LOCUS       sequence-418301-        7142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 for strain-specific barcode DNA tag with bc157 through Tn7 
            bacterial insertion, Kanamycin 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 7142)
  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 7142)
  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: pBC130
FEATURES             Location/Qualifiers
     source          1..7142
                     /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)."
     terminator      complement(681..933)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(953..1504)
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(1546..1725)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     protein_bind    complement(1803..1850)
                     /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(1877..1882,1883..1898,1899..1905)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1913..1929
                     /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             1952..2635
                     /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     2547..2570
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      2649..2743
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     mobile_element  3146..3311
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        3354..3799
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3800..3886,3887..5221)
                     /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      5304..5398
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5519..5538)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5530..6345
                     /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            6512..6620
                     /note="incP origin of transfer"
     rep_origin      6631..7019
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(7050..7136)
                     /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 aatgccggtc gatctagtaa catagatgac accgcgcgcg ataatttatc
      721 ctagtttgcg cgctatattt tgttttctat cgcgtattaa atgtataatt gcgggactct
      781 aatcataaaa acccatctca taaataacgt catgcattac atgttaatta ttacatgctt
      841 aacgtaattc aacagaaatt atatgataat catcgcaaga ccggcaacag gattcaatct
      901 taagaaactt tattgccaaa tgtttgaacg atctgcttga ctctaggggt catcagattt
      961 cggtgacggg caggaccgga cggggcggca ccggcaggct gaagtccagc tgccagaaac
     1021 ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg cagcatgccg cggggggcat
     1081 atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg cagcccgatg acagcgacca
     1141 cgctcttgaa gccctgtgcc tccagggact tcagcaggtg ggtgtagagc gtggagccca
     1201 gtcccgtccg ctggtggcgg ggggagacgt acacggtcga ctcggccgtc cagtcgtagg
     1261 cgttgcgtgc cttccaggga cccgcgtagg cgatgccggc gacctcgccg tccacctcgg
     1321 cgacgagcca gggatagcgc tcccgcagac ggacgaggtc gtccgtccac tcctgcggtt
     1381 cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat gtagtggttg acgatggtgc
     1441 agaccgccgg catgtccgcc tcggtggcac ggcggatgtc ggccgggcgt cgttctgggc
     1501 tcatggtaga tcccctcgat cgagttgaga gtgaatatga gactctaatt ggataccgag
     1561 gggaatttat ggaacgtcag tggagcattt ttgacaagaa atatttgcta gctgatagtg
     1621 accttaggcg acttttgaac gcgcaataat ggtttctgac gtatgtgctt agctcattaa
     1681 actccagaaa cccgcggctc agtggctcct tcaacgttgc ggttctgtca gttccaaacg
     1741 taaaacggct tgtcccgcgt catcggcggg ggtcataacg tgactccctt aattctcagc
     1801 ttgaagttcc tatactttct agagaatagg aacttcggaa taggtacttc cgctactaaa
     1861 ttcgagcggg gaattcttga caattaatca tcggctcgta taatgtgtgg aattgtgagc
     1921 ggataacaat tttcacacag gaaacagcta aatggtgagc aagggcgagg aggtcatcaa
     1981 ggagttcatg cgcttcaagg agcacatgga gggctccgtg aacggccacg agttcgagat
     2041 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaggc tgaaggtgac
     2101 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagatca tgtacggctc
     2161 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     2221 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     2281 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aaggtgcgcg gcaccaactt
     2341 cccctccgac ggccccgtaa tgcagaaaaa gaccatgggc tgggaggcct cctccgagcg
     2401 gatgtacccc gaggacggcg ccctgaaggg cgagatgaag atgaggctga ggctgaagga
     2461 cggcggccac tacgacgccg aggtcaagac cacctacatg gccaagaagc ccgtgcagct
     2521 gcccggcgcc tacaagaccg acatcaagct ggacatcacc tcccacaacg aggactacac
     2581 catcgtggaa cagtacgagc gcgccgaggg ccgccactcc accggcgccg cttaattagc
     2641 tgagcttgga ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt
     2701 cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatctcccgc tttacgaatt
     2761 cccatgggga gcatgcggag ttacgtcatc cccaccttcc gctgcgttct tcatcgatgc
     2821 ccgcttagtg atatgcttaa aactccaagc acagccttct ccccatgcaa ctgtgtccta
     2881 tatagctacg ttaggtgttc attaatgtcg gatcacctaa tgagggacaa ccaaggaccc
     2941 aaatgtgtaa aggaacacgt gaataggatc agttagtcat taatgggtct gacgtttcac
     3001 ttccagcctt gcggttacat ggggatgctg gttgtcattg cccctccttc ctctaagtga
     3061 taaggttact tcctctaaat gaccaagcag ggtatctaat cctgttaacg ctcgctcaga
