LOCUS       sequence-418298-        7391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc140 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 7391)
  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 7391)
  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: pBC113
FEATURES             Location/Qualifiers
     source          1..7391
                     /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  3395..3560
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3603..4048
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4049..4135,4136..5470)
                     /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      5553..5647
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5768..5787)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5779..6594
                     /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            6761..6869
                     /note="incP origin of transfer"
     rep_origin      6880..7268
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7299..7385)
                     /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 tctccccatg gcgcagtaag ctctactcgc
     3121 cagcgttaac tccattatgc tagattcgag aactcgctca gttgtacttg ccattgtgaa
     3181 acgtcagacc cattatggcg cagtaagctc tactcgcagc gttaactcca ttatggcgcc
     3241 tcggcgagga gtgggatata tctccgcatg gggatgctgg ttgtcattgc ccctccttcc
     3301 tctaagtgat aaggttactt cctctaaatg accaagcagg gtatctaatc ctgttaacgc
     3361 tcgctcagag aattcgcatg cggagtcgag ggagaaccag ataagtgaaa tctagttcca
     3421 aactattttg tcatttttaa ttttcgtatt agcttacgac gctacaccca gttcccatct
     3481 attttgtcac tcttccctaa ataatcctta aaaactccat ttccacccct cccagttccc
     3541 aactattttg tccgcccaca cgctgagcga attccacgct tgtggaattc ctcgagggag
     3601 ttcacatata cctgccgttc actattattt agtgaaatga gatattatga tattttctga
     3661 attgtgatta aaaaggcaac tttatgccca tgcaacagaa actataaaaa atacagagaa
     3721 tgaaaagaaa cagatagatt ttttagttct ttaggcccgt agtctgcaaa tccttttatg
     3781 attttctatc aaacaaaaga ggaaaataga ccagttgcaa tccaaacgag agtctaatag
     3841 aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat aaagcaggca agacctaaaa
     3901 tgtgtaaagg gcaaagtgta tactttggcg tcacccctta catattttag gtcttttttt
     3961 attgtgcgta actaacttgc catcttcaaa caggagggct ggaagaagca gaccgctaac
     4021 acagtacata aaaaaggaga catgaacgat gaacatcaaa aagtttgcaa aacaagcaac
     4081 agtattaacc tttactaccg cactgctggc aggaggcgca actcaagcgt ttgcgaaaga
     4141 aacgaaccaa aagccatata aggaaacata cggcatttcc catattacac gccatgatat
     4201 gctgcaaatc cctgaacagc aaaaaaatga aaaatatcaa gttcctgaat tcgattcgtc
     4261 cacaattaaa aatatctctt ctgcaaaagg cctggacgtt tgggacagct ggccattaca
     4321 aaacgctgac ggcactgtcg caaactatca cggctaccac atcgtctttg cattagccgg
     4381 agatcctaaa aatgcggatg acacatcgat ttacatgttc tatcaaaaag tcggcgaaac
     4441 ttctattgac agctggaaaa acgctggccg cgtctttaaa gacagcgaca aattcgatgc
     4501 aaatgattct atcctaaaag accaaacaca agaatggtca ggttcagcca catttacatc
     4561 tgacggaaaa atccgtttat tctacactga tttctccggt aaacattacg gcaaacaaac
     4621 actgacaact gcacaagtta acgtatcagc atcagacagc tctttgaaca tcaacggtgt
     4681 agaggattat aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg tacagcagtt
     4741 catcgatgaa ggcaactaca gctcaggcga caaccatacg ctgagagatc ctcactacgt
     4801 agaagataaa ggccacaaat acttagtatt tgaagcaaac actggaactg aagatggcta
     4861 ccaaggcgaa gaatctttat ttaacaaagc atactatggc aaaagcacat cattcttccg
     4921 tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg
     4981 cgctctcggt atgattgagc taaacgatga ttacacactg aaaaaagtga tgaaaccgct
     5041 gattgcatct aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg
     5101 caaatggtac ctgttcactg actcccgcgg atcaaaaatg acgattgacg gcattacgtc
     5161 taacgatatt tacatgcttg gttatgtttc taattcttta actggcccat acaagccgct
     5221 gaacaaaact ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa cctttactta
     5281 ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa gctatatgac
     5341 aaacagagga ttctacgcag acaaacaatc aacgtttgcg ccaagcttcc tgctgaacat
     5401 caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac aattaacagt
     5461 taacaaataa attcgctgag cgaggatgca catgtgaccg agggaagctt gcggccgcgt
     5521 cgtgactggg aaaaccctgg cgactagtct tggactcctg ttgatagatc cagtaatgac
     5581 ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt tttttattgg
     5641 tgagaatcca ggggtcccca ataattacga tttaaatttg tgtctcaaaa tctctgatgt
     5701 tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac
     5761 agtaatacaa ggggtgttat gagccatatt cagcgtgaaa cgagctgtag ccgtccgcgt
     5821 ctgaacagca acatggatgc ggatctgtat ggctataaat gggcgcgtga taacgtgggt
     5881 cagagcggcg cgaccattta tcgtctgtat ggcaaaccgg atgcgccgga actgtttctg
     5941 aaacatggca aaggcagcgt ggcgaacgat gtgaccgatg aaatggtgcg tctgaactgg
     6001 ctgaccgaat ttatgccgct gccgaccatt aaacatttta ttcgcacccc ggatgatgcg
     6061 tggctgctga ccaccgcgat tccgggcaaa accgcgtttc aggtgctgga agaatatccg
     6121 gatagcggcg aaaacattgt ggatgcgctg gccgtgtttc tgcgtcgtct gcatagcatt
     6181 ccggtgtgca actgcccgtt taacagcgat cgtgtgtttc gtctggccca ggcgcagagc
     6241 cgtatgaaca acggcctggt ggatgcgagc gattttgatg atgaacgtaa cggctggccg
     6301 gtggaacagg tgtggaaaga aatgcataaa ctgctgccgt ttagcccgga tagcgtggtg
     6361 acccacggcg attttagcct ggataacctg attttcgatg aaggcaaact gattggctgc
     6421 attgatgtgg gccgtgtggg cattgcggat cgttatcagg atctggccat tctgtggaac
     6481 tgcctgggcg aatttagccc gagcctgcaa aaacgtctgt ttcagaaata tggcattgat
     6541 aatccggata tgaacaaact gcaatttcat ctgatgctgg atgaattttt ctaataatta
     6601 attggaccgc ggtccgcgcg ttgtcctttt ccgctgcata accctgcttc ggggtcatta
     6661 tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg
     6721 ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag
     6781 gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca
     6841 acgggaatcc tgctctgcga ggctggccgt aggccggccg atctgaagat cagcagttca
     6901 acctgttgat agtacgtact aagctctcat gtttcacgta ctaagctctc atgtttaacg
     6961 tactaagctc tcatgtttaa cgaactaaac cctcatggct aacgtactaa gctctcatgg
     7021 ctaacgtact aagctctcat gtttcacgta ctaagctctc atgtttgaac aataaaatta
     7081 atataaatca gcaacttaaa tagcctctaa ggttttaagt tttataagaa aaaaaagaat
     7141 atataaggct tttaaagcct ttaaggttta acggttgtgg acaacaagcc agggatgtaa
     7201 cgcactgaga agcccttaga gcctctcaaa gcaattttga gtgacacagg aacacttaac
     7261 ggctgacatg gggcgcgccc agctgtctag ggcggcggat ttgtcctact caggagagcg
     7321 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     7381 atttgatgcc t
//
