LOCUS       sequence-406226-        8454 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 empty backbone for strain-specific barcode DNA tag through 
            Tn7 bacterial insertion, Kanamycin selectable marker and tagBFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8454)
  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 8454)
  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: pBCC101
FEATURES             Location/Qualifiers
     source          1..8454
                     /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)."
     terminator      2671..2765
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        join(2914..2919,2920..2937,2938..2944)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2998..3657
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3064..3083)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3999..4304
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4183..4202
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     mobile_element  4458..4623
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        4666..5111
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(5112..5198,5199..6533)
                     /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      6616..6710
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(6831..6850)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6842..7657
                     /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            7824..7932
                     /note="incP origin of transfer"
     rep_origin      7943..8331
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(8362..8448)
                     /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 aaatgtctga attgattaaa gagaatatgc
     1981 atatgaagtt gtatatggaa ggtactgttg ataaccacca ttttaaatgt acatctgagg
     2041 gagaaggtaa gccttacgaa ggtactcaaa caatgcgtat taaagtagtt gagggaggtc
     2101 ctttaccatt cgcttttgac atattggcaa cttcattctt atatggatct aagacattta
     2161 ttaaccatac tcaaggtata ccagatttct ttaaacaatc atttcctgaa ggattcacat
     2221 gggagagagt aacaacttat gaagatggtg gagttttgac tgctactcag gatacatctt
     2281 tacaagacgg ttgtttgatt tataatgtta agattcgtgg agtaaacttc acttctaatg
     2341 gtccagttat gcagaagaaa acattgggat gggaggcatt cactgagaca ttataccctg
     2401 ctgatggtgg attggaaggt cgtaacgaca tggcattgaa attagtagga ggttcacact
     2461 tgatagctaa tattaagact acatatcgtt ctaagaaacc agctaaaaac ttgaaaatgc
     2521 ctggtgttta ctatgttgat tatcgtttag aaagaattaa ggaagcaaat aacgaaactt
     2581 acgttgagca acatgaagta gctgttgcac gttattgtga cttgccttct aagttaggac
     2641 acaaattgaa cgcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct
     2701 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     2761 agaatctccc gctttacgaa ttcccatggg gagcatgcgg agttacgtca tccccacctt
     2821 ccgctgcgtt cttcatcgat gcccgcttag tgatatgctt aaaactccaa gcacagcctt
     2881 ctccccgggg cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     2941 aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga agctaaaatg
     3001 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     3061 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     3121 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     3181 attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag
     3241 ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg
     3301 ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca catatattcg
     3361 caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat
     3421 atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc
     3481 aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac
     3541 aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc
     3601 ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaaacg
     3661 cgtggatccg gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc
     3721 ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg tatgctatga
     3781 agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga
     3841 tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc
     3901 cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat
     3961 gaacggctct tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct
     4021 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
     4081 ccggacgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc
     4141 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata
     4201 tggccagtgt gccggtatcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
     4261 atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta
     4321 tacacagccc ccggggatgc tggttgtcat tgcccctcct tcctctaagt gataaggtta
     4381 cttcctctaa atgaccaagc agggtatcta atcctgttaa cgctcgctca gagaattcgc
     4441 atgcggagtc gagggagaac cagataagtg aaatctagtt ccaaactatt ttgtcatttt
     4501 taattttcgt attagcttac gacgctacac ccagttccca tctattttgt cactcttccc
     4561 taaataatcc ttaaaaactc catttccacc cctcccagtt cccaactatt ttgtccgccc
     4621 acacgctgag cgaattccac gcttgtggaa ttcctcgagg gagttcacat atacctgccg
     4681 ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc
     4741 aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag
     4801 attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa
     4861 agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta
     4921 aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt
     4981 gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact
     5041 tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg
     5101 agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta
     5161 ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat
     5221 ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac
     5281 agcaaaaaaa tgaaaaatat caagttcctg aattcgattc gtccacaatt aaaaatatct
     5341 cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg
     5401 tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg
     5461 atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga
     5521 aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa
     5581 aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt
     5641 tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag
     5701 ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa
     5761 tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact
     5821 acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca
     5881 aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt
     5941 tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac
     6001 ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg
     6061 agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag
     6121 taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca
     6181 ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc
     6241 ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg
     6301 tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac
     6361 ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg
     6421 cagacaaaca atcaacgttt gcgccaagct tcctgctgaa catcaaaggc aagaaaacat
     6481 ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaattcgct
     6541 gagcgaggat gcacatgtga ccgagggaag cttgcggccg cgtcgtgact gggaaaaccc
     6601 tggcgactag tcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg
     6661 atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaggggtcc
     6721 ccaataatta cgatttaaat ttgtgtctca aaatctctga tgttacattg cacaagataa
     6781 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     6841 tatgagccat attcagcgtg aaacgagctg tagccgtccg cgtctgaaca gcaacatgga
     6901 tgcggatctg tatggctata aatgggcgcg tgataacgtg ggtcagagcg gcgcgaccat
     6961 ttatcgtctg tatggcaaac cggatgcgcc ggaactgttt ctgaaacatg gcaaaggcag
     7021 cgtggcgaac gatgtgaccg atgaaatggt gcgtctgaac tggctgaccg aatttatgcc
     7081 gctgccgacc attaaacatt ttattcgcac cccggatgat gcgtggctgc tgaccaccgc
     7141 gattccgggc aaaaccgcgt ttcaggtgct ggaagaatat ccggatagcg gcgaaaacat
     7201 tgtggatgcg ctggccgtgt ttctgcgtcg tctgcatagc attccggtgt gcaactgccc
     7261 gtttaacagc gatcgtgtgt ttcgtctggc ccaggcgcag agccgtatga acaacggcct
     7321 ggtggatgcg agcgattttg atgatgaacg taacggctgg ccggtggaac aggtgtggaa
     7381 agaaatgcat aaactgctgc cgtttagccc ggatagcgtg gtgacccacg gcgattttag
     7441 cctggataac ctgattttcg atgaaggcaa actgattggc tgcattgatg tgggccgtgt
     7501 gggcattgcg gatcgttatc aggatctggc cattctgtgg aactgcctgg gcgaatttag
     7561 cccgagcctg caaaaacgtc tgtttcagaa atatggcatt gataatccgg atatgaacaa
     7621 actgcaattt catctgatgc tggatgaatt tttctaataa ttaattggac cgcggtccgc
     7681 gcgttgtcct tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta
     7741 tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct
     7801 tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg
     7861 tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg
     7921 cgaggctggc cgtaggccgg ccgatctgaa gatcagcagt tcaacctgtt gatagtacgt
     7981 actaagctct catgtttcac gtactaagct ctcatgttta acgtactaag ctctcatgtt
     8041 taacgaacta aaccctcatg gctaacgtac taagctctca tggctaacgt actaagctct
     8101 catgtttcac gtactaagct ctcatgtttg aacaataaaa ttaatataaa tcagcaactt
     8161 aaatagcctc taaggtttta agttttataa gaaaaaaaag aatatataag gcttttaaag
     8221 cctttaaggt ttaacggttg tggacaacaa gccagggatg taacgcactg agaagccctt
     8281 agagcctctc aaagcaattt tgagtgacac aggaacactt aacggctgac atggggcgcg
     8341 cccagctgtc tagggcggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     8401 gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat gcct
//
