LOCUS       sequence-418899-        6926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc135 through Tn7 
            bacterial insertion, Gentamicin selectable marker and far-red-FP 
            fluorescent marker mPlum.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6926)
  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 6926)
  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: pBC108
FEATURES             Location/Qualifiers
     source          1..6926
                     /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)."
     CDS             1741..2424
                     /codon_start=1
                     /product="far-red monomeric derivative of DsRed fluorescent
                     protein (Wang et al., 2004)"
                     /label=mPlum
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKEHMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     RLKVTKGGPLPFAWDILSPQIMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGEFIYKVKVRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEMK
                     MRLRLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRH
                     STGAA"
     primer_bind     2336..2359
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      2438..2532
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  2930..3095
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3138..3583
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3584..3670,3671..5005)
                     /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      5088..5182
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5303..5322)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5314..6129
                     /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            6296..6404
                     /note="incP origin of transfer"
     rep_origin      6415..6803
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6834..6920)
                     /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 atggtgagca agggcgagga ggtcatcaag gagttcatgc gcttcaagga gcacatggag
     1801 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1861 ggcacccaga ccgccaggct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     1921 atcctgtccc ctcagatcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     1981 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     2041 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     2101 tacaaggtga aggtgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaaaaag
     2161 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     2221 gagatgaaga tgaggctgag gctgaaggac ggcggccact acgacgccga ggtcaagacc
     2281 acctacatgg ccaagaagcc cgtgcagctg cccggcgcct acaagaccga catcaagctg
     2341 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg cgccgagggc
     2401 cgccactcca ccggcgccgc ttaattagct gagcttggac tcctgttgat agatccagta
     2461 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     2521 attggtgaga atctcccgct ttacgaattc ccatggggag catgcggagt tacgtcatcc
     2581 ccaccttccg ctgcgttctt catcgatgcc cgcttagtga tatgcttaaa actccaagca
     2641 cagccttctc ccctgtacga atctcctact atggtcttca accgtgtcat tgcggagata
     2701 tatcccactc ctcgccgagg ctgccattat gaacacctaa cgtagctata taggacacag
     2761 ttgcatttgg caagtacaac tgagcgagtt ctcgaatcta gcatggggat gctggttgtc
     2821 attgcccctc cttcctctaa gtgataaggt tacttcctct aaatgaccaa gcagggtatc
     2881 taatcctgtt aacgctcgct cagagaattc gcatgcggag tcgagggaga accagataag
     2941 tgaaatctag ttccaaacta ttttgtcatt tttaattttc gtattagctt acgacgctac
     3001 acccagttcc catctatttt gtcactcttc cctaaataat ccttaaaaac tccatttcca
     3061 cccctcccag ttcccaacta ttttgtccgc ccacacgctg agcgaattcc acgcttgtgg
     3121 aattcctcga gggagttcac atatacctgc cgttcactat tatttagtga aatgagatat
     3181 tatgatattt tctgaattgt gattaaaaag gcaactttat gcccatgcaa cagaaactat
     3241 aaaaaataca gagaatgaaa agaaacagat agatttttta gttctttagg cccgtagtct
     3301 gcaaatcctt ttatgatttt ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa
     3361 acgagagtct aatagaatga ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc
     3421 aggcaagacc taaaatgtgt aaagggcaaa gtgtatactt tggcgtcacc ccttacatat
     3481 tttaggtctt tttttattgt gcgtaactaa cttgccatct tcaaacagga gggctggaag
