LOCUS       sequence-418898-        6888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc133 through Tn7 
            bacterial insertion, Gentamicin 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 6888)
  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 6888)
  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: pBC106
FEATURES             Location/Qualifiers
     source          1..6888
                     /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)."
     CDS             1741..2424
                     /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     2336..2359
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      2438..2532
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  2892..3057
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3100..3545
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3546..3632,3633..4967)
                     /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      5050..5144
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5265..5284)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5276..6091
                     /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            6258..6366
                     /note="incP origin of transfer"
     rep_origin      6377..6765
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6796..6882)
                     /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 aattcttgac aattaatcat
     1681 cggctcgtat aatgtgtgga attgtgagcg gataacaatt ttcacacagg aaacagctaa
     1741 atggtgagca agggcgagga ggtcatcaag gagttcatgc gcttcaagga gcacatggag
     1801 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1861 ggcacccaga ccgccaggct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     1921 atcctgtccc ctcagatcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     1981 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     2041 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     2101 tacaaggtga aggtgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaaaaag
     2161 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     2221 gagatgaaga tgaggctgag gctgaaggac ggcggccact acgacgccga ggtcaagacc
     2281 acctacatgg ccaagaagcc cgtgcagctg cccggcgcct acaagaccga catcaagctg
     2341 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg cgccgagggc
     2401 cgccactcca ccggcgccgc ttaattagct gagcttggac tcctgttgat agatccagta
     2461 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     2521 attggtgaga atctcccgct ttacgaattc ccatggggag catgcggagt tacgtcatcc
     2581 ccaccttccg ctgcgttctt catcgatgcc cgcttagtga tatgcttaaa actccaagca
     2641 cagccttctc cccatggagt taacgctggc gagtagagct tactgcgcca taatggcagc
     2701 ctcggcgagg agtgggatat atctccgcat tgcaagtaca actgagcgag ttctcgaatc
     2761 tagcatgggg atgctggttg tcattgcccc tccttcctct aagtgataag gttacttcct
     2821 ctaaatgacc aagcagggta tctaatcctg ttaacgctcg ctcagagaat tcgcatgcgg
     2881 agtcgaggga gaaccagata agtgaaatct agttccaaac tattttgtca tttttaattt
     2941 tcgtattagc ttacgacgct acacccagtt cccatctatt ttgtcactct tccctaaata
     3001 atccttaaaa actccatttc cacccctccc agttcccaac tattttgtcc gcccacacgc
     3061 tgagcgaatt ccacgcttgt ggaattcctc gagggagttc acatatacct gccgttcact
     3121 attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa aggcaacttt
     3181 atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag atagattttt
     3241 tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga
     3301 aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc
     3361 gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac
     3421 tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact aacttgccat
     3481 cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa aaggagacat
     3541 gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt actaccgcac
     3601 tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag ccatataagg
     3661 aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct gaacagcaaa
     3721 aaaatgaaaa atatcaagtt cctgaattcg attcgtccac aattaaaaat atctcttctg
     3781 caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc actgtcgcaa
     3841 actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat gcggatgaca
     3901 catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg
     3961 ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc
     4021 aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc cgtttattct
     4081 acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca caagttaacg
     4141 tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa tcaatctttg
     4201 acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc aactacagct
     4261 caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc cacaaatact
     4321 tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa tctttattta
     4381 acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa aaacttctgc
     4441 aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg attgagctaa
     4501 acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac acagtaacag
     4561 atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg ttcactgact
     4621 cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac atgcttggtt
     4681 atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc cttgtgttaa
     4741 aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct gtacctcaag
     4801 cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc tacgcagaca
     4861 aacaatcaac gtttgcgcca agcttcctgc tgaacatcaa aggcaagaaa acatctgttg
     4921 tcaaagacag catccttgaa caaggacaat taacagttaa caaataaatt cgctgagcga
     4981 ggatgcacat gtgaccgagg gaagcttgcg gccgcgtcgt gactgggaaa accctggcga
     5041 ctagtcttgg actcctgttg atagatccag taatgacctc agaactccat ctggatttgt
     5101 tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaggg gtccccaata
     5161 attacgattt aaatttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     5221 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     5281 ccatattcag cgtgaaacga gctgtagccg tccgcgtctg aacagcaaca tggatgcgga
     5341 tctgtatggc tataaatggg cgcgtgataa cgtgggtcag agcggcgcga ccatttatcg
     5401 tctgtatggc aaaccggatg cgccggaact gtttctgaaa catggcaaag gcagcgtggc
     5461 gaacgatgtg accgatgaaa tggtgcgtct gaactggctg accgaattta tgccgctgcc
     5521 gaccattaaa cattttattc gcaccccgga tgatgcgtgg ctgctgacca ccgcgattcc
     5581 gggcaaaacc gcgtttcagg tgctggaaga atatccggat agcggcgaaa acattgtgga
     5641 tgcgctggcc gtgtttctgc gtcgtctgca tagcattccg gtgtgcaact gcccgtttaa
     5701 cagcgatcgt gtgtttcgtc tggcccaggc gcagagccgt atgaacaacg gcctggtgga
     5761 tgcgagcgat tttgatgatg aacgtaacgg ctggccggtg gaacaggtgt ggaaagaaat
     5821 gcataaactg ctgccgttta gcccggatag cgtggtgacc cacggcgatt ttagcctgga
     5881 taacctgatt ttcgatgaag gcaaactgat tggctgcatt gatgtgggcc gtgtgggcat
     5941 tgcggatcgt tatcaggatc tggccattct gtggaactgc ctgggcgaat ttagcccgag
     6001 cctgcaaaaa cgtctgtttc agaaatatgg cattgataat ccggatatga acaaactgca
     6061 atttcatctg atgctggatg aatttttcta ataattaatt ggaccgcggt ccgcgcgttg
     6121 tccttttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca
     6181 tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt
     6241 atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc
     6301 ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc
     6361 tggccgtagg ccggccgatc tgaagatcag cagttcaacc tgttgatagt acgtactaag
     6421 ctctcatgtt tcacgtacta agctctcatg tttaacgtac taagctctca tgtttaacga
     6481 actaaaccct catggctaac gtactaagct ctcatggcta acgtactaag ctctcatgtt
     6541 tcacgtacta agctctcatg tttgaacaat aaaattaata taaatcagca acttaaatag
     6601 cctctaaggt tttaagtttt ataagaaaaa aaagaatata taaggctttt aaagccttta
     6661 aggtttaacg gttgtggaca acaagccagg gatgtaacgc actgagaagc ccttagagcc
     6721 tctcaaagca attttgagtg acacaggaac acttaacggc tgacatgggg cgcgcccagc
     6781 tgtctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa
     6841 acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcct
//
