LOCUS       sequence-420659-        7412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc182 through Tn7 
            bacterial insertion, Spectinomycin and Streptomycin selectable 
            marker and tagBFP fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7412)
  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 7412)
  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: pBC155
FEATURES             Location/Qualifiers
     source          1..7412
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      308..402
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      504..589
                     /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"
     terminator      949..1036
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    complement(1069..1116)
                     /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(1157..1162,1163..1179,1180..1185)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             1375..1908
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     protein_bind    complement(2038..2085)
                     /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(2113..2118,2119..2134,2135..2141)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2149..2165
                     /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      2918..3012
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3416..3581
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3624..4069
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4070..4156,4157..5491)
                     /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      5574..5668
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5789..5808)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5800..6615
                     /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            6782..6890
                     /note="incP origin of transfer"
     rep_origin      6901..7289
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7320..7406)
                     /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 taggacaaay 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 acacgggaat cctgctctgc gaggctggcc gtaggccggc cgatctgaag
      901 atcagccccc ttcaacgcct ggggtaatga ctctctagct tgcggcatca aataaaacga
      961 aaggctcagg tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     1021 cctgagtagg acaaatccgc ccgccgctgc aagaattcaa gcttggagga agttcctata
     1081 ctttctagag aataggaact tcggaatagg aacttcaaga tcccctgatt ccctttgtca
     1141 acagcaatgg atcgaattga cataagcctg ttcggttcgt aaactgtaat gcaagtagcg
     1201 tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt
     1261 ggcggttttc atggcttgtt atgactgttt ttttgtnaca gtctatgcct cgggcatcca
     1321 agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta
     1381 cgcagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt aggtggctca
     1441 agtatgggca tcattcgcac atgtaggctc ggccctgacc aagtcaaatc catgcgggct
     1501 gctcttgatc ttttcggtcg tgagttcgga gacgtagcca cctactccca acatcagccg
     1561 gactccgatt acctcgggaa cttgctccgt agtaagacat tcatcgcgct tgctgccttc
     1621 gaccaagaag cggttgttgg cgctctcgcg gcttacgttc tgcccaagtt tgagcagccg
     1681 cgtagtgaga tctatatcta tgatctcgca gtctccggcg agcaccggag gcagggcatt
     1741 gccaccgcgc tcatcaatct cctcaagcat gaggccaacg cgcttggtgc ttatgtgatc
     1801 tacgtgcaag cagattacgg tgacgatccc gcagtggctc tctatacaaa gttgggcata
     1861 cgggaagaag tgatgcactt tgatatcgac ccaagtaccg ccacctaaca attcgttcaa
     1921 gccgagatcg gcttcccggc cgcggagttg ttcggtaaat tgtcacaacg ccgcggccaa
     1981 ttcgatctag aattccattg agtaagtttt taagcacatc agcttcaaaa gcgctctgaa
     2041 gttcctatac tttctagaga ataggaactt cggaataggt acttccgcta ctagaattcg
     2101 agcggggaat ttttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat
     2161 aacaattttc acacaggaaa cagctaaatg gtgagtaaag gtgaagagtt gattaaagag
     2221 aacatgcata tgaagttata catggaggga actgtcaata atcaccactt taagtgtaca
     2281 tcagagggtg agggaaagcc atacgaggga acccaaacta tgagaatcaa agtagtggag
     2341 ggaggtcctc ttccatttgc ttttgatata ctagcaacaa gtttcatgta tggttccagg
     2401 accttcatta accatactca gggaatccct gacttcttta aacagtcttt tcctgaaggt
     2461 tttacatggg agagggttac cacttacgag gacggtggag tcttgacagc aacccaggac
     2521 acttcattac aagatggatg cctaatatac aatgtgaaaa ttaggggtgt gaatttccct
     2581 agtaacggac cagttatgca gaagaaaaca ctaggttggg aagctaatac tgaaatgttg
     2641 taccctgccg acggaggttt agaaggtaga tccgacatgg cccttaagct agtcggaggt
     2701 ggacacttga tctgtaactt taaaaccaca tataggtcta agaagccagc aaagaatcta
     2761 aaaatgcctg gtgtttacta tgtggaccat agactagaaa ggataaaaga agcagacaaa
     2821 gaaacttacg tggagcaaca tgaggtcgcc gtcgctaggt attgcgactt accttccaag
     2881 ctaggtcaca aattgaacgc ttaattagct gagcttggac tcctgttgat agatccagta
     2941 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     3001 attggtgaga atctcccgct ttacgaattc ccatggggag catgcggagt tacgtcatcc
     3061 ccaccttccg ctgcgttctt catcgatgcc cgcttagtga tatgcttaaa actccaagca
     3121 cagccttctc ccctgcggag atatatccca ctcctcgccg aggctgccat aatgcggaga
     3181 tatatcccac tcctcgccga ggctgccatt aatggcaagt acaactgagc gagttctcga
     3241 atctagcatt aatgcgaaac gcttgacgtc agccgacggt tgtaggcatt ggggatgctg
