgca gctgcccggc
     2761 gcctacaaga ccgacatcaa gctggacatc acctcccaca acgaggacta caccatcgtg
     2821 gaacagtacg agcgcgccga gggccgccac tccaccggcg ccgcttaatt agctgagctt
     2881 ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg
     2941 ctcggttgcc gccgggcgtt ttttattggt gagaatctcc cgctttacga attcccatgg
     3001 ggagcatgcg gagttacgtc atccccacct tccgctgcgt tcttcatcga tgcccgctta
     3061 gtgatatgct taaaactcca agcacagcct tctccccaat gtaaccgcaa ggctggaagt
     3121 gaaacgtcag acccaaatga tcgccagcag gaggccgaac tgtctagtca tcataatggc
     3181 agcctcggcg aggagtggga tatatctccg cataatgcta gattcgagaa ctcgctcagt
     3241 tgtacttgcc aggggatgct ggttgtcatt gcccctcctt cctctaagtg ataaggttac
     3301 ttcctctaaa tgaccaagca gggtatctaa tcctgttaac gctcgctcag agaattcgca
     3361 tgcggagtcg agggagaacc agataagtga aatctagttc caaactattt tgtcattttt
     3421 aattttcgta ttagcttacg acgctacacc cagttcccat ctattttgtc actcttccct
     3481 aaataatcct taaaaactcc atttccaccc ctcccagttc ccaactattt tgtccgccca
     3541 cacgctgagc gaattccacg cttgtggaat tcctcgaggg agttcacata tacctgccgt
     3601 tcactattat ttagtgaaat gagatattat gatattttct gaattgtgat taaaaaggca
     3661 actttatgcc catgcaacag aaactataaa aaatacagag aatgaaaaga aacagataga
     3721 ttttttagtt ctttaggccc gtagtctgca aatcctttta tgattttcta tcaaacaaaa
     3781 gaggaaaata gaccagttgc aatccaaacg agagtctaat agaatgaggt cgaaaagtaa
     3841 atcgcgcggg tttgttactg ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg
     3901 tatactttgg cgtcacccct tacatatttt aggtcttttt ttattgtgcg taactaactt
     3961 gccatcttca aacaggaggg ctggaagaag cagaccgcta acacagtaca taaaaaagga
     4021 gacatgaacg atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac
     4081 cgcactgctg gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata
     4141 taaggaaaca tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca
     4201 gcaaaaaaat gaaaaatatc aagttcctga attcgattcg tccacaatta aaaatatctc
     4261 ttctgcaaaa ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt
     4321 cgcaaactat cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga
     4381 tgacacatcg atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa
     4441 aaacgctggc cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa
     4501 agaccaaaca caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt
     4561 attctacact gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt
     4621 taacgtatca gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat
     4681 ctttgacggt gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta
     4741 cagctcaggc gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa
     4801 atacttagta tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt
     4861 atttaacaaa gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact
     4921 tctgcaaagc gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga
     4981 gctaaacgat gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt
     5041 aacagatgaa attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac
     5101 tgactcccgc ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct
     5161 tggttatgtt tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt
     5221 gttaaaaatg gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc
     5281 tcaagcgaaa ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc
     5341 agacaaacaa tcaacgtttg cgccaagctt cctgctgaac atcaaaggca agaaaacatc
     5401 tgttgtcaaa gacagcatcc ttgaacaagg acaattaaca gttaacaaat aaattcgctg
     5461 agcgaggatg cacatgtgac cgagggaagc ttgcggccgc gtcgtgactg ggaaaaccct
     5521 ggcgactagt cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga
     5581 tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc caggggtccc
     5641 caataattac gatttaaatt tgtgtctcaa aatctctgat gttacattgc acaagataaa
     5701 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     5761 atgagccata ttcagcgtga aacgagctgt agccgtccgc gtctgaacag caacatggat
     5821 gcggatctgt atggctataa atgggcgcgt gataacgtgg gtcagagcgg cgcgaccatt
     5881 tatcgtctgt atggcaaacc ggatgcgccg gaactgtttc tgaaacatgg caaaggcagc
     5941 gtggcgaacg atgtgaccga tgaaatggtg cgtctgaact ggctgaccga atttatgccg
     6001 ctgccgacca ttaaacattt tattcgcacc ccggatgatg cgtggctgct gaccaccgcg
     6061 attccgggca aaaccgcgtt tcaggtgctg gaagaatatc cggatagcgg cgaaaacatt
     6121 gtggatgcgc tggccgtgtt tctgcgtcgt ctgcatagca ttccggtgtg caactgcccg
     6181 tttaacagcg atcgtgtgtt tcgtctggcc caggcgcaga gccgtatgaa caacggcctg
     6241 gtggatgcga gcgattttga tgatgaacgt aacggctggc cggtggaaca ggtgtggaaa
     6301 gaaatgcata aactgctgcc gtttagcccg gatagcgtgg tgacccacgg cgattttagc
     6361 ctggataacc tgattttcga tgaaggcaaa ctgattggct gcattgatgt gggccgtgtg
     6421 ggcattgcgg atcgttatca ggatctggcc attctgtgga actgcctggg cgaatttagc
     6481 ccgagcctgc aaaaacgtct gtttcagaaa tatggcattg ataatccgga tatgaacaaa
     6541 ctgcaatttc atctgatgct ggatgaattt ttctaataat taattggacc gcggtccgcg
     6601 cgttgtcctt ttccgctgca taaccctgct tcggggtcat tatagcgatt ttttcggtat
     6661 atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta gactttcctt
     6721 ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc gcgagcgggt
     6781 gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat cctgctctgc
     6841 gaggctggcc gtaggccggc cgatctgaag atcagcagtt caacctgttg atagtacgta
     6901 ctaagctctc atgtttcacg tactaagctc tcatgtttaa cgtactaagc tctcatgttt
     6961 aacgaactaa accctcatgg ctaacgtact aagctctcat ggctaacgta ctaagctctc
     7021 atgtttcacg tactaagctc tcatgtttga acaataaaat taatataaat cagcaactta
     7081 aatagcctct aaggttttaa gttttataag aaaaaaaaga atatataagg cttttaaagc
     7141 ctttaaggtt taacggttgt ggacaacaag ccagggatgt aacgcactga gaagccctta
     7201 gagcctctca aagcaatttt gagtgacaca ggaacactta acggctgaca tggggcgcgc
     7261 ccagctgtct agggcggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag
     7321 ataaaacgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cct
//
