LOCUS       sequence-420657-        6965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc097 through Tn7 
            bacterial insertion, Gentamicin selectable marker and tagRFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6965)
  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 6965)
  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: pBC070
FEATURES             Location/Qualifiers
     source          1..6965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      282..376
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      478..564
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    complement(597..644)
                     /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(685..690,691..707,708..713)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             902..1435
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     protein_bind    complement(1565..1612)
                     /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(1640..1645,1646..1661,1662..1668)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1676..1692
                     /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      2445..2539
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  2943..3108
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3151..3596
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3597..3683,3684..5018)
                     /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      5101..5195
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5316..5335)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5327..6142
                     /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            6309..6417
                     /note="incP origin of transfer"
     rep_origin      6428..6816
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6847..6933)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 acaggaggcc gcctaggccg cggccgcgcg aatttgccga attcggatcc ggagggagtg
       61 tgggcggaca ataaagtctt aaactgaaca aaatagatct aaactatgac aataaagtct
      121 taaactagac agaatagttg taaactgaaa tcagtccagt tatgctgtga aaaagcatac
      181 tggacttttg ttatggctaa agcaaactct tcattttctg aagtgcaaat tgcccgtcgt
      241 attaaagagg ggcgtggcgc tgcaagaatt caagcttgga ggactcctgt tgatagatcc
      301 agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt
      361 ttttattggt gagaatccaa gctagactgc gatgagtggc agggcggggc gtaatttctt
      421 aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt gaggcatcaa
      481 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
      541 aacgctctcc tgagtaggac aaatccgccc gccgctgcaa gaattcaagc ttggaggaag
      601 ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaagatc ccctgattcc
      661 ctttgtcaac agcaatggat cgaattgaca taagcctgtt cggttcgtaa actgtaatgc
      721 aagtagcgta tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac
      781 ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttgtacagtc tatgcctcgg
      841 gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac
      901 gatgttacgc agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttagg
      961 tggctcaagt atgggcatca ttcgcacatg taggctcggc cctgaccaag tcaaatccat
     1021 gcgggctgct cttgatcttt tcggtcgtga gttcggagac gtagccacct actcccaaca
     1081 tcagccggac tccgattacc tcgggaactt gctccgtagt aagacattca tcgcgcttgc
     1141 tgccttcgac caagaagcgg ttgttggcgc tctcgcggct tacgttctgc ccaagtttga
     1201 gcagccgcgt agtgagatct atatctatga tctcgcagtc tccggcgagc accggaggca
     1261 gggcattgcc accgcgctca tcaatctcct caagcatgag gccaacgcgc ttggtgctta
