LOCUS       sequence-418906-        7139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc158 through Tn7 
            bacterial insertion, Kanamycin 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 7139)
  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 7139)
  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: pBC131
FEATURES             Location/Qualifiers
     source          1..7139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      297..391
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      493..579
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    complement(612..659)
                     /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)."
     terminator      complement(670..922)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(942..1493)
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(1535..1714)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     protein_bind    complement(1792..1839)
                     /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(1867..1872,1873..1888,1889..1895)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1903..1919
                     /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             1942..2625
                     /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     2537..2560
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      2639..2733
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3132..3297
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3340..3785
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3786..3872,3873..5207)
                     /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      5290..5384
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5505..5524)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5516..6331
                     /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            6498..6606
                     /note="incP origin of transfer"
     rep_origin      6617..7005
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7036..7122)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ggataacaat ttcacacagg aggccgccta ggccgcggcc gcgcgaattt gccgaattcg
       61 gatccggagg gagtgtgggc ggacaataaa gtcttaaact gaacaaaata gatctaaact
      121 atgacaataa agtcttaaac tagacagaat agttgtaaac tgaaatcagt ccagttatgc
      181 tgtgaaaaag catactggac ttttgttatg gctaaagcaa actcttcatt ttctgaagtg
      241 caaattgccc gtcgtattaa agaggggcgt ggcgctgcaa gaattcaagc ttggaggact
      301 cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg
      361 ttgccgccgg gcgtttttta ttggtgagaa tccaagctag actgcgatga gtggcagggc
      421 ggggcgtaat ttcttaaggc agttattggt gcccttaaac gcctggggta atgactctct
      481 agcttgaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
      541 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgcccgccgc tgcaagaatt
      601 caagcttgga ggaagttcct atactttcta gagaatagga acttcggaat aggaacttca
      661 atgccggtcg atctagtaac atagatgaca ccgcgcgcga taatttatcc tagtttgcgc
      721 gctatatttt gttttctatc gcgtattaaa tgtataattg cgggactcta atcataaaaa
      781 cccatctcat aaataacgtc atgcattaca tgttaattat tacatgctta acgtaattca
      841 acagaaatta tatgataatc atcgcaagac cggcaacagg attcaatctt aagaaacttt
      901 attgccaaat gtttgaacga tctgcttgac tctaggggtc atcagatttc ggtgacgggc
      961 aggaccggac ggggcggcac cggcaggctg aagtccagct gccagaaacc cacgtcatgc
     1021 cagttcccgt gcttgaagcc ggccgcccgc agcatgccgc ggggggcata tccgagcgcc
     1081 tcgtgcatgc gcacgctcgg gtcgttgggc agcccgatga cagcgaccac gctcttgaag
     1141 ccctgtgcct ccagggactt cagcaggtgg gtgtagagcg tggagcccag tcccgtccgc
     1201 tggtggcggg gggagacgta cacggtcgac tcggccgtcc agtcgtaggc gttgcgtgcc
     1261 ttccagggac ccgcgtaggc gatgccggcg acctcgccgt ccacctcggc gacgagccag
     1321 ggatagcgct cccgcagacg gacgaggtcg tccgtccact cctgcggttc ctgcggctcg
     1381 gtacggaagt tgaccgtgct tgtctcgatg tagtggttga cgatggtgca gaccgccggc
     1441 atgtccgcct cggtggcacg gcggatgtcg gccgggcgtc gttctgggct catggtagat
     1501 cccctcgatc gagttgagag tgaatatgag actctaattg gataccgagg ggaatttatg
     1561 gaacgtcagt ggagcatttt tgacaagaaa tatttgctag ctgatagtga ccttaggcga
     1621 cttttgaacg cgcaataatg gtttctgacg tatgtgctta gctcattaaa ctccagaaac
     1681 ccgcggctca gtggctcctt caacgttgcg gttctgtcag ttccaaacgt aaaacggctt
     1741 gtcccgcgtc atcggcgggg gtcataacgt gactccctta attctcagct tgaagttcct
     1801 atactttcta gagaatagga acttcggaat aggtacttcc gctactagaa ttcgagcggg
     1861 gaattcttga caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat
     1921 tttcacacag gaaacagcta aatggtgagc aagggcgagg aggtcatcaa ggagttcatg
     1981 cgcttcaagg agcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     2041 ggcgagggcc gcccctacga gggcacccag accgccaggc tgaaggtgac caagggtggc
     2101 cccctgccct tcgcctggga catcctgtcc cctcagatca tgtacggctc caaggcctac
     2161 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     2221 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     2281 ctgcaggacg gcgagttcat ctacaaggtg aaggtgcgcg gcaccaactt cccctccgac
     2341 ggccccgtaa tgcagaaaaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     2401 gaggacggcg ccctgaaggg cgagatgaag atgaggctga ggctgaagga cggcggccac
     2461 tacgacgccg aggtcaagac cacctacatg gccaagaagc ccgtgcagct gcccggcgcc
     2521 tacaagaccg acatcaagct ggacatcacc tcccacaacg aggactacac catcgtggaa
     2581 cagtacgagc gcgccgaggg ccgccactcc accggcgccg cttaattagc tgagcttgga
     2641 ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc
     2701 ggttgccgcc gggcgttttt tattggtgag aatctcccgc tttacgaatt cccatgggga
