LOCUS       sequence-418911-        7224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc151 through Tn7 
            bacterial insertion, Kanamycin selectable marker and GFP fluorescent
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7224)
  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 7224)
  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: pBC124
FEATURES             Location/Qualifiers
     source          1..7224
                     /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)."
     terminator      complement(681..933)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(953..1504)
                     /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(1546..1725)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     protein_bind    complement(1803..1850)
                     /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(1878..1883,1884..1899,1900..1906)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1914..1930
                     /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             1953..2666
                     /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(1983..2011)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2606..2626
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2689..2783
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3228..3393
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3436..3881
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3882..3968,3969..5303)
                     /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      5386..5480
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5601..5620)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5612..6427
                     /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            6594..6702
                     /note="incP origin of transfer"
     rep_origin      6713..7101
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7132..7218)
                     /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 aatgccggtc gatctagtaa catagatgac accgcgcgcg ataatttatc
      721 ctagtttgcg cgctatattt tgttttctat cgcgtattaa atgtataatt gcgggactct
      781 aatcataaaa acccatctca taaataacgt catgcattac atgttaatta ttacatgctt
      841 aacgtaattc aacagaaatt atatgataat catcgcaaga ccggcaacag gattcaatct
      901 taagaaactt tattgccaaa tgtttgaacg atctgcttga ctctaggggt catcagattt
      961 cggtgacggg caggaccgga cggggcggca ccggcaggct gaagtccagc tgccagaaac
     1021 ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg cagcatgccg cggggggcat
     1081 atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg cagcccgatg acagcgacca
     1141 cgctcttgaa gccctgtgcc tccagggact tcagcaggtg ggtgtagagc gtggagccca
     1201 gtcccgtccg ctggtggcgg ggggagacgt acacggtcga ctcggccgtc cagtcgtagg
     1261 cgttgcgtgc cttccaggga cccgcgtagg cgatgccggc gacctcgccg tccacctcgg
     1321 cgacgagcca gggatagcgc tcccgcagac ggacgaggtc gtccgtccac tcctgcggtt
     1381 cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat gtagtggttg acgatggtgc
     1441 agaccgccgg catgtccgcc tcggtggcac ggcggatgtc ggccgggcgt cgttctgggc
     1501 tcatggtaga tcccctcgat cgagttgaga gtgaatatga gactctaatt ggataccgag
     1561 gggaatttat ggaacgtcag tggagcattt ttgacaagaa atatttgcta gctgatagtg
     1621 accttaggcg acttttgaac gcgcaataat ggtttctgac gtatgtgctt agctcattaa
     1681 actccagaaa cccgcggctc agtggctcct tcaacgttgc ggttctgtca gttccaaacg
