LOCUS       sequence-419974-        7367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc141 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 7367)
  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 7367)
  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: pBC114
FEATURES             Location/Qualifiers
     source          1..7367
                     /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  3371..3536
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3579..4024
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4025..4111,4112..5446)
                     /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      5529..5623
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5744..5763)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5755..6570
                     /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            6737..6845
                     /note="incP origin of transfer"
     rep_origin      6856..7244
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7275..7361)
                     /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 aacacctaac gtagctatat
     3121 aggacacagt tgaatgaaca cctaacgtag ctatatagga cacagttgct gcctacaacc
     3181 gtcggctgac gtcaagcgtt tcgcataatg cctacaaccg tcggctgacg tcaagcgttt
     3241 cgcatgggga tgctggttgt cattgcccct ccttcctcta agtgataagg ttacttcctc
     3301 taaatgacca agcagggtat ctaatcctgt taacgctcgc tcagagaatt cgcatgcgga
     3361 gtcgagggag aaccagataa gtgaaatcta gttccaaact attttgtcat ttttaatttt
     3421 cgtattagct tacgacgcta cacccagttc ccatctattt tgtcactctt ccctaaataa
     3481 tccttaaaaa ctccatttcc acccctccca gttcccaact attttgtccg cccacacgct
     3541 gagcgaattc cacgcttgtg gaattcctcg agggagttca catatacctg ccgttcacta
     3601 ttatttagtg aaatgagata ttatgatatt ttctgaattg tgattaaaaa ggcaacttta
     3661 tgcccatgca acagaaacta taaaaaatac agagaatgaa aagaaacaga tagatttttt
     3721 agttctttag gcccgtagtc tgcaaatcct tttatgattt tctatcaaac aaaagaggaa
     3781 aatagaccag ttgcaatcca aacgagagtc taatagaatg aggtcgaaaa gtaaatcgcg
     3841 cgggtttgtt actgataaag caggcaagac ctaaaatgtg taaagggcaa agtgtatact
     3901 ttggcgtcac cccttacata ttttaggtct ttttttattg tgcgtaacta acttgccatc
     3961 ttcaaacagg agggctggaa gaagcagacc gctaacacag tacataaaaa aggagacatg
     4021 aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta ttaaccttta ctaccgcact
     4081 gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg aaccaaaagc catataagga
     4141 aacatacggc atttcccata ttacacgcca tgatatgctg caaatccctg aacagcaaaa
     4201 aaatgaaaaa tatcaagttc ctgaattcga ttcgtccaca attaaaaata tctcttctgc
     4261 aaaaggcctg gacgtttggg acagctggcc attacaaaac gctgacggca ctgtcgcaaa
     4321 ctatcacggc taccacatcg tctttgcatt agccggagat cctaaaaatg cggatgacac
     4381 atcgatttac atgttctatc aaaaagtcgg cgaaacttct attgacagct ggaaaaacgc
     4441 tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat gattctatcc taaaagacca
     4501 aacacaagaa tggtcaggtt cagccacatt tacatctgac ggaaaaatcc gtttattcta
     4561 cactgatttc tccggtaaac attacggcaa acaaacactg acaactgcac aagttaacgt
     4621 atcagcatca gacagctctt tgaacatcaa cggtgtagag gattataaat caatctttga
     4681 cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc gatgaaggca actacagctc
     4741 aggcgacaac catacgctga gagatcctca ctacgtagaa gataaaggcc acaaatactt
