LOCUS       sequence-418908-        6962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc100 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 6962)
  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 6962)
  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: pBC073
FEATURES             Location/Qualifiers
     source          1..6962
                     /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)."
     terminator      2471..2565
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  2966..3131
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3174..3619
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3620..3706,3707..5041)
                     /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      5124..5218
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5339..5358)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5350..6165
                     /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            6332..6440
                     /note="incP origin of transfer"
     rep_origin      6451..6839
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6870..6956)
                     /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 aatttttgac aattaatcat
     1681 cggctcgtat aatgtgtgga attgtgagcg gataacaatt ttcacacagg aaacagctaa
     1741 atggtgagta aaggtgaaga gttgattaaa gagaacatgc atatgaagtt atacatggag
     1801 ggaactgtca ataatcacca ctttaagtgt acatcagagg gtgagggaaa gccatacgag
     1861 ggaacccaaa ctatgagaat caaagtagtg gagggaggtc ctcttccatt tgcttttgat
     1921 atactagcaa caagtttcat gtatggttcc aggaccttca ttaaccatac tcagggaatc
     1981 cctgacttct ttaaacagtc ttttcctgaa ggttttacat gggagagggt taccacttac
     2041 gaggacggtg gagtcttgac agcaacccag gacacttcat tacaagatgg atgcctaata
     2101 tacaatgtga aaattagggg tgtgaatttc cctagtaacg gaccagttat gcagaagaaa
     2161 acactaggtt gggaagctaa tactgaaatg ttgtaccctg ccgacggagg tttagaaggt
     2221 agatccgaca tggcccttaa gctagtcgga ggtggacact tgatctgtaa ctttaaaacc
     2281 acatataggt ctaagaagcc agcaaagaat ctaaaaatgc ctggtgttta ctatgtggac
     2341 catagactag aaaggataaa agaagcagac aaagaaactt acgtggagca acatgaggtc
     2401 gccgtcgcta ggtattgcga cttaccttcc aagctaggtc acaaattgaa cgcttaatta
     2461 gctgagcttg gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg
     2521 ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatctccc gctttacgaa
     2581 ttcccatggg gagcatgcgg agttacgtca tccccacctt ccgctgcgtt cttcatcgat
     2641 gcccgcttag tgatatgctt aaaactccaa gcacagcctt ctccccatga tcgccagcag
     2701 gaggccgaac tgctagtcat cattatgggt ctgacgtttc acttccagcc ttgcggttac
     2761 attatgacta actgatccta ttcacgtgtt cctttacaca ttatggcaag tacaactgag
     2821 cgagttctcg aatctagcat ggggatgctg gttgtcattg cccctccttc ctctaagtga
     2881 taaggttact tcctctaaat gaccaagcag ggtatctaat cctgttaacg ctcgctcaga
     2941 gaattcgcat gcggagtcga gggagaacca gataagtgaa atctagttcc aaactatttt
     3001 gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca
     3061 ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt
     3121 gtccgcccac acgctgagcg aattccacgc ttgtggaatt cctcgaggga gttcacatat
     3181 acctgccgtt cactattatt tagtgaaatg agatattatg atattttctg aattgtgatt
     3241 aaaaaggcaa ctttatgccc atgcaacaga aactataaaa aatacagaga atgaaaagaa
     3301 acagatagat tttttagttc tttaggcccg tagtctgcaa atccttttat gattttctat
     3361 caaacaaaag aggaaaatag accagttgca atccaaacga gagtctaata gaatgaggtc
     3421 gaaaagtaaa tcgcgcgggt ttgttactga taaagcaggc aagacctaaa atgtgtaaag
     3481 ggcaaagtgt atactttggc gtcacccctt acatatttta ggtctttttt tattgtgcgt
     3541 aactaacttg ccatcttcaa acaggagggc tggaagaagc agaccgctaa cacagtacat
     3601 aaaaaaggag acatgaacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac
     3661 ctttactacc gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca
     3721 aaagccatat aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat
     3781 ccctgaacag caaaaaaatg aaaaatatca agttcctgaa ttcgattcgt ccacaattaa
