LOCUS       sequence-417285-        6974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc189 through Tn7 
            bacterial insertion, Gentamicin selectable marker and AmCyan1 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6974)
  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 6974)
  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: pBC162
FEATURES             Location/Qualifiers
     source          1..6974
                     /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)."
     regulatory      1807..1816
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1813..2505
                     /codon_start=1
                     /product="Anemonia majano cyan fluorescent protein"
                     /label=AmCyan
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTST
                     FKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYEDG
                     GVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKMTTGWDPSFEKMTVCDGILKGDV
                     TAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHAVEHRIARTDLDKGGNSVQLTEHAVAH
                     ITSVVPFA"
     terminator      2519..2613
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  2978..3143
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3186..3631
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3632..3718,3719..5053)
                     /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      5136..5230
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5351..5370)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5362..6177
                     /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            6344..6452
                     /note="incP origin of transfer"
     rep_origin      6463..6851
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6882..6968)
                     /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 atgaccatga ttacgccaag cttgcatgcc tgcaggtcga ctctagagga tccacgggta
     1801 ccggtcgcca ccatggctct ttcaaacaag tttatcggag atgacatgaa aatgacctac
     1861 catatggatg gctgtgtcaa tgggcattac tttaccgtca aaggtgaagg cagcgggaag
     1921 ccatacgaag ggacgcagac ctcgactttt aaagtcacca tggccaacgg tgggcccctt
     1981 gcattctcct ttgacatact atctacagtg ttcatgtatg gaaatcgatg ctttactgcg
     2041 tatcctacca gtatgcccga ctatttcaaa caagcatttc ctgacggaat gtcatatgaa
     2101 aggactttta cctatgaaga tggaggagtt gctacagcca gttgggaaat aagccttaaa
     2161 ggcaactgct ttgagcacaa atccacgttt catggagtga actttcctgc tgatggacct
     2221 gtgatggcga agatgacaac tggttgggac ccatcttttg agaaaatgac tgtctgcgat
     2281 ggaatattga agggtgatgt caccgcgttc ctcatgctgc aaggaggtgg caattacaga
     2341 tgccaattcc acacttctta caagacaaaa aaaccggtga cgatgccacc aaaccatgcg
     2401 gtggaacatc gcattgcgag gaccgacctt gacaaaggtg gcaacagtgt tcagctgacg
     2461 gagcacgctg ttgcacatat aacctctgtt gtccctttcg cttaattagc tgagcttgga
     2521 ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc
     2581 ggttgccgcc gggcgttttt tattggtgag aatctcccgc tttacgaatt cccatgggga
     2641 gcatgcggag ttacgtcatc cccaccttcc gctgcgttct tcatcgatgc ccgcttagtg
     2701 atatgcttaa aactccaagc acagccttct ccccatgcgg agatatatcc cactcctcgc
     2761 cgaggctgcc attaatgaca cggttgaaga ccatagtagg agattcgtac attatgccta
     2821 caaccgtcgg ctgacgtcaa gcgtttcgca ttggggatgc tggttgtcat tgcccctcct
     2881 tcctctaagt gataaggtta cttcctctaa atgaccaagc agggtatcta atcctgttaa
     2941 cgctcgctca gagaattcgc atgcggagtc gagggagaac cagataagtg aaatctagtt
     3001 ccaaactatt ttgtcatttt taattttcgt attagcttac gacgctacac ccagttccca
     3061 tctattttgt cactcttccc taaataatcc ttaaaaactc catttccacc cctcccagtt
     3121 cccaactatt ttgtccgccc acacgctgag cgaattccac gcttgtggaa ttcctcgagg
     3181 gagttcacat atacctgccg ttcactatta tttagtgaaa tgagatatta tgatattttc
     3241 tgaattgtga ttaaaaaggc aactttatgc ccatgcaaca gaaactataa aaaatacaga
     3301 gaatgaaaag aaacagatag attttttagt tctttaggcc cgtagtctgc aaatcctttt
     3361 atgattttct atcaaacaaa agaggaaaat agaccagttg caatccaaac gagagtctaa
     3421 tagaatgagg tcgaaaagta aatcgcgcgg gtttgttact gataaagcag gcaagaccta
     3481 aaatgtgtaa agggcaaagt gtatactttg gcgtcacccc ttacatattt taggtctttt
     3541 tttattgtgc gtaactaact tgccatcttc aaacaggagg gctggaagaa gcagaccgct