     1741 taaaacggct tgtcccgcgt catcggcggg ggtcataacg tgactccctt aattctcagc
     1801 ttgaagttcc tatactttct agagaatagg aacttcggaa taggtacttc cgctactaga
     1861 attcgagcgg ggaattcttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
     1921 cggataacaa ttttcacaca ggaaacagct aaatgcgtaa aggagaagaa cttttcactg
     1981 gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca
     2041 gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta
     2101 ctggaaaact acctgttcca tggccaacac ttgtcactac tttcggttat ggtgttcaat
     2161 gctttgcgag atacccagat catatgaaac agcatgactt tttcaagagt gtcatgcccg
     2221 aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac aagacacgtg
     2281 ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt
     2341 ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac tcacacaatg
     2401 tatacatcat ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa attagacaca
     2461 acattgaaga tggaagcgtt caactagcag accattatca acaaaatact ccaattggcg
     2521 atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc ctttcgaaag
     2581 atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta
     2641 cacatggcat ggatgaacta tacaaatgag cttaattagc tgagcttgga ctcctgttga
     2701 tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc
     2761 gggcgttttt tattggtgag aatctcccgc tttacgaatt cccatgggga gcatgcggag
     2821 ttacgtcatc cccaccttcc gctgcgttct tcatcgatgc ccgcttagtg atatgcttaa
     2881 aactccaagc acagccttct ccccaatgcg aaacgcttga cgtcagccga cggttgtagg
     2941 cattaatggc agcctcggcg aggagtggga tatatctccg cattaatgaa cacctaacgt
     3001 agctatatag gacacagttg cattaatgac acggttgaag accatagtag gagattcgta
     3061 catttgcaac tgtgtcctat atagctacgt taggtgttca ttggggatgc tggttgtcat
     3121 tgcccctcct tcctctaagt gataaggtta cttcctctaa atgaccaagc agggtatcta
     3181 atcctgttaa cgctcgctca gagaattcgc atgcggagtc gagggagaac cagataagtg
     3241 aaatctagtt ccaaactatt ttgtcatttt taattttcgt attagcttac gacgctacac
     3301 ccagttccca tctattttgt cactcttccc taaataatcc ttaaaaactc catttccacc
     3361 cctcccagtt cccaactatt ttgtccgccc acacgctgag cgaattccac gcttgtggaa
     3421 ttcctcgagg gagttcacat atacctgccg ttcactatta tttagtgaaa tgagatatta
     3481 tgatattttc tgaattgtga ttaaaaaggc aactttatgc ccatgcaaca gaaactataa
     3541 aaaatacaga gaatgaaaag aaacagatag attttttagt tctttaggcc cgtagtctgc
     3601 aaatcctttt atgattttct atcaaacaaa agaggaaaat agaccagttg caatccaaac
     3661 gagagtctaa tagaatgagg tcgaaaagta aatcgcgcgg gtttgttact gataaagcag
     3721 gcaagaccta aaatgtgtaa agggcaaagt gtatactttg gcgtcacccc ttacatattt
     3781 taggtctttt tttattgtgc gtaactaact tgccatcttc aaacaggagg gctggaagaa
     3841 gcagaccgct aacacagtac ataaaaaagg agacatgaac gatgaacatc aaaaagtttg
     3901 caaaacaagc aacagtatta acctttacta ccgcactgct ggcaggaggc gcaactcaag
     3961 cgtttgcgaa agaaacgaac caaaagccat ataaggaaac atacggcatt tcccatatta
     4021 cacgccatga tatgctgcaa atccctgaac agcaaaaaaa tgaaaaatat caagttcctg
     4081 aattcgattc gtccacaatt aaaaatatct cttctgcaaa aggcctggac gtttgggaca
     4141 gctggccatt acaaaacgct gacggcactg tcgcaaacta tcacggctac cacatcgtct
     4201 ttgcattagc cggagatcct aaaaatgcgg atgacacatc gatttacatg ttctatcaaa
     4261 aagtcggcga aacttctatt gacagctgga aaaacgctgg ccgcgtcttt aaagacagcg
     4321 acaaattcga tgcaaatgat tctatcctaa aagaccaaac acaagaatgg tcaggttcag
     4381 ccacatttac atctgacgga aaaatccgtt tattctacac tgatttctcc ggtaaacatt
     4441 acggcaaaca aacactgaca actgcacaag ttaacgtatc agcatcagac agctctttga
