LOCUS       sequence-419253-        6927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc132 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 6927)
  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 6927)
  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: pBC105
FEATURES             Location/Qualifiers
     source          1..6927
                     /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  2931..3096
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3139..3584
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3585..3671,3672..5006)
                     /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      5089..5183
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5304..5323)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5315..6130
                     /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            6297..6405
                     /note="incP origin of transfer"
     rep_origin      6416..6804
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6835..6921)
                     /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 cccatgctag attcgagaac tcgctcagtt gtacttgcca aatgctagat
     2701 tcgagaactc gctcagttgt acttgccatt aatgcgaaac gcttgacgtc agccgacggt
     2761 tgtaggaatg atcgccagca ggaggccgaa ctgtctagtc atcatgggga tgctggttgt
     2821 cattgcccct ccttcctcta agtgataagg ttacttcctc taaatgacca agcagggtat
     2881 ctaatcctgt taacgctcgc tcagagaatt cgcatgcgga gtcgagggag aaccagataa
     2941 gtgaaatcta gttccaaact attttgtcat ttttaatttt cgtattagct tacgacgcta
     3001 cacccagttc ccatctattt tgtcactctt ccctaaataa tccttaaaaa ctccatttcc
     3061 acccctccca gttcccaact attttgtccg cccacacgct gagcgaattc cacgcttgtg
     3121 gaattcctcg agggagttca catatacctg ccgttcacta ttatttagtg aaatgagata
     3181 ttatgatatt ttctgaattg tgattaaaaa ggcaacttta tgcccatgca acagaaacta
     3241 taaaaaatac agagaatgaa aagaaacaga tagatttttt agttctttag gcccgtagtc
     3301 tgcaaatcct tttatgattt tctatcaaac aaaagaggaa aatagaccag ttgcaatcca
     3361 aacgagagtc taatagaatg aggtcgaaaa gtaaatcgcg cgggtttgtt actgataaag
     3421 caggcaagac ctaaaatgtg taaagggcaa agtgtatact ttggcgtcac cccttacata
     3481 ttttaggtct ttttttattg tgcgtaacta acttgccatc ttcaaacagg agggctggaa
     3541 gaagcagacc gctaacacag tacataaaaa aggagacatg aacgatgaac atcaaaaagt
     3601 ttgcaaaaca agcaacagta ttaaccttta ctaccgcact gctggcagga ggcgcaactc
     3661 aagcgtttgc gaaagaaacg aaccaaaagc catataagga aacatacggc atttcccata
     3721 ttacacgcca tgatatgctg caaatccctg aacagcaaaa aaatgaaaaa tatcaagttc
     3781 ctgaattcga ttcgtccaca attaaaaata tctcttctgc aaaaggcctg gacgtttggg
     3841 acagctggcc attacaaaac gctgacggca ctgtcgcaaa ctatcacggc taccacatcg
     3901 tctttgcatt agccggagat cctaaaaatg cggatgacac atcgatttac atgttctatc
     3961 aaaaagtcgg cgaaacttct attgacagct ggaaaaacgc tggccgcgtc tttaaagaca
     4021 gcgacaaatt cgatgcaaat gattctatcc taaaagacca aacacaagaa tggtcaggtt
     4081 cagccacatt tacatctgac ggaaaaatcc gtttattcta cactgatttc tccggtaaac
     4141 attacggcaa acaaacactg acaactgcac aagttaacgt atcagcatca gacagctctt
     4201 tgaacatcaa cggtgtagag gattataaat caatctttga cggtgacgga aaaacgtatc
     4261 aaaatgtaca gcagttcatc gatgaaggca actacagctc aggcgacaac catacgctga