     3601 aacacagtac ataaaaaagg agacatgaac gatgaacatc aaaaagtttg caaaacaagc
     3661 aacagtatta acctttacta ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa
     3721 agaaacgaac caaaagccat ataaggaaac atacggcatt tcccatatta cacgccatga
     3781 tatgctgcaa atccctgaac agcaaaaaaa tgaaaaatat caagttcctg aattcgattc
     3841 gtccacaatt aaaaatatct cttctgcaaa aggcctggac gtttgggaca gctggccatt
     3901 acaaaacgct gacggcactg tcgcaaacta tcacggctac cacatcgtct ttgcattagc
     3961 cggagatcct aaaaatgcgg atgacacatc gatttacatg ttctatcaaa aagtcggcga
     4021 aacttctatt gacagctgga aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga
     4081 tgcaaatgat tctatcctaa aagaccaaac acaagaatgg tcaggttcag ccacatttac
     4141 atctgacgga aaaatccgtt tattctacac tgatttctcc ggtaaacatt acggcaaaca
     4201 aacactgaca actgcacaag ttaacgtatc agcatcagac agctctttga acatcaacgg
     4261 tgtagaggat tataaatcaa tctttgacgg tgacggaaaa acgtatcaaa atgtacagca
     4321 gttcatcgat gaaggcaact acagctcagg cgacaaccat acgctgagag atcctcacta
     4381 cgtagaagat aaaggccaca aatacttagt atttgaagca aacactggaa ctgaagatgg
     4441 ctaccaaggc gaagaatctt tatttaacaa agcatactat ggcaaaagca catcattctt
     4501 ccgtcaagaa agtcaaaaac ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa
     4561 cggcgctctc ggtatgattg agctaaacga tgattacaca ctgaaaaaag tgatgaaacc
     4621 gctgattgca tctaacacag taacagatga aattgaacgc gcgaacgtct ttaaaatgaa
     4681 cggcaaatgg tacctgttca ctgactcccg cggatcaaaa atgacgattg acggcattac
     4741 gtctaacgat atttacatgc ttggttatgt ttctaattct ttaactggcc catacaagcc
     4801 gctgaacaaa actggccttg tgttaaaaat ggatcttgat cctaacgatg taacctttac
     4861 ttactcacac ttcgctgtac ctcaagcgaa aggaaacaat gtcgtgatta caagctatat
     4921 gacaaacaga ggattctacg cagacaaaca atcaacgttt gcgccaagct tcctgctgaa
     4981 catcaaaggc aagaaaacat ctgttgtcaa agacagcatc cttgaacaag gacaattaac
     5041 agttaacaaa taaattcgct gagcgaggat gcacatgtga ccgagggaag cttgcggccg
     5101 cgtcgtgact gggaaaaccc tggcgactag tcttggactc ctgttgatag atccagtaat
     5161 gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat
     5221 tggtgagaat ccaggggtcc ccaataatta cgatttaaat ttgtgtctca aaatctctga
     5281 tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata
     5341 aacagtaata caaggggtgt tatgagccat attcagcgtg aaacgagctg tagccgtccg
     5401 cgtctgaaca gcaacatgga tgcggatctg tatggctata aatgggcgcg tgataacgtg
     5461 ggtcagagcg gcgcgaccat ttatcgtctg tatggcaaac cggatgcgcc ggaactgttt
     5521 ctgaaacatg gcaaaggcag cgtggcgaac gatgtgaccg atgaaatggt gcgtctgaac
     5581 tggctgaccg aatttatgcc gctgccgacc attaaacatt ttattcgcac cccggatgat
     5641 gcgtggctgc tgaccaccgc gattccgggc aaaaccgcgt ttcaggtgct ggaagaatat
     5701 ccggatagcg gcgaaaacat tgtggatgcg ctggccgtgt ttctgcgtcg tctgcatagc
     5761 attccggtgt gcaactgccc gtttaacagc gatcgtgtgt ttcgtctggc ccaggcgcag
     5821 agccgtatga acaacggcct ggtggatgcg agcgattttg atgatgaacg taacggctgg
     5881 ccggtggaac aggtgtggaa agaaatgcat aaactgctgc cgtttagccc ggatagcgtg
     5941 gtgacccacg gcgattttag cctggataac ctgattttcg atgaaggcaa actgattggc
     6001 tgcattgatg tgggccgtgt gggcattgcg gatcgttatc aggatctggc cattctgtgg
     6061 aactgcctgg gcgaatttag cccgagcctg caaaaacgtc tgtttcagaa atatggcatt
     6121 gataatccgg atatgaacaa actgcaattt catctgatgc tggatgaatt tttctaataa
     6181 ttaattggac cgcggtccgc gcgttgtcct tttccgctgc ataaccctgc ttcggggtca
     6241 ttatagcgat tttttcggta tatccatcct ttttcgcacg atatacagga ttttgccaaa
     6301 gggttcgtgt agactttcct tggtgtatcc aacggcgtca gccgggcagg ataggtgaag
     6361 taggcccacc cgcgagcggg tgttccttct tcactgtccc ttattcgcac ctggcggtgc
     6421 tcaacgggaa tcctgctctg cgaggctggc cgtaggccgg ccgatctgaa gatcagcagt
     6481 tcaacctgtt gatagtacgt actaagctct catgtttcac gtactaagct ctcatgttta
     6541 acgtactaag ctctcatgtt taacgaacta aaccctcatg gctaacgtac taagctctca
     6601 tggctaacgt actaagctct catgtttcac gtactaagct ctcatgtttg aacaataaaa
     6661 ttaatataaa tcagcaactt aaatagcctc taaggtttta agttttataa gaaaaaaaag
     6721 aatatataag gcttttaaag cctttaaggt ttaacggttg tggacaacaa gccagggatg
     6781 taacgcactg agaagccctt agagcctctc aaagcaattt tgagtgacac aggaacactt
     6841 aacggctgac atggggcgcg cccagctgtc tagggcggcg gatttgtcct actcaggaga
     6901 gcgttcaccg acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt
     6961 tttatttgat gcct
//
