LOCUS       sequence-419248-        6931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc134 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 6931)
  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 6931)
  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: pBC107
FEATURES             Location/Qualifiers
     source          1..6931
                     /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  2935..3100
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3143..3588
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3589..3675,3676..5010)
                     /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      5093..5187
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5308..5327)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5319..6134
                     /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            6301..6409
                     /note="incP origin of transfer"
     rep_origin      6420..6808
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6839..6925)
                     /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 cccatgacta actgatccta ttcacgtgtt cctttacaca ttatgacacg
     2701 gttgaagacc atagtaggag attcgtacat taatgatgac tagacagttc ggcctcctgc
     2761 tggcgatcat ttgaacacct aacgtagcta tataggacac agttgcattg gggatgctgg
     2821 ttgtcattgc ccctccttcc tctaagtgat aaggttactt cctctaaatg accaagcagg
     2881 gtatctaatc ctgttaacgc tcgctcagag aattcgcatg cggagtcgag ggagaaccag
     2941 ataagtgaaa tctagttcca aactattttg tcatttttaa ttttcgtatt agcttacgac
     3001 gctacaccca gttcccatct attttgtcac tcttccctaa ataatcctta aaaactccat
     3061 ttccacccct cccagttccc aactattttg tccgcccaca cgctgagcga attccacgct
     3121 tgtggaattc ctcgagggag ttcacatata cctgccgttc actattattt agtgaaatga
     3181 gatattatga tattttctga attgtgatta aaaaggcaac tttatgccca tgcaacagaa
     3241 actataaaaa atacagagaa tgaaaagaaa cagatagatt ttttagttct ttaggcccgt
     3301 agtctgcaaa tccttttatg attttctatc aaacaaaaga ggaaaataga ccagttgcaa
     3361 tccaaacgag agtctaatag aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat
     3421 aaagcaggca agacctaaaa tgtgtaaagg gcaaagtgta tactttggcg tcacccctta
     3481 catattttag gtcttttttt attgtgcgta actaacttgc catcttcaaa caggagggct
     3541 ggaagaagca gaccgctaac acagtacata aaaaaggaga catgaacgat gaacatcaaa
     3601 aagtttgcaa aacaagcaac agtattaacc tttactaccg cactgctggc aggaggcgca
     3661 actcaagcgt ttgcgaaaga aacgaaccaa aagccatata aggaaacata cggcatttcc
     3721 catattacac gccatgatat gctgcaaatc cctgaacagc aaaaaaatga aaaatatcaa
     3781 gttcctgaat tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg cctggacgtt
     3841 tgggacagct ggccattaca aaacgctgac ggcactgtcg caaactatca cggctaccac
     3901 atcgtctttg cattagccgg agatcctaaa aatgcggatg acacatcgat ttacatgttc
     3961 tatcaaaaag tcggcgaaac ttctattgac agctggaaaa acgctggccg cgtctttaaa
     4021 gacagcgaca aattcgatgc aaatgattct atcctaaaag accaaacaca agaatggtca
     4081 ggttcagcca catttacatc tgacggaaaa atccgtttat tctacactga tttctccggt
     4141 aaacattacg gcaaacaaac actgacaact gcacaagtta acgtatcagc atcagacagc
     4201 tctttgaaca tcaacggtgt agaggattat aaatcaatct ttgacggtga cggaaaaacg
     4261 tatcaaaatg tacagcagtt catcgatgaa ggcaactaca gctcaggcga caaccatacg
     4321 ctgagagatc ctcactacgt agaagataaa ggccacaaat acttagtatt tgaagcaaac
     4381 actggaactg aagatggcta ccaaggcgaa gaatctttat ttaacaaagc atactatggc
     4441 aaaagcacat cattcttccg tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc
     4501 acggctgagt tagcaaacgg cgctctcggt atgattgagc taaacgatga ttacacactg
     4561 aaaaaagtga tgaaaccgct gattgcatct aacacagtaa cagatgaaat tgaacgcgcg
     4621 aacgtcttta aaatgaacgg caaatggtac ctgttcactg actcccgcgg atcaaaaatg
     4681 acgattgacg gcattacgtc taacgatatt tacatgcttg gttatgtttc taattcttta
     4741 actggcccat acaagccgct gaacaaaact ggccttgtgt taaaaatgga tcttgatcct
     4801 aacgatgtaa cctttactta ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc
     4861 gtgattacaa gctatatgac aaacagagga ttctacgcag acaaacaatc aacgtttgcg
     4921 ccaagcttcc tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt
     4981 gaacaaggac aattaacagt taacaaataa attcgctgag cgaggatgca catgtgaccg
     5041 agggaagctt gcggccgcgt cgtgactggg aaaaccctgg cgactagtct tggactcctg
     5101 ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc
     5161 cgccgggcgt tttttattgg tgagaatcca ggggtcccca ataattacga tttaaatttg
     5221 tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa
     5281 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt cagcgtgaaa
     5341 cgagctgtag ccgtccgcgt ctgaacagca acatggatgc ggatctgtat ggctataaat
     5401 gggcgcgtga taacgtgggt cagagcggcg cgaccattta tcgtctgtat ggcaaaccgg
     5461 atgcgccgga actgtttctg aaacatggca aaggcagcgt ggcgaacgat gtgaccgatg
     5521 aaatggtgcg tctgaactgg ctgaccgaat ttatgccgct gccgaccatt aaacatttta
     5581 ttcgcacccc ggatgatgcg tggctgctga ccaccgcgat tccgggcaaa accgcgtttc
     5641 aggtgctgga agaatatccg gatagcggcg aaaacattgt ggatgcgctg gccgtgtttc
     5701 tgcgtcgtct gcatagcatt ccggtgtgca actgcccgtt taacagcgat cgtgtgtttc
     5761 gtctggccca ggcgcagagc cgtatgaaca acggcctggt ggatgcgagc gattttgatg
     5821 atgaacgtaa cggctggccg gtggaacagg tgtggaaaga aatgcataaa ctgctgccgt
     5881 ttagcccgga tagcgtggtg acccacggcg attttagcct ggataacctg attttcgatg
     5941 aaggcaaact gattggctgc attgatgtgg gccgtgtggg cattgcggat cgttatcagg
     6001 atctggccat tctgtggaac tgcctgggcg aatttagccc gagcctgcaa aaacgtctgt
     6061 ttcagaaata tggcattgat aatccggata tgaacaaact gcaatttcat ctgatgctgg
     6121 atgaattttt ctaataatta attggaccgc ggtccgcgcg ttgtcctttt ccgctgcata
     6181 accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt tcgcacgata
     6241 tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc
     6301 gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta
     6361 ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgt aggccggccg
     6421 atctgaagat cagcagttca acctgttgat agtacgtact aagctctcat gtttcacgta
     6481 ctaagctctc atgtttaacg tactaagctc tcatgtttaa cgaactaaac cctcatggct
     6541 aacgtactaa gctctcatgg ctaacgtact aagctctcat gtttcacgta ctaagctctc
     6601 atgtttgaac aataaaatta atataaatca gcaacttaaa tagcctctaa ggttttaagt
     6661 tttataagaa aaaaaagaat atataaggct tttaaagcct ttaaggttta acggttgtgg
     6721 acaacaagcc agggatgtaa cgcactgaga agcccttaga gcctctcaaa gcaattttga
     6781 gtgacacagg aacacttaac ggctgacatg gggcgcgccc agctgtctag ggcggcggat
     6841 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     6901 tcgactgagc ctttcgtttt atttgatgcc t
//
