LOCUS       sequence-420633-        6884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc131 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 6884)
  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 6884)
  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: pBC104
FEATURES             Location/Qualifiers
     source          1..6884
                     /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  2888..3053
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3096..3541
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3542..3628,3629..4963)
                     /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      5046..5140
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5261..5280)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5272..6087
                     /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            6254..6362
                     /note="incP origin of transfer"
     rep_origin      6373..6761
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6792..6878)
                     /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 gacagttcgg cctcctgctg gcgatcatac taactgatcc
     2701 tattcacgtg ttcctttaca cataatgcct acaaccgtcg gctgacgtca agcgtttcgc
     2761 atggggatgc tggttgtcat tgcccctcct tcctctaagt gataaggtta cttcctctaa
     2821 atgaccaagc agggtatcta atcctgttaa cgctcgctca gagaattcgc atgcggagtc
     2881 gagggagaac cagataagtg aaatctagtt ccaaactatt ttgtcatttt taattttcgt
     2941 attagcttac gacgctacac ccagttccca tctattttgt cactcttccc taaataatcc
     3001 ttaaaaactc catttccacc cctcccagtt cccaactatt ttgtccgccc acacgctgag
     3061 cgaattccac gcttgtggaa ttcctcgagg gagttcacat atacctgccg ttcactatta
     3121 tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc aactttatgc
     3181 ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag attttttagt
     3241 tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa agaggaaaat
     3301 agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta aatcgcgcgg
     3361 gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt gtatactttg
     3421 gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact tgccatcttc
     3481 aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg agacatgaac
     3541 gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta ccgcactgct
     3601 ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat ataaggaaac
     3661 atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac agcaaaaaaa
     3721 tgaaaaatat caagttcctg aattcgattc gtccacaatt aaaaatatct cttctgcaaa
     3781 aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg tcgcaaacta
     3841 tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg atgacacatc
     3901 gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga aaaacgctgg
     3961 ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa aagaccaaac
     4021 acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt tattctacac
     4081 tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag ttaacgtatc
     4141 agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa tctttgacgg
     4201 tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact acagctcagg
     4261 cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca aatacttagt
     4321 atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt tatttaacaa
     4381 agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac ttctgcaaag
     4441 cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg agctaaacga
     4501 tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag taacagatga
     4561 aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca ctgactcccg
     4621 cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc ttggttatgt
     4681 ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg tgttaaaaat
     4741 ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac ctcaagcgaa
     4801 aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg cagacaaaca
     4861 atcaacgttt gcgccaagct tcctgctgaa catcaaaggc aagaaaacat ctgttgtcaa
     4921 agacagcatc cttgaacaag gacaattaac agttaacaaa taaattcgct gagcgaggat
     4981 gcacatgtga ccgagggaag cttgcggccg cgtcgtgact gggaaaaccc tggcgactag
     5041 tcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag
     5101 aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaggggtcc ccaataatta
     5161 cgatttaaat ttgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
     5221 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     5281 attcagcgtg aaacgagctg tagccgtccg cgtctgaaca gcaacatgga tgcggatctg
     5341 tatggctata aatgggcgcg tgataacgtg ggtcagagcg gcgcgaccat ttatcgtctg
     5401 tatggcaaac cggatgcgcc ggaactgttt ctgaaacatg gcaaaggcag cgtggcgaac
     5461 gatgtgaccg atgaaatggt gcgtctgaac tggctgaccg aatttatgcc gctgccgacc
     5521 attaaacatt ttattcgcac cccggatgat gcgtggctgc tgaccaccgc gattccgggc
     5581 aaaaccgcgt ttcaggtgct ggaagaatat ccggatagcg gcgaaaacat tgtggatgcg
     5641 ctggccgtgt ttctgcgtcg tctgcatagc attccggtgt gcaactgccc gtttaacagc
     5701 gatcgtgtgt ttcgtctggc ccaggcgcag agccgtatga acaacggcct ggtggatgcg
     5761 agcgattttg atgatgaacg taacggctgg ccggtggaac aggtgtggaa agaaatgcat
     5821 aaactgctgc cgtttagccc ggatagcgtg gtgacccacg gcgattttag cctggataac
     5881 ctgattttcg atgaaggcaa actgattggc tgcattgatg tgggccgtgt gggcattgcg
     5941 gatcgttatc aggatctggc cattctgtgg aactgcctgg gcgaatttag cccgagcctg
     6001 caaaaacgtc tgtttcagaa atatggcatt gataatccgg atatgaacaa actgcaattt
     6061 catctgatgc tggatgaatt tttctaataa ttaattggac cgcggtccgc gcgttgtcct
     6121 tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta tatccatcct
     6181 ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct tggtgtatcc
     6241 aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg tgttccttct
     6301 tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg cgaggctggc
     6361 cgtaggccgg ccgatctgaa gatcagcagt tcaacctgtt gatagtacgt actaagctct
     6421 catgtttcac gtactaagct ctcatgttta acgtactaag ctctcatgtt taacgaacta
     6481 aaccctcatg gctaacgtac taagctctca tggctaacgt actaagctct catgtttcac
     6541 gtactaagct ctcatgtttg aacaataaaa ttaatataaa tcagcaactt aaatagcctc
     6601 taaggtttta agttttataa gaaaaaaaag aatatataag gcttttaaag cctttaaggt
     6661 ttaacggttg tggacaacaa gccagggatg taacgcactg agaagccctt agagcctctc
     6721 aaagcaattt tgagtgacac aggaacactt aacggctgac atggggcgcg cccagctgtc
     6781 tagggcggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga
     6841 aaggcccagt ctttcgactg agcctttcgt tttatttgat gcct
//