     3841 aaatatctct tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacgctga
     3901 cggcactgtc gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa
     3961 aaatgcggat gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga
     4021 cagctggaaa aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc
     4081 tatcctaaaa gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa
     4141 aatccgttta ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac
     4201 tgcacaagtt aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta
     4261 taaatcaatc tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga
     4321 aggcaactac agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa
     4381 aggccacaaa tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga
     4441 agaatcttta tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag
     4501 tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg
     4561 tatgattgag ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc
     4621 taacacagta acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta
     4681 cctgttcact gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat
     4741 ttacatgctt ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac
     4801 tggccttgtg ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt
     4861 cgctgtacct caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg
     4921 attctacgca gacaaacaat caacgtttgc gccaagcttc ctgctgaaca tcaaaggcaa
     4981 gaaaacatct gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata
     5041 aattcgctga gcgaggatgc acatgtgacc gagggaagct tgcggccgcg tcgtgactgg
     5101 gaaaaccctg gcgactagtc ttggactcct gttgatagat ccagtaatga cctcagaact
     5161 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     5221 aggggtcccc aataattacg atttaaattt gtgtctcaaa atctctgatg ttacattgca
     5281 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     5341 aggggtgtta tgagccatat tcagcgtgaa acgagctgta gccgtccgcg tctgaacagc
     5401 aacatggatg cggatctgta tggctataaa tgggcgcgtg ataacgtggg tcagagcggc
     5461 gcgaccattt atcgtctgta tggcaaaccg gatgcgccgg aactgtttct gaaacatggc
     5521 aaaggcagcg tggcgaacga tgtgaccgat gaaatggtgc gtctgaactg gctgaccgaa
     5581 tttatgccgc tgccgaccat taaacatttt attcgcaccc cggatgatgc gtggctgctg
     5641 accaccgcga ttccgggcaa aaccgcgttt caggtgctgg aagaatatcc ggatagcggc
     5701 gaaaacattg tggatgcgct ggccgtgttt ctgcgtcgtc tgcatagcat tccggtgtgc
     5761 aactgcccgt ttaacagcga tcgtgtgttt cgtctggccc aggcgcagag ccgtatgaac
     5821 aacggcctgg tggatgcgag cgattttgat gatgaacgta acggctggcc ggtggaacag
     5881 gtgtggaaag aaatgcataa actgctgccg tttagcccgg atagcgtggt gacccacggc
     5941 gattttagcc tggataacct gattttcgat gaaggcaaac tgattggctg cattgatgtg
     6001 ggccgtgtgg gcattgcgga tcgttatcag gatctggcca ttctgtggaa ctgcctgggc
     6061 gaatttagcc cgagcctgca aaaacgtctg tttcagaaat atggcattga taatccggat
     6121 atgaacaaac tgcaatttca tctgatgctg gatgaatttt tctaataatt aattggaccg
     6181 cggtccgcgc gttgtccttt tccgctgcat aaccctgctt cggggtcatt atagcgattt
     6241 tttcggtata tccatccttt ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag
     6301 actttccttg gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta ggcccacccg
     6361 cgagcgggtg ttccttcttc actgtccctt attcgcacct ggcggtgctc aacgggaatc
     6421 ctgctctgcg aggctggccg taggccggcc gatctgaaga tcagcagttc aacctgttga
     6481 tagtacgtac taagctctca tgtttcacgt actaagctct catgtttaac gtactaagct
     6541 ctcatgttta acgaactaaa ccctcatggc taacgtacta agctctcatg gctaacgtac
     6601 taagctctca tgtttcacgt actaagctct catgtttgaa caataaaatt aatataaatc
     6661 agcaacttaa atagcctcta aggttttaag ttttataaga aaaaaaagaa tatataaggc
     6721 ttttaaagcc tttaaggttt aacggttgtg gacaacaagc cagggatgta acgcactgag
     6781 aagcccttag agcctctcaa agcaattttg agtgacacag gaacacttaa cggctgacat
     6841 ggggcgcgcc cagctgtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac
     6901 aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
     6961 ct
//