     1321 tgtgatctac gtgcaagcag attacggtga cgatcccgca gtggctctct atacaaagtt
     1381 gggcatacgg gaagaagtga tgcactttga tatcgaccca agtaccgcca cctaacaatt
     1441 cgttcaagcc gagatcggct tcccggccgc ggagttgttc ggtaaattgt cacaacgccg
     1501 cggccaattc gatctagaat tccattgagt aagtttttaa gcacatcagc ttcaaaagcg
     1561 ctctgaagtt cctatacttt ctagagaata ggaacttcgg aataggtact tccgctacta
     1621 gaattcgagc ggggaatttt tgacaattaa tcatcggctc gtataatgtg tggaattgtg
     1681 agcggataac aattttcaca caggaaacag ctaaatggtg agtaaaggtg aagagttgat
     1741 taaagagaac atgcatatga agttatacat ggagggaact gtcaataatc accactttaa
     1801 gtgtacatca gagggtgagg gaaagccata cgagggaacc caaactatga gaatcaaagt
     1861 agtggaggga ggtcctcttc catttgcttt tgatatacta gcaacaagtt tcatgtatgg
     1921 ttccaggacc ttcattaacc atactcaggg aatccctgac ttctttaaac agtcttttcc
     1981 tgaaggtttt acatgggaga gggttaccac ttacgaggac ggtggagtct tgacagcaac
     2041 ccaggacact tcattacaag atggatgcct aatatacaat gtgaaaatta ggggtgtgaa
     2101 tttccctagt aacggaccag ttatgcagaa gaaaacacta ggttgggaag ctaatactga
     2161 aatgttgtac cctgccgacg gaggtttaga aggtagatcc gacatggccc ttaagctagt
     2221 cggaggtgga cacttgatct gtaactttaa aaccacatat aggtctaaga agccagcaaa
     2281 gaatctaaaa atgcctggtg tttactatgt ggaccataga ctagaaagga taaaagaagc
     2341 agacaaagaa acttacgtgg agcaacatga ggtcgccgtc gctaggtatt gcgacttacc
     2401 ttccaagcta ggtcacaaat tgaacgctta attagctgag cttggactcc tgttgataga
     2461 tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt gccgccgggc
     2521 gttttttatt ggtgagaatc tcccgcttta cgaattccca tggggagcat gcggagttac
     2581 gtcatcccca ccttccgctg cgttcttcat cgatgcccgc ttagtgatat gcttaaaact
     2641 ccaagcacag ccttctcccc tgcggagata tatcccactc ctcgccgagg ctgccataat
     2701 gcggagatat atcccactcc tcgccgaggc tgccattaat ggcaagtaca actgagcgag
     2761 ttctcgaatc tagcattaat gcgaaacgct tgacgtcagc cgacggttgt aggcattggg
     2821 gatgctggtt gtcattgccc ctccttcctc taagtgataa ggttacttcc tctaaatgac
     2881 caagcagggt atctaatcct gttaacgctc gctcagagaa ttcgcatgcg gagtcgaggg
     2941 agaaccagat aagtgaaatc tagttccaaa ctattttgtc atttttaatt ttcgtattag
     3001 cttacgacgc tacacccagt tcccatctat tttgtcactc ttccctaaat aatccttaaa
     3061 aactccattt ccacccctcc cagttcccaa ctattttgtc cgcccacacg ctgagcgaat
     3121 tccacgcttg tggaattcct cgagggagtt cacatatacc tgccgttcac tattatttag
     3181 tgaaatgaga tattatgata ttttctgaat tgtgattaaa aaggcaactt tatgcccatg
     3241 caacagaaac tataaaaaat acagagaatg aaaagaaaca gatagatttt ttagttcttt
     3301 aggcccgtag tctgcaaatc cttttatgat tttctatcaa acaaaagagg aaaatagacc
     3361 agttgcaatc caaacgagag tctaatagaa tgaggtcgaa aagtaaatcg cgcgggtttg
     3421 ttactgataa agcaggcaag acctaaaatg tgtaaagggc aaagtgtata ctttggcgtc
     3481 accccttaca tattttaggt ctttttttat tgtgcgtaac taacttgcca tcttcaaaca
     3541 ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca tgaacgatga
     3601 acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca ctgctggcag
     3661 gaggcgcaac tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag gaaacatacg
     3721 gcatttccca tattacacgc catgatatgc tgcaaatccc tgaacagcaa aaaaatgaaa
     3781 aatatcaagt tcctgaattc gattcgtcca caattaaaaa tatctcttct gcaaaaggcc
     3841 tggacgtttg ggacagctgg ccattacaaa acgctgacgg cactgtcgca aactatcacg
     3901 gctaccacat cgtctttgca ttagccggag atcctaaaaa tgcggatgac acatcgattt
     3961 acatgttcta tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac gctggccgcg
     4021 tctttaaaga cagcgacaaa ttcgatgcaa atgattctat cctaaaagac caaacacaag
     4081 aatggtcagg ttcagccaca tttacatctg acggaaaaat ccgtttattc tacactgatt
