LOCUS       sequence-417235-        7009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc188 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 7009)
  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 7009)
  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: pBC161
FEATURES             Location/Qualifiers
     source          1..7009
                     /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  3013..3178
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3221..3666
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3667..3753,3754..5088)
                     /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      5171..5265
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5386..5405)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5397..6212
                     /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            6379..6487
                     /note="incP origin of transfer"
     rep_origin      6498..6886
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6917..7003)
                     /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 cccccgaaac gcttgacgtc agccgacggt
     2761 tgtaggcatt aatgactaac tgatcctatt cagtgttcct ttacacatta atgaacacct
     2821 aacgtagcta tataggacac agttgcatta atggagttaa cgctggcgag tagagcttac
     2881 tgcgccaggg gatgctggtt gtcattgccc ctccttcctc taagtgataa ggttacttcc
     2941 tctaaatgac caagcagggt atctaatcct gttaacgctc gctcagagaa ttcgcatgcg
     3001 gagtcgaggg agaaccagat aagtgaaatc tagttccaaa ctattttgtc atttttaatt
     3061 ttcgtattag cttacgacgc tacacccagt tcccatctat tttgtcactc ttccctaaat
     3121 aatccttaaa aactccattt ccacccctcc cagttcccaa ctattttgtc cgcccacacg
     3181 ctgagcgaat tccacgcttg tggaattcct cgagggagtt cacatatacc tgccgttcac
     3241 tattatttag tgaaatgaga tattatgata ttttctgaat tgtgattaaa aaggcaactt
     3301 tatgcccatg caacagaaac tataaaaaat acagagaatg aaaagaaaca gatagatttt
     3361 ttagttcttt aggcccgtag tctgcaaatc cttttatgat tttctatcaa acaaaagagg
     3421 aaaatagacc agttgcaatc caaacgagag tctaatagaa tgaggtcgaa aagtaaatcg
     3481 cgcgggtttg ttactgataa agcaggcaag acctaaaatg tgtaaagggc aaagtgtata
     3541 ctttggcgtc accccttaca tattttaggt ctttttttat tgtgcgtaac taacttgcca
     3601 tcttcaaaca ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca
     3661 tgaacgatga acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca
     3721 ctgctggcag gaggcgcaac tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag
     3781 gaaacatacg gcatttccca tattacacgc catgatatgc tgcaaatccc tgaacagcaa
     3841 aaaaatgaaa aatatcaagt tcctgaattc gattcgtcca caattaaaaa tatctcttct
     3901 gcaaaaggcc tggacgtttg ggacagctgg ccattacaaa acgctgacgg cactgtcgca
     3961 aactatcacg gctaccacat cgtctttgca ttagccggag atcctaaaaa tgcggatgac
     4021 acatcgattt acatgttcta tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac
     4081 gctggccgcg tctttaaaga cagcgacaaa ttcgatgcaa atgattctat cctaaaagac
     4141 caaacacaag aatggtcagg ttcagccaca tttacatctg acggaaaaat ccgtttattc
     4201 tacactgatt tctccggtaa acattacggc aaacaaacac tgacaactgc acaagttaac
     4261 gtatcagcat cagacagctc tttgaacatc aacggtgtag aggattataa atcaatcttt
     4321 gacggtgacg gaaaaacgta tcaaaatgta cagcagttca tcgatgaagg caactacagc
     4381 tcaggcgaca accatacgct gagagatcct cactacgtag aagataaagg ccacaaatac
     4441 ttagtatttg aagcaaacac tggaactgaa gatggctacc aaggcgaaga atctttattt
     4501 aacaaagcat actatggcaa aagcacatca ttcttccgtc aagaaagtca aaaacttctg
     4561 caaagcgata aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat gattgagcta
     4621 aacgatgatt acacactgaa aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca
     4681 gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca aatggtacct gttcactgac
     4741 tcccgcggat caaaaatgac gattgacggc attacgtcta acgatattta catgcttggt
     4801 tatgtttcta attctttaac tggcccatac aagccgctga acaaaactgg ccttgtgtta
     4861 aaaatggatc ttgatcctaa cgatgtaacc tttacttact cacacttcgc tgtacctcaa
     4921 gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa acagaggatt ctacgcagac
     4981 aaacaatcaa cgtttgcgcc aagcttcctg ctgaacatca aaggcaagaa aacatctgtt
     5041 gtcaaagaca gcatccttga acaaggacaa ttaacagtta acaaataaat tcgctgagcg
     5101 aggatgcaca tgtgaccgag ggaagcttgc ggccgcgtcg tgactgggaa aaccctggcg
     5161 actagtcttg gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg
     5221 ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatccagg ggtccccaat
     5281 aattacgatt taaatttgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     5341 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     5401 gccatattca gcgtgaaacg agctgtagcc gtccgcgtct gaacagcaac atggatgcgg
     5461 atctgtatgg ctataaatgg gcgcgtgata acgtgggtca gagcggcgcg accatttatc
     5521 gtctgtatgg caaaccggat gcgccggaac tgtttctgaa acatggcaaa ggcagcgtgg
     5581 cgaacgatgt gaccgatgaa atggtgcgtc tgaactggct gaccgaattt atgccgctgc
     5641 cgaccattaa acattttatt cgcaccccgg atgatgcgtg gctgctgacc accgcgattc
     5701 cgggcaaaac cgcgtttcag gtgctggaag aatatccgga tagcggcgaa aacattgtgg
     5761 atgcgctggc cgtgtttctg cgtcgtctgc atagcattcc ggtgtgcaac tgcccgttta
     5821 acagcgatcg tgtgtttcgt ctggcccagg cgcagagccg tatgaacaac ggcctggtgg
     5881 atgcgagcga ttttgatgat gaacgtaacg gctggccggt ggaacaggtg tggaaagaaa
     5941 tgcataaact gctgccgttt agcccggata gcgtggtgac ccacggcgat tttagcctgg
     6001 ataacctgat tttcgatgaa ggcaaactga ttggctgcat tgatgtgggc cgtgtgggca
     6061 ttgcggatcg ttatcaggat ctggccattc tgtggaactg cctgggcgaa tttagcccga
     6121 gcctgcaaaa acgtctgttt cagaaatatg gcattgataa tccggatatg aacaaactgc
     6181 aatttcatct gatgctggat gaatttttct aataattaat tggaccgcgg tccgcgcgtt
     6241 gtccttttcc gctgcataac cctgcttcgg ggtcattata gcgatttttt cggtatatcc
     6301 atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact ttccttggtg
     6361 tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc
     6421 cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg
     6481 ctggccgtag gccggccgat ctgaagatca gcagttcaac ctgttgatag tacgtactaa
     6541 gctctcatgt ttcacgtact aagctctcat gtttaacgta ctaagctctc atgtttaacg
     6601 aactaaaccc tcatggctaa cgtactaagc tctcatggct aacgtactaa gctctcatgt
     6661 ttcacgtact aagctctcat gtttgaacaa taaaattaat ataaatcagc aacttaaata
     6721 gcctctaagg ttttaagttt tataagaaaa aaaagaatat ataaggcttt taaagccttt
     6781 aaggtttaac ggttgtggac aacaagccag ggatgtaacg cactgagaag cccttagagc
     6841 ctctcaaagc aattttgagt gacacaggaa cacttaacgg ctgacatggg gcgcgcccag
     6901 ctgtctaggg cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa
     6961 aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcct
//