     3541 aagcagaccg ctaacacagt acataaaaaa ggagacatga acgatgaaca tcaaaaagtt
     3601 tgcaaaacaa gcaacagtat taacctttac taccgcactg ctggcaggag gcgcaactca
     3661 agcgtttgcg aaagaaacga accaaaagcc atataaggaa acatacggca tttcccatat
     3721 tacacgccat gatatgctgc aaatccctga acagcaaaaa aatgaaaaat atcaagttcc
     3781 tgaattcgat tcgtccacaa ttaaaaatat ctcttctgca aaaggcctgg acgtttggga
     3841 cagctggcca ttacaaaacg ctgacggcac tgtcgcaaac tatcacggct accacatcgt
     3901 ctttgcatta gccggagatc ctaaaaatgc ggatgacaca tcgatttaca tgttctatca
     3961 aaaagtcggc gaaacttcta ttgacagctg gaaaaacgct ggccgcgtct ttaaagacag
     4021 cgacaaattc gatgcaaatg attctatcct aaaagaccaa acacaagaat ggtcaggttc
     4081 agccacattt acatctgacg gaaaaatccg tttattctac actgatttct ccggtaaaca
     4141 ttacggcaaa caaacactga caactgcaca agttaacgta tcagcatcag acagctcttt
     4201 gaacatcaac ggtgtagagg attataaatc aatctttgac ggtgacggaa aaacgtatca
     4261 aaatgtacag cagttcatcg atgaaggcaa ctacagctca ggcgacaacc atacgctgag
     4321 agatcctcac tacgtagaag ataaaggcca caaatactta gtatttgaag caaacactgg
     4381 aactgaagat ggctaccaag gcgaagaatc tttatttaac aaagcatact atggcaaaag
     4441 cacatcattc ttccgtcaag aaagtcaaaa acttctgcaa agcgataaaa aacgcacggc
     4501 tgagttagca aacggcgctc tcggtatgat tgagctaaac gatgattaca cactgaaaaa
     4561 agtgatgaaa ccgctgattg catctaacac agtaacagat gaaattgaac gcgcgaacgt
     4621 ctttaaaatg aacggcaaat ggtacctgtt cactgactcc cgcggatcaa aaatgacgat
     4681 tgacggcatt acgtctaacg atatttacat gcttggttat gtttctaatt ctttaactgg
     4741 cccatacaag ccgctgaaca aaactggcct tgtgttaaaa atggatcttg atcctaacga
     4801 tgtaaccttt acttactcac acttcgctgt acctcaagcg aaaggaaaca atgtcgtgat
     4861 tacaagctat atgacaaaca gaggattcta cgcagacaaa caatcaacgt ttgcgccaag
     4921 cttcctgctg aacatcaaag gcaagaaaac atctgttgtc aaagacagca tccttgaaca
     4981 aggacaatta acagttaaca aataaattcg ctgagcgagg atgcacatgt gaccgaggga
     5041 agcttgcggc cgcgtcgtga ctgggaaaac cctggcgact agtcttggac tcctgttgat
     5101 agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg
     5161 ggcgtttttt attggtgaga atccaggggt ccccaataat tacgatttaa atttgtgtct
     5221 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg
     5281 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcagcg tgaaacgagc
     5341 tgtagccgtc cgcgtctgaa cagcaacatg gatgcggatc tgtatggcta taaatgggcg
     5401 cgtgataacg tgggtcagag cggcgcgacc atttatcgtc tgtatggcaa accggatgcg
     5461 ccggaactgt ttctgaaaca tggcaaaggc agcgtggcga acgatgtgac cgatgaaatg
     5521 gtgcgtctga actggctgac cgaatttatg ccgctgccga ccattaaaca ttttattcgc
     5581 accccggatg atgcgtggct gctgaccacc gcgattccgg gcaaaaccgc gtttcaggtg
     5641 ctggaagaat atccggatag cggcgaaaac attgtggatg cgctggccgt gtttctgcgt
     5701 cgtctgcata gcattccggt gtgcaactgc ccgtttaaca gcgatcgtgt gtttcgtctg
     5761 gcccaggcgc agagccgtat gaacaacggc ctggtggatg cgagcgattt tgatgatgaa
     5821 cgtaacggct ggccggtgga acaggtgtgg aaagaaatgc ataaactgct gccgtttagc
     5881 ccggatagcg tggtgaccca cggcgatttt agcctggata acctgatttt cgatgaaggc
     5941 aaactgattg gctgcattga tgtgggccgt gtgggcattg cggatcgtta tcaggatctg
     6001 gccattctgt ggaactgcct gggcgaattt agcccgagcc tgcaaaaacg tctgtttcag
     6061 aaatatggca ttgataatcc ggatatgaac aaactgcaat ttcatctgat gctggatgaa
     6121 tttttctaat aattaattgg accgcggtcc gcgcgttgtc cttttccgct gcataaccct
     6181 gcttcggggt cattatagcg attttttcgg tatatccatc ctttttcgca cgatatacag
     6241 gattttgcca aagggttcgt gtagactttc cttggtgtat ccaacggcgt cagccgggca
     6301 ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc
     6361 acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccgtaggcc ggccgatctg
     6421 aagatcagca gttcaacctg ttgatagtac gtactaagct ctcatgtttc acgtactaag
     6481 ctctcatgtt taacgtacta agctctcatg tttaacgaac taaaccctca tggctaacgt
     6541 actaagctct catggctaac gtactaagct ctcatgtttc acgtactaag ctctcatgtt
     6601 tgaacaataa aattaatata aatcagcaac ttaaatagcc tctaaggttt taagttttat
     6661 aagaaaaaaa agaatatata aggcttttaa agcctttaag gtttaacggt tgtggacaac
     6721 aagccaggga tgtaacgcac tgagaagccc ttagagcctc tcaaagcaat tttgagtgac
     6781 acaggaacac ttaacggctg acatggggcg cgcccagctg tctagggcgg cggatttgtc
     6841 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
     6901 tgagcctttc gttttatttg atgcct
//