     2761 gcatgcggag ttacgtcatc cccaccttcc gctgcgttct tcatcgatgc ccgcttagtg
     2821 atatgcttaa aactccaagc acagccttct cccccggatc acctaatgag ggacaaccaa
     2881 ggacccatta tgatgactag acagttcggc ctcctgctgg cgatcattaa tgtacgaatc
     2941 tcctactatg gtcttcaacc gtgtcataat gaacacctaa cgtagctata taggacacag
     3001 ttgcatgggg atgctggttg tcattgcccc tccttcctct aagtgataag gttacttcct
     3061 ctaaatgacc aagcagggta tctaatcctg ttaacgctcg ctcagagaat tcgcatgcgg
     3121 agtcgaggga gaaccagata agtgaaatct agttccaaac tattttgtca tttttaattt
     3181 tcgtattagc ttacgacgct acacccagtt cccatctatt ttgtcactct tccctaaata
     3241 atccttaaaa actccatttc cacccctccc agttcccaac tattttgtcc gcccacacgc
     3301 tgagcgaatt ccacgcttgt ggaattcctc gagggagttc acatatacct gccgttcact
     3361 attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa aggcaacttt
     3421 atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag atagattttt
     3481 tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga
     3541 aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc
     3601 gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac
     3661 tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact aacttgccat
     3721 cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa aaggagacat
     3781 gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt actaccgcac
     3841 tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag ccatataagg
     3901 aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct gaacagcaaa
     3961 aaaatgaaaa atatcaagtt cctgaattcg attcgtccac aattaaaaat atctcttctg
     4021 caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc actgtcgcaa
     4081 actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat gcggatgaca
     4141 catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg
     4201 ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc
     4261 aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc cgtttattct
     4321 acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca caagttaacg
     4381 tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa tcaatctttg
     4441 acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc aactacagct
     4501 caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc cacaaatact
     4561 tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa tctttattta
     4621 acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa aaacttctgc
     4681 aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg attgagctaa
     4741 acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac acagtaacag
     4801 atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg ttcactgact
     4861 cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac atgcttggtt
     4921 atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc cttgtgttaa
     4981 aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct gtacctcaag
     5041 cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc tacgcagaca
     5101 aacaatcaac gtttgcgcca agcttcctgc tgaacatcaa aggcaagaaa acatctgttg
     5161 tcaaagacag catccttgaa caaggacaat taacagttaa caaataaatt cgctgagcga
     5221 ggatgcacat gtgaccgagg gaagcttgcg gccgcgtcgt gactgggaaa accctggcga
     5281 ctagtcttgg actcctgttg atagatccag taatgacctc agaactccat ctggatttgt
     5341 tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaggg gtccccaata
     5401 attacgattt aaatttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     5461 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     5521 ccatattcag cgtgaaacga gctgtagccg tccgcgtctg aacagcaaca tggatgcgga
     5581 tctgtatggc tataaatggg cgcgtgataa cgtgggtcag agcggcgcga ccatttatcg
     5641 tctgtatggc aaaccggatg cgccggaact gtttctgaaa catggcaaag gcagcgtggc
     5701 gaacgatgtg accgatgaaa tggtgcgtct gaactggctg accgaattta tgccgctgcc
     5761 gaccattaaa cattttattc gcaccccgga tgatgcgtgg ctgctgacca ccgcgattcc
     5821 gggcaaaacc gcgtttcagg tgctggaaga atatccggat agcggcgaaa acattgtgga
     5881 tgcgctggcc gtgtttctgc gtcgtctgca tagcattccg gtgtgcaact gcccgtttaa
     5941 cagcgatcgt gtgtttcgtc tggcccaggc gcagagccgt atgaacaacg gcctggtgga
     6001 tgcgagcgat tttgatgatg aacgtaacgg ctggccggtg gaacaggtgt ggaaagaaat
     6061 gcataaactg ctgccgttta gcccggatag cgtggtgacc cacggcgatt ttagcctgga
     6121 taacctgatt ttcgatgaag gcaaactgat tggctgcatt gatgtgggcc gtgtgggcat
     6181 tgcggatcgt tatcaggatc tggccattct gtggaactgc ctgggcgaat ttagcccgag
     6241 cctgcaaaaa cgtctgtttc agaaatatgg cattgataat ccggatatga acaaactgca
     6301 atttcatctg atgctggatg aatttttcta ataattaatt ggaccgcggt ccgcgcgttg
     6361 tccttttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca
     6421 tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt
     6481 atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc
     6541 ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc
     6601 tggccgtagg ccggccgatc tgaagatcag cagttcaacc tgttgatagt acgtactaag
     6661 ctctcatgtt tcacgtacta agctctcatg tttaacgtac taagctctca tgtttaacga
     6721 actaaaccct catggctaac gtactaagct ctcatggcta acgtactaag ctctcatgtt
     6781 tcacgtacta agctctcatg tttgaacaat aaaattaata taaatcagca acttaaatag
     6841 cctctaaggt tttaagtttt ataagaaaaa aaagaatata taaggctttt aaagccttta
     6901 aggtttaacg gttgtggaca acaagccagg gatgtaacgc actgagaagc ccttagagcc
     6961 tctcaaagca attttgagtg acacaggaac acttaacggc tgacatgggg cgcgcccagc
     7021 tgtctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa
     7081 acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgccttt aattaaagc
//