     4501 acatcaacgg tgtagaggat tataaatcaa tctttgacgg tgacggaaaa acgtatcaaa
     4561 atgtacagca gttcatcgat gaaggcaact acagctcagg cgacaaccat acgctgagag
     4621 atcctcacta cgtagaagat aaaggccaca aatacttagt atttgaagca aacactggaa
     4681 ctgaagatgg ctaccaaggc gaagaatctt tatttaacaa agcatactat ggcaaaagca
     4741 catcattctt ccgtcaagaa agtcaaaaac ttctgcaaag cgataaaaaa cgcacggctg
     4801 agttagcaaa cggcgctctc ggtatgattg agctaaacga tgattacaca ctgaaaaaag
     4861 tgatgaaacc gctgattgca tctaacacag taacagatga aattgaacgc gcgaacgtct
     4921 ttaaaatgaa cggcaaatgg tacctgttca ctgactcccg cggatcaaaa atgacgattg
     4981 acggcattac gtctaacgat atttacatgc ttggttatgt ttctaattct ttaactggcc
     5041 catacaagcc gctgaacaaa actggccttg tgttaaaaat ggatcttgat cctaacgatg
     5101 taacctttac ttactcacac ttcgctgtac ctcaagcgaa aggaaacaat gtcgtgatta
     5161 caagctatat gacaaacaga ggattctacg cagacaaaca atcaacgttt gcgccaagct
     5221 tcctgctgaa catcaaaggc aagaaaacat ctgttgtcaa agacagcatc cttgaacaag
     5281 gacaattaac agttaacaaa taaattcgct gagcgaggat gcacatgtga ccgagggaag
     5341 cttgcggccg cgtcgtgact gggaaaaccc tggcgactag tcttggactc ctgttgatag
     5401 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
     5461 cgttttttat tggtgagaat ccaggggtcc ccaataatta cgatttaaat ttgtgtctca
     5521 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
     5581 tgcttacata aacagtaata caaggggtgt tatgagccat attcagcgtg aaacgagctg
     5641 tagccgtccg cgtctgaaca gcaacatgga tgcggatctg tatggctata aatgggcgcg
     5701 tgataacgtg ggtcagagcg gcgcgaccat ttatcgtctg tatggcaaac cggatgcgcc
     5761 ggaactgttt ctgaaacatg gcaaaggcag cgtggcgaac gatgtgaccg atgaaatggt
     5821 gcgtctgaac tggctgaccg aatttatgcc gctgccgacc attaaacatt ttattcgcac
     5881 cccggatgat gcgtggctgc tgaccaccgc gattccgggc aaaaccgcgt ttcaggtgct
     5941 ggaagaatat ccggatagcg gcgaaaacat tgtggatgcg ctggccgtgt ttctgcgtcg
     6001 tctgcatagc attccggtgt gcaactgccc gtttaacagc gatcgtgtgt ttcgtctggc
     6061 ccaggcgcag agccgtatga acaacggcct ggtggatgcg agcgattttg atgatgaacg
     6121 taacggctgg ccggtggaac aggtgtggaa agaaatgcat aaactgctgc cgtttagccc
     6181 ggatagcgtg gtgacccacg gcgattttag cctggataac ctgattttcg atgaaggcaa
     6241 actgattggc tgcattgatg tgggccgtgt gggcattgcg gatcgttatc aggatctggc
     6301 cattctgtgg aactgcctgg gcgaatttag cccgagcctg caaaaacgtc tgtttcagaa
     6361 atatggcatt gataatccgg atatgaacaa actgcaattt catctgatgc tggatgaatt
     6421 tttctaataa ttaattggac cgcggtccgc gcgttgtcct tttccgctgc ataaccctgc
     6481 ttcggggtca ttatagcgat tttttcggta tatccatcct ttttcgcacg atatacagga
     6541 ttttgccaaa gggttcgtgt agactttcct tggtgtatcc aacggcgtca gccgggcagg
     6601 ataggtgaag taggcccacc cgcgagcggg tgttccttct tcactgtccc ttattcgcac
     6661 ctggcggtgc tcaacgggaa tcctgctctg cgaggctggc cgtaggccgg ccgatctgaa
     6721 gatcagcagt tcaacctgtt gatagtacgt actaagctct catgtttcac gtactaagct
     6781 ctcatgttta acgtactaag ctctcatgtt taacgaacta aaccctcatg gctaacgtac
     6841 taagctctca tggctaacgt actaagctct catgtttcac gtactaagct ctcatgtttg
     6901 aacaataaaa ttaatataaa tcagcaactt aaatagcctc taaggtttta agttttataa
     6961 gaaaaaaaag aatatataag gcttttaaag cctttaaggt ttaacggttg tggacaacaa
     7021 gccagggatg taacgcactg agaagccctt agagcctctc aaagcaattt tgagtgacac
     7081 aggaacactt aacggctgac atggggcgcg cccagctgtc tagggcggcg gatttgtcct
     7141 actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt ctttcgactg
     7201 agcctttcgt tttatttgat gcct
//