     4801 agtatttgaa gcaaacactg gaactgaaga tggctaccaa ggcgaagaat ctttatttaa
     4861 caaagcatac tatggcaaaa gcacatcatt cttccgtcaa gaaagtcaaa aacttctgca
     4921 aagcgataaa aaacgcacgg ctgagttagc aaacggcgct ctcggtatga ttgagctaaa
     4981 cgatgattac acactgaaaa aagtgatgaa accgctgatt gcatctaaca cagtaacaga
     5041 tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa tggtacctgt tcactgactc
     5101 ccgcggatca aaaatgacga ttgacggcat tacgtctaac gatatttaca tgcttggtta
     5161 tgtttctaat tctttaactg gcccatacaa gccgctgaac aaaactggcc ttgtgttaaa
     5221 aatggatctt gatcctaacg atgtaacctt tacttactca cacttcgctg tacctcaagc
     5281 gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac agaggattct acgcagacaa
     5341 acaatcaacg tttgcgccaa gcttcctgct gaacatcaaa ggcaagaaaa catctgttgt
     5401 caaagacagc atccttgaac aaggacaatt aacagttaac aaataaattc gctgagcgag
     5461 gatgcacatg tgaccgaggg aagcttgcgg ccgcgtcgtg actgggaaaa ccctggcgac
     5521 tagtcttgga ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt
     5581 cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccagggg tccccaataa
     5641 ttacgattta aatttgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
     5701 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     5761 catattcagc gtgaaacgag ctgtagccgt ccgcgtctga acagcaacat ggatgcggat
     5821 ctgtatggct ataaatgggc gcgtgataac gtgggtcaga gcggcgcgac catttatcgt
     5881 ctgtatggca aaccggatgc gccggaactg tttctgaaac atggcaaagg cagcgtggcg
     5941 aacgatgtga ccgatgaaat ggtgcgtctg aactggctga ccgaatttat gccgctgccg
     6001 accattaaac attttattcg caccccggat gatgcgtggc tgctgaccac cgcgattccg
     6061 ggcaaaaccg cgtttcaggt gctggaagaa tatccggata gcggcgaaaa cattgtggat
     6121 gcgctggccg tgtttctgcg tcgtctgcat agcattccgg tgtgcaactg cccgtttaac
     6181 agcgatcgtg tgtttcgtct ggcccaggcg cagagccgta tgaacaacgg cctggtggat
     6241 gcgagcgatt ttgatgatga acgtaacggc tggccggtgg aacaggtgtg gaaagaaatg
     6301 cataaactgc tgccgtttag cccggatagc gtggtgaccc acggcgattt tagcctggat
     6361 aacctgattt tcgatgaagg caaactgatt ggctgcattg atgtgggccg tgtgggcatt
     6421 gcggatcgtt atcaggatct ggccattctg tggaactgcc tgggcgaatt tagcccgagc
     6481 ctgcaaaaac gtctgtttca gaaatatggc attgataatc cggatatgaa caaactgcaa
     6541 tttcatctga tgctggatga atttttctaa taattaattg gaccgcggtc cgcgcgttgt
     6601 ccttttccgc tgcataaccc tgcttcgggg tcattatagc gattttttcg gtatatccat
     6661 cctttttcgc acgatataca ggattttgcc aaagggttcg tgtagacttt ccttggtgta
     6721 tccaacggcg tcagccgggc aggataggtg aagtaggccc acccgcgagc gggtgttcct
     6781 tcttcactgt cccttattcg cacctggcgg tgctcaacgg gaatcctgct ctgcgaggct
     6841 ggccgtaggc cggccgatct gaagatcagc agttcaacct gttgatagta cgtactaagc
     6901 tctcatgttt cacgtactaa gctctcatgt ttaacgtact aagctctcat gtttaacgaa
     6961 ctaaaccctc atggctaacg tactaagctc tcatggctaa cgtactaagc tctcatgttt
     7021 cacgtactaa gctctcatgt ttgaacaata aaattaatat aaatcagcaa cttaaatagc
     7081 ctctaaggtt ttaagtttta taagaaaaaa aagaatatat aaggctttta aagcctttaa
     7141 ggtttaacgg ttgtggacaa caagccaggg atgtaacgca ctgagaagcc cttagagcct
     7201 ctcaaagcaa ttttgagtga cacaggaaca cttaacggct gacatggggc gcgcccagct
     7261 gtctagggcg gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa
     7321 cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcct
//