     3121 gaattcgcat gcggagtcga gggagaacca gataagtgaa atctagttcc aaactatttt
     3181 gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca
     3241 ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt
     3301 gtccgcccac acgctgagcg aattccacgc ttgtggaatt cctcgaggga gttcacatat
     3361 acctgccgtt cactattatt tagtgaaatg agatattatg atattttctg aattgtgatt
     3421 aaaaaggcaa ctttatgccc atgcaacaga aactataaaa aatacagaga atgaaaagaa
     3481 acagatagat tttttagttc tttaggcccg tagtctgcaa atccttttat gattttctat
     3541 caaacaaaag aggaaaatag accagttgca atccaaacga gagtctaata gaatgaggtc
     3601 gaaaagtaaa tcgcgcgggt ttgttactga taaagcaggc aagacctaaa atgtgtaaag
     3661 ggcaaagtgt atactttggc gtcacccctt acatatttta ggtctttttt tattgtgcgt
     3721 aactaacttg ccatcttcaa acaggagggc tggaagaagc agaccgctaa cacagtacat
     3781 aaaaaaggag acatgaacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac
     3841 ctttactacc gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca
     3901 aaagccatat aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat
     3961 ccctgaacag caaaaaaatg aaaaatatca agttcctgaa ttcgattcgt ccacaattaa
     4021 aaatatctct tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacgctga
     4081 cggcactgtc gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa
     4141 aaatgcggat gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga
     4201 cagctggaaa aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc
     4261 tatcctaaaa gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa
     4321 aatccgttta ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac
     4381 tgcacaagtt aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta
     4441 taaatcaatc tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga
     4501 aggcaactac agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa
     4561 aggccacaaa tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga
     4621 agaatcttta tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag
     4681 tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg
     4741 tatgattgag ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc
     4801 taacacagta acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta
     4861 cctgttcact gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat
     4921 ttacatgctt ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac
     4981 tggccttgtg ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt
     5041 cgctgtacct caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg
     5101 attctacgca gacaaacaat caacgtttgc gccaagcttc ctgctgaaca tcaaaggcaa
     5161 gaaaacatct gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata
     5221 aattcgctga gcgaggatgc acatgtgacc gagggaagct tgcggccgcg tcgtgactgg
     5281 gaaaaccctg gcgactagtc ttggactcct gttgatagat ccagtaatga cctcagaact
     5341 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     5401 aggggtcccc aataattacg atttaaattt gtgtctcaaa atctctgatg ttacattgca
     5461 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     5521 aggggtgtta tgagccatat tcagcgtgaa acgagctgta gccgtccgcg tctgaacagc
     5581 aacatggatg cggatctgta tggctataaa tgggcgcgtg ataacgtggg tcagagcggc
     5641 gcgaccattt atcgtctgta tggcaaaccg gatgcgccgg aactgtttct gaaacatggc
     5701 aaaggcagcg tggcgaacga tgtgaccgat gaaatggtgc gtctgaactg gctgaccgaa
     5761 tttatgccgc tgccgaccat taaacatttt attcgcaccc cggatgatgc gtggctgctg
     5821 accaccgcga ttccgggcaa aaccgcgttt caggtgctgg aagaatatcc ggatagcggc
     5881 gaaaacattg tggatgcgct ggccgtgttt ctgcgtcgtc tgcatagcat tccggtgtgc
     5941 aactgcccgt ttaacagcga tcgtgtgttt cgtctggccc aggcgcagag ccgtatgaac
     6001 aacggcctgg tggatgcgag cgattttgat gatgaacgta acggctggcc ggtggaacag
     6061 gtgtggaaag aaatgcataa actgctgccg tttagcccgg atagcgtggt gacccacggc
     6121 gattttagcc tggataacct gattttcgat gaaggcaaac tgattggctg cattgatgtg
     6181 ggccgtgtgg gcattgcgga tcgttatcag gatctggcca ttctgtggaa ctgcctgggc
     6241 gaatttagcc cgagcctgca aaaacgtctg tttcagaaat atggcattga taatccggat
     6301 atgaacaaac tgcaatttca tctgatgctg gatgaatttt tctaataatt aattggaccg
     6361 cggtccgcgc gttgtccttt tccgctgcat aaccctgctt cggggtcatt atagcgattt
     6421 tttcggtata tccatccttt ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag
     6481 actttccttg gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta ggcccacccg
     6541 cgagcgggtg ttccttcttc actgtccctt attcgcacct ggcggtgctc aacgggaatc
     6601 ctgctctgcg aggctggccg taggccggcc gatctgaaga tcagcagttc aacctgttga
     6661 tagtacgtac taagctctca tgtttcacgt actaagctct catgtttaac gtactaagct
     6721 ctcatgttta acgaactaaa ccctcatggc taacgtacta agctctcatg gctaacgtac
     6781 taagctctca tgtttcacgt actaagctct catgtttgaa caataaaatt aatataaatc
     6841 agcaacttaa atagcctcta aggttttaag ttttataaga aaaaaaagaa tatataaggc
     6901 ttttaaagcc tttaaggttt aacggttgtg gacaacaagc cagggatgta acgcactgag
     6961 aagcccttag agcctctcaa agcaattttg agtgacacag gaacacttaa cggctgacat
     7021 ggggcgcgcc cagctgtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac
     7081 aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
     7141 ct
//