     4321 gagatcctca ctacgtagaa gataaaggcc acaaatactt agtatttgaa gcaaacactg
     4381 gaactgaaga tggctaccaa ggcgaagaat ctttatttaa caaagcatac tatggcaaaa
     4441 gcacatcatt cttccgtcaa gaaagtcaaa aacttctgca aagcgataaa aaacgcacgg
     4501 ctgagttagc aaacggcgct ctcggtatga ttgagctaaa cgatgattac acactgaaaa
     4561 aagtgatgaa accgctgatt gcatctaaca cagtaacaga tgaaattgaa cgcgcgaacg
     4621 tctttaaaat gaacggcaaa tggtacctgt tcactgactc ccgcggatca aaaatgacga
     4681 ttgacggcat tacgtctaac gatatttaca tgcttggtta tgtttctaat tctttaactg
     4741 gcccatacaa gccgctgaac aaaactggcc ttgtgttaaa aatggatctt gatcctaacg
     4801 atgtaacctt tacttactca cacttcgctg tacctcaagc gaaaggaaac aatgtcgtga
     4861 ttacaagcta tatgacaaac agaggattct acgcagacaa acaatcaacg tttgcgccaa
     4921 gcttcctgct gaacatcaaa ggcaagaaaa catctgttgt caaagacagc atccttgaac
     4981 aaggacaatt aacagttaac aaataaattc gctgagcgag gatgcacatg tgaccgaggg
     5041 aagcttgcgg ccgcgtcgtg actgggaaaa ccctggcgac tagtcttgga ctcctgttga
     5101 tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc
     5161 gggcgttttt tattggtgag aatccagggg tccccaataa ttacgattta aatttgtgtc
     5221 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
     5281 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcagc gtgaaacgag
     5341 ctgtagccgt ccgcgtctga acagcaacat ggatgcggat ctgtatggct ataaatgggc
     5401 gcgtgataac gtgggtcaga gcggcgcgac catttatcgt ctgtatggca aaccggatgc
     5461 gccggaactg tttctgaaac atggcaaagg cagcgtggcg aacgatgtga ccgatgaaat
     5521 ggtgcgtctg aactggctga ccgaatttat gccgctgccg accattaaac attttattcg
     5581 caccccggat gatgcgtggc tgctgaccac cgcgattccg ggcaaaaccg cgtttcaggt
     5641 gctggaagaa tatccggata gcggcgaaaa cattgtggat gcgctggccg tgtttctgcg
     5701 tcgtctgcat agcattccgg tgtgcaactg cccgtttaac agcgatcgtg tgtttcgtct
     5761 ggcccaggcg cagagccgta tgaacaacgg cctggtggat gcgagcgatt ttgatgatga
     5821 acgtaacggc tggccggtgg aacaggtgtg gaaagaaatg cataaactgc tgccgtttag
     5881 cccggatagc gtggtgaccc acggcgattt tagcctggat aacctgattt tcgatgaagg
     5941 caaactgatt ggctgcattg atgtgggccg tgtgggcatt gcggatcgtt atcaggatct
     6001 ggccattctg tggaactgcc tgggcgaatt tagcccgagc ctgcaaaaac gtctgtttca
     6061 gaaatatggc attgataatc cggatatgaa caaactgcaa tttcatctga tgctggatga
     6121 atttttctaa taattaattg gaccgcggtc cgcgcgttgt ccttttccgc tgcataaccc
     6181 tgcttcgggg tcattatagc gattttttcg gtatatccat cctttttcgc acgatataca
     6241 ggattttgcc aaagggttcg tgtagacttt ccttggtgta tccaacggcg tcagccgggc
     6301 aggataggtg aagtaggccc acccgcgagc gggtgttcct tcttcactgt cccttattcg
     6361 cacctggcgg tgctcaacgg gaatcctgct ctgcgaggct ggccgtaggc cggccgatct
     6421 gaagatcagc agttcaacct gttgatagta cgtactaagc tctcatgttt cacgtactaa
     6481 gctctcatgt ttaacgtact aagctctcat gtttaacgaa ctaaaccctc atggctaacg
     6541 tactaagctc tcatggctaa cgtactaagc tctcatgttt cacgtactaa gctctcatgt
     6601 ttgaacaata aaattaatat aaatcagcaa cttaaatagc ctctaaggtt ttaagtttta
     6661 taagaaaaaa aagaatatat aaggctttta aagcctttaa ggtttaacgg ttgtggacaa
     6721 caagccaggg atgtaacgca ctgagaagcc cttagagcct ctcaaagcaa ttttgagtga
     6781 cacaggaaca cttaacggct gacatggggc gcgcccagct gtctagggcg gcggatttgt
     6841 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga
     6901 ctgagccttt cgttttattt gatgcct
//
