LOCUS       sequence-418909-        7413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc146 through Tn7 
            bacterial insertion, Tetracyline selectable marker and GFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7413)
  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 7413)
  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: pBC119
FEATURES             Location/Qualifiers
     source          1..7413
                     /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..2898
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSVMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2215..2243)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2838..2858
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2921..3015
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3417..3582
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3625..4070
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4071..4157,4158..5492)
                     /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      5575..5669
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5790..5809)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5801..6616
                     /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            6783..6891
                     /note="incP origin of transfer"
     rep_origin      6902..7290
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7321..7407)
                     /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 ctaaatgcgt aaaggagaag aacttttcac tggagttgtc
     2221 ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag
     2281 ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa
     2341 ctacctgttc catggccaac acttgtcact actttcggtt atggtgttca atgctttgcg
     2401 agatacccag atcatatgaa acagcatgac tttttcaaga gtgtcatgcc cgaaggttat
     2461 gtacaggaaa gaactatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc
     2521 aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa
     2581 gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa tgtatacatc
     2641 atggcagaca aacaaaagaa tggaatcaaa gttaacttca aaattagaca caacattgaa
     2701 gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct
     2761 gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa agatcccaac
     2821 gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc
     2881 atggatgaac tatacaaatg agcttaatta gctgagcttg gactcctgtt gatagatcca
     2941 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     3001 tttattggtg agaatctccc gctttacgaa ttcccatggg gagcatgcgg agttacgtca
     3061 tccccacctt ccgctgcgtt cttcatcgat gcccgcttag tgatatgctt aaaactccaa
     3121 gcacagcctt ctccccatga tgactagaca gttcggcctc ctgctggcga tcattgtacg
     3181 aatctcctac tatggtcttc aaccgtgtca ttaatgcaac tgtgtcctat atagctacgt
     3241 taggtgttca ttaatgccta caaccgtcgg ctgacgtcaa gcgtttcgca tggggatgct
     3301 ggttgtcatt gcccctcctt cctctaagtg ataaggttac ttcctctaaa tgaccaagca
     3361 gggtatctaa tcctgttaac gctcgctcag agaattcgca tgcggagtcg agggagaacc
     3421 agataagtga aatctagttc caaactattt tgtcattttt aattttcgta ttagcttacg
     3481 acgctacacc cagttcccat ctattttgtc actcttccct aaataatcct taaaaactcc
     3541 atttccaccc ctcccagttc ccaactattt tgtccgccca cacgctgagc gaattccacg
     3601 cttgtggaat tcctcgaggg agttcacata tacctgccgt tcactattat ttagtgaaat
     3661 gagatattat gatattttct gaattgtgat taaaaaggca actttatgcc catgcaacag
     3721 aaactataaa aaatacagag aatgaaaaga aacagataga ttttttagtt ctttaggccc
     3781 gtagtctgca aatcctttta tgattttcta tcaaacaaaa gaggaaaata gaccagttgc
     3841 aatccaaacg agagtctaat agaatgaggt cgaaaagtaa atcgcgcggg tttgttactg
     3901 ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg tatactttgg cgtcacccct
     3961 tacatatttt aggtcttttt ttattgtgcg taactaactt gccatcttca aacaggaggg
     4021 ctggaagaag cagaccgcta acacagtaca taaaaaagga gacatgaacg atgaacatca
     4081 aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg gcaggaggcg
     4141 caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca tacggcattt
     4201 cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat gaaaaatatc
     4261 aagttcctga attcgattcg tccacaatta aaaatatctc ttctgcaaaa ggcctggacg
     4321 tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat cacggctacc
     4381 acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg atttacatgt
     4441 tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc cgcgtcttta
     4501 aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca caagaatggt
     4561 caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact gatttctccg
     4621 gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca gcatcagaca
     4681 gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt gacggaaaaa
     4741 cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc gacaaccata
     4801 cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta tttgaagcaa
     4861 acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa gcatactatg
     4921 gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc gataaaaaac
     4981 gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat gattacacac
     5041 tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa attgaacgcg
     5101 cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc ggatcaaaaa
     5161 tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt tctaattctt
     5221 taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg gatcttgatc
     5281 ctaacgatgt aacctttact tactcacact tcgctgtacc tcaagcgaaa ggaaacaatg
     5341 tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa tcaacgtttg
     5401 cgccaagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa gacagcatcc
     5461 ttgaacaagg acaattaaca gttaacaaat aaattcgctg agcgaggatg cacatgtgac
     5521 cgagggaagc ttgcggccgc gtcgtgactg ggaaaaccct ggcgactagt cttggactcc
     5581 tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt
     5641 gccgccgggc gttttttatt ggtgagaatc caggggtccc caataattac gatttaaatt
     5701 tgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     5761 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcagcgtga
     5821 aacgagctgt agccgtccgc gtctgaacag caacatggat gcggatctgt atggctataa
     5881 atgggcgcgt gataacgtgg gtcagagcgg cgcgaccatt tatcgtctgt atggcaaacc
     5941 ggatgcgccg gaactgtttc tgaaacatgg caaaggcagc gtggcgaacg atgtgaccga
     6001 tgaaatggtg cgtctgaact ggctgaccga atttatgccg ctgccgacca ttaaacattt
     6061 tattcgcacc ccggatgatg cgtggctgct gaccaccgcg attccgggca aaaccgcgtt
     6121 tcaggtgctg gaagaatatc cggatagcgg cgaaaacatt gtggatgcgc tggccgtgtt
     6181 tctgcgtcgt ctgcatagca ttccggtgtg caactgcccg tttaacagcg atcgtgtgtt
     6241 tcgtctggcc caggcgcaga gccgtatgaa caacggcctg gtggatgcga gcgattttga
     6301 tgatgaacgt aacggctggc cggtggaaca ggtgtggaaa gaaatgcata aactgctgcc
     6361 gtttagcccg gatagcgtgg tgacccacgg cgattttagc ctggataacc tgattttcga
     6421 tgaaggcaaa ctgattggct gcattgatgt gggccgtgtg ggcattgcgg atcgttatca
     6481 ggatctggcc attctgtgga actgcctggg cgaatttagc ccgagcctgc aaaaacgtct
     6541 gtttcagaaa tatggcattg ataatccgga tatgaacaaa ctgcaatttc atctgatgct
     6601 ggatgaattt ttctaataat taattggacc gcggtccgcg cgttgtcctt ttccgctgca
     6661 taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga
     6721 tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag
     6781 ccgggcagga taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct
     6841 tattcgcacc tggcggtgct caacgggaat cctgctctgc gaggctggcc gtaggccggc
     6901 cgatctgaag atcagcagtt caacctgttg atagtacgta ctaagctctc atgtttcacg
     6961 tactaagctc tcatgtttaa cgtactaagc tctcatgttt aacgaactaa accctcatgg
     7021 ctaacgtact aagctctcat ggctaacgta ctaagctctc atgtttcacg tactaagctc
     7081 tcatgtttga acaataaaat taatataaat cagcaactta aatagcctct aaggttttaa
     7141 gttttataag aaaaaaaaga atatataagg cttttaaagc ctttaaggtt taacggttgt
     7201 ggacaacaag ccagggatgt aacgcactga gaagccctta gagcctctca aagcaatttt
     7261 gagtgacaca ggaacactta acggctgaca tggggcgcgc ccagctgtct agggcggcgg
     7321 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     7381 tttcgactga gcctttcgtt ttatttgatg cct
//