     3301 gttgtcattg cccctccttc ctctaagtga taaggttact tcctctaaat gaccaagcag
     3361 ggtatctaat cctgttaacg ctcgctcaga gaattcgcat gcggagtcga gggagaacca
     3421 gataagtgaa atctagttcc aaactatttt gtcattttta attttcgtat tagcttacga
     3481 cgctacaccc agttcccatc tattttgtca ctcttcccta aataatcctt aaaaactcca
     3541 tttccacccc tcccagttcc caactatttt gtccgcccac acgctgagcg aattccacgc
     3601 ttgtggaatt cctcgaggga gttcacatat acctgccgtt cactattatt tagtgaaatg
     3661 agatattatg atattttctg aattgtgatt aaaaaggcaa ctttatgccc atgcaacaga
     3721 aactataaaa aatacagaga atgaaaagaa acagatagat tttttagttc tttaggcccg
     3781 tagtctgcaa atccttttat gattttctat caaacaaaag aggaaaatag accagttgca
     3841 atccaaacga gagtctaata gaatgaggtc gaaaagtaaa tcgcgcgggt ttgttactga
     3901 taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt atactttggc gtcacccctt
     3961 acatatttta ggtctttttt tattgtgcgt aactaacttg ccatcttcaa acaggagggc
     4021 tggaagaagc agaccgctaa cacagtacat aaaaaaggag acatgaacga tgaacatcaa
     4081 aaagtttgca aaacaagcaa cagtattaac ctttactacc gcactgctgg caggaggcgc
     4141 aactcaagcg tttgcgaaag aaacgaacca aaagccatat aaggaaacat acggcatttc
     4201 ccatattaca cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca
     4261 agttcctgaa ttcgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt
     4321 ttgggacagc tggccattac aaaacgctga cggcactgtc gcaaactatc acggctacca
     4381 catcgtcttt gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt
     4441 ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa
     4501 agacagcgac aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc
     4561 aggttcagcc acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg
     4621 taaacattac ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag catcagacag
     4681 ctctttgaac atcaacggtg tagaggatta taaatcaatc tttgacggtg acggaaaaac
     4741 gtatcaaaat gtacagcagt tcatcgatga aggcaactac agctcaggcg acaaccatac
     4801 gctgagagat cctcactacg tagaagataa aggccacaaa tacttagtat ttgaagcaaa
     4861 cactggaact gaagatggct accaaggcga agaatcttta tttaacaaag catactatgg
     4921 caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg
     4981 cacggctgag ttagcaaacg gcgctctcgg tatgattgag ctaaacgatg attacacact
     5041 gaaaaaagtg atgaaaccgc tgattgcatc taacacagta acagatgaaa ttgaacgcgc
     5101 gaacgtcttt aaaatgaacg gcaaatggta cctgttcact gactcccgcg gatcaaaaat
     5161 gacgattgac ggcattacgt ctaacgatat ttacatgctt ggttatgttt ctaattcttt
     5221 aactggccca tacaagccgc tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc
     5281 taacgatgta acctttactt actcacactt cgctgtacct caagcgaaag gaaacaatgt
     5341 cgtgattaca agctatatga caaacagagg attctacgca gacaaacaat caacgtttgc
     5401 gccaagcttc ctgctgaaca tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct
     5461 tgaacaagga caattaacag ttaacaaata aattcgctga gcgaggatgc acatgtgacc
     5521 gagggaagct tgcggccgcg tcgtgactgg gaaaaccctg gcgactagtc ttggactcct
     5581 gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg
     5641 ccgccgggcg ttttttattg gtgagaatcc aggggtcccc aataattacg atttaaattt
     5701 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     5761 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcagcgtgaa
     5821 acgagctgta gccgtccgcg tctgaacagc aacatggatg cggatctgta tggctataaa
     5881 tgggcgcgtg ataacgtggg tcagagcggc gcgaccattt atcgtctgta tggcaaaccg
     5941 gatgcgccgg aactgtttct gaaacatggc aaaggcagcg tggcgaacga tgtgaccgat
     6001 gaaatggtgc gtctgaactg gctgaccgaa tttatgccgc tgccgaccat taaacatttt
     6061 attcgcaccc cggatgatgc gtggctgctg accaccgcga ttccgggcaa aaccgcgttt
     6121 caggtgctgg aagaatatcc ggatagcggc gaaaacattg tggatgcgct ggccgtgttt
     6181 ctgcgtcgtc tgcatagcat tccggtgtgc aactgcccgt ttaacagcga tcgtgtgttt
     6241 cgtctggccc aggcgcagag ccgtatgaac aacggcctgg tggatgcgag cgattttgat
     6301 gatgaacgta acggctggcc ggtggaacag gtgtggaaag aaatgcataa actgctgccg
     6361 tttagcccgg atagcgtggt gacccacggc gattttagcc tggataacct gattttcgat
     6421 gaaggcaaac tgattggctg cattgatgtg ggccgtgtgg gcattgcgga tcgttatcag
     6481 gatctggcca ttctgtggaa ctgcctgggc gaatttagcc cgagcctgca aaaacgtctg
     6541 tttcagaaat atggcattga taatccggat atgaacaaac tgcaatttca tctgatgctg
     6601 gatgaatttt tctaataatt aattggaccg cggtccgcgc gttgtccttt tccgctgcat
     6661 aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt ttcgcacgat
     6721 atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa cggcgtcagc
     6781 cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc actgtccctt
     6841 attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg taggccggcc
     6901 gatctgaaga tcagcagttc aacctgttga tagtacgtac taagctctca tgtttcacgt
     6961 actaagctct catgtttaac gtactaagct ctcatgttta acgaactaaa ccctcatggc
     7021 taacgtacta agctctcatg gctaacgtac taagctctca tgtttcacgt actaagctct
     7081 catgtttgaa caataaaatt aatataaatc agcaacttaa atagcctcta aggttttaag
     7141 ttttataaga aaaaaaagaa tatataaggc ttttaaagcc tttaaggttt aacggttgtg
     7201 gacaacaagc cagggatgta acgcactgag aagcccttag agcctctcaa agcaattttg
     7261 agtgacacag gaacacttaa cggctgacat ggggcgcgcc cagctgtcta gggcggcgga
     7321 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
     7381 ttcgactgag cctttcgttt tatttgatgc ct
//