     4141 tctccggtaa acattacggc aaacaaacac tgacaactgc acaagttaac gtatcagcat
     4201 cagacagctc tttgaacatc aacggtgtag aggattataa atcaatcttt gacggtgacg
     4261 gaaaaacgta tcaaaatgta cagcagttca tcgatgaagg caactacagc tcaggcgaca
     4321 accatacgct gagagatcct cactacgtag aagataaagg ccacaaatac ttagtatttg
     4381 aagcaaacac tggaactgaa gatggctacc aaggcgaaga atctttattt aacaaagcat
     4441 actatggcaa aagcacatca ttcttccgtc aagaaagtca aaaacttctg caaagcgata
     4501 aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat gattgagcta aacgatgatt
     4561 acacactgaa aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca gatgaaattg
     4621 aacgcgcgaa cgtctttaaa atgaacggca aatggtacct gttcactgac tcccgcggat
     4681 caaaaatgac gattgacggc attacgtcta acgatattta catgcttggt tatgtttcta
     4741 attctttaac tggcccatac aagccgctga acaaaactgg ccttgtgtta aaaatggatc
     4801 ttgatcctaa cgatgtaacc tttacttact cacacttcgc tgtacctcaa gcgaaaggaa
     4861 acaatgtcgt gattacaagc tatatgacaa acagaggatt ctacgcagac aaacaatcaa
     4921 cgtttgcgcc aagcttcctg ctgaacatca aaggcaagaa aacatctgtt gtcaaagaca
     4981 gcatccttga acaaggacaa ttaacagtta acaaataaat tcgctgagcg aggatgcaca
     5041 tgtgaccgag ggaagcttgc ggccgcgtcg tgactgggaa aaccctggcg actagtcttg
     5101 gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc
     5161 tcggttgccg ccgggcgttt tttattggtg agaatccagg ggtccccaat aattacgatt
     5221 taaatttgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     5281 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     5341 gcgtgaaacg agctgtagcc gtccgcgtct gaacagcaac atggatgcgg atctgtatgg
     5401 ctataaatgg gcgcgtgata acgtgggtca gagcggcgcg accatttatc gtctgtatgg
     5461 caaaccggat gcgccggaac tgtttctgaa acatggcaaa ggcagcgtgg cgaacgatgt
     5521 gaccgatgaa atggtgcgtc tgaactggct gaccgaattt atgccgctgc cgaccattaa
     5581 acattttatt cgcaccccgg atgatgcgtg gctgctgacc accgcgattc cgggcaaaac
     5641 cgcgtttcag gtgctggaag aatatccgga tagcggcgaa aacattgtgg atgcgctggc
     5701 cgtgtttctg cgtcgtctgc atagcattcc ggtgtgcaac tgcccgttta acagcgatcg
     5761 tgtgtttcgt ctggcccagg cgcagagccg tatgaacaac ggcctggtgg atgcgagcga
     5821 ttttgatgat gaacgtaacg gctggccggt ggaacaggtg tggaaagaaa tgcataaact
     5881 gctgccgttt agcccggata gcgtggtgac ccacggcgat tttagcctgg ataacctgat
     5941 tttcgatgaa ggcaaactga ttggctgcat tgatgtgggc cgtgtgggca ttgcggatcg
     6001 ttatcaggat ctggccattc tgtggaactg cctgggcgaa tttagcccga gcctgcaaaa
     6061 acgtctgttt cagaaatatg gcattgataa tccggatatg aacaaactgc aatttcatct
     6121 gatgctggat gaatttttct aataattaat tggaccgcgg tccgcgcgtt gtccttttcc
     6181 gctgcataac cctgcttcgg ggtcattata gcgatttttt cggtatatcc atcctttttc
     6241 gcacgatata caggattttg ccaaagggtt cgtgtagact ttccttggtg tatccaacgg
     6301 cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc cttcttcact
     6361 gtcccttatt cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg ctggccgtag
     6421 gccggccgat ctgaagatca gcagttcaac ctgttgatag tacgtactaa gctctcatgt
     6481 ttcacgtact aagctctcat gtttaacgta ctaagctctc atgtttaacg aactaaaccc
     6541 tcatggctaa cgtactaagc tctcatggct aacgtactaa gctctcatgt ttcacgtact
     6601 aagctctcat gtttgaacaa taaaattaat ataaatcagc aacttaaata gcctctaagg
     6661 ttttaagttt tataagaaaa aaaagaatat ataaggcttt taaagccttt aaggtttaac
     6721 ggttgtggac aacaagccag ggatgtaacg cactgagaag cccttagagc ctctcaaagc
     6781 aattttgagt gacacaggaa cacttaacgg ctgacatggg gcgcgcccag ctgtctaggg
     6841 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     6901 ccagtctttc gactgagcct ttcgttttat ttgatgcctt taattaaagc ggataacaat
     6961 ttcac
//
