LOCUS       sequence-419245-        8466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 empty backbone for strain-specific barcode DNA tag through 
            Tn7 bacterial insertion, Kanamycin selectable marker and tagRFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8466)
  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 8466)
  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: pBCC099
FEATURES             Location/Qualifiers
     source          1..8466
                     /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(1878..1883,1884..1899,1900..1906)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1914..1930
                     /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      2683..2777
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        join(2926..2931,2932..2949,2950..2956)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3010..3669
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3076..3095)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4011..4316
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4195..4214
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     mobile_element  4470..4635
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        4678..5123
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(5124..5210,5211..6545)
                     /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      6628..6722
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(6843..6862)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6854..7669
                     /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            7836..7944
                     /note="incP origin of transfer"
     rep_origin      7955..8343
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(8374..8460)
                     /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 cgctactaga
     1861 attcgagcgg ggaatttttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
     1921 cggataacaa ttttcacaca ggaaacagct aaatggtgag taaaggtgaa gagttgatta
     1981 aagagaacat gcatatgaag ttatacatgg agggaactgt caataatcac cactttaagt
     2041 gtacatcaga gggtgaggga aagccatacg agggaaccca aactatgaga atcaaagtag
     2101 tggagggagg tcctcttcca tttgcttttg atatactagc aacaagtttc atgtatggtt
     2161 ccaggacctt cattaaccat actcagggaa tccctgactt ctttaaacag tcttttcctg
     2221 aaggttttac atgggagagg gttaccactt acgaggacgg tggagtcttg acagcaaccc
     2281 aggacacttc attacaagat ggatgcctaa tatacaatgt gaaaattagg ggtgtgaatt
     2341 tccctagtaa cggaccagtt atgcagaaga aaacactagg ttgggaagct aatactgaaa
     2401 tgttgtaccc tgccgacgga ggtttagaag gtagatccga catggccctt aagctagtcg
     2461 gaggtggaca cttgatctgt aactttaaaa ccacatatag gtctaagaag ccagcaaaga
     2521 atctaaaaat gcctggtgtt tactatgtgg accatagact agaaaggata aaagaagcag
     2581 acaaagaaac ttacgtggag caacatgagg tcgccgtcgc taggtattgc gacttacctt
     2641 ccaagctagg tcacaaattg aacgcttaat tagctgagct tggactcctg ttgatagatc
     2701 cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt
     2761 tttttattgg tgagaatctc ccgctttacg aattcccatg gggagcatgc ggagttacgt
     2821 catccccacc ttccgctgcg ttcttcatcg atgcccgctt agtgatatgc ttaaaactcc
     2881 aagcacagcc ttctccccgg ggcggccgca ttaggcaccc caggctttac actttatgct
     2941 tccggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc aggagctaag
     3001 gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat
     3061 cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt
     3121 cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg
     3181 gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg
     3241 aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag
     3301 caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta
     3361 cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg
     3421 tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat
     3481 ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat
     3541 acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat
     3601 ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc
     3661 ggggcgtaaa cgcgtggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc
     3721 gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga
     3781 ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca
     3841 aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg
     3901 tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg
     3961 gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa atgcagttta
     4021 aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata
     4081 ttattgacac gcccggacga cggatggtga tccccctggc cagtgcacgt ctgctgtcag
     4141 ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga
     4201 tgaccaccga tatggccagt gtgccggtat ccgttatcgg ggaagaagtg gctgatctca
     4261 gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt
     4321 caggctccct tatacacagc ccccggggat gctggttgtc attgcccctc cttcctctaa
     4381 gtgataaggt tacttcctct aaatgaccaa gcagggtatc taatcctgtt aacgctcgct
     4441 cagagaattc gcatgcggag tcgagggaga accagataag tgaaatctag ttccaaacta
     4501 ttttgtcatt tttaattttc gtattagctt acgacgctac acccagttcc catctatttt
     4561 gtcactcttc cctaaataat ccttaaaaac tccatttcca cccctcccag ttcccaacta
     4621 ttttgtccgc ccacacgctg agcgaattcc acgcttgtgg aattcctcga gggagttcac
     4681 atatacctgc cgttcactat tatttagtga aatgagatat tatgatattt tctgaattgt
     4741 gattaaaaag gcaactttat gcccatgcaa cagaaactat aaaaaataca gagaatgaaa
     4801 agaaacagat agatttttta gttctttagg cccgtagtct gcaaatcctt ttatgatttt
     4861 ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa acgagagtct aatagaatga
     4921 ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc aggcaagacc taaaatgtgt
     4981 aaagggcaaa gtgtatactt tggcgtcacc ccttacatat tttaggtctt tttttattgt
     5041 gcgtaactaa cttgccatct tcaaacagga gggctggaag aagcagaccg ctaacacagt
     5101 acataaaaaa ggagacatga acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat
     5161 taacctttac taccgcactg ctggcaggag gcgcaactca agcgtttgcg aaagaaacga
     5221 accaaaagcc atataaggaa acatacggca tttcccatat tacacgccat gatatgctgc
     5281 aaatccctga acagcaaaaa aatgaaaaat atcaagttcc tgaattcgat tcgtccacaa
     5341 ttaaaaatat ctcttctgca aaaggcctgg acgtttggga cagctggcca ttacaaaacg
     5401 ctgacggcac tgtcgcaaac tatcacggct accacatcgt ctttgcatta gccggagatc
     5461 ctaaaaatgc ggatgacaca tcgatttaca tgttctatca aaaagtcggc gaaacttcta
     5521 ttgacagctg gaaaaacgct ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg
     5581 attctatcct aaaagaccaa acacaagaat ggtcaggttc agccacattt acatctgacg
     5641 gaaaaatccg tttattctac actgatttct ccggtaaaca ttacggcaaa caaacactga
     5701 caactgcaca agttaacgta tcagcatcag acagctcttt gaacatcaac ggtgtagagg
     5761 attataaatc aatctttgac ggtgacggaa aaacgtatca aaatgtacag cagttcatcg
     5821 atgaaggcaa ctacagctca ggcgacaacc atacgctgag agatcctcac tacgtagaag
     5881 ataaaggcca caaatactta gtatttgaag caaacactgg aactgaagat ggctaccaag
     5941 gcgaagaatc tttatttaac aaagcatact atggcaaaag cacatcattc ttccgtcaag
     6001 aaagtcaaaa acttctgcaa agcgataaaa aacgcacggc tgagttagca aacggcgctc
     6061 tcggtatgat tgagctaaac gatgattaca cactgaaaaa agtgatgaaa ccgctgattg
     6121 catctaacac agtaacagat gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat
     6181 ggtacctgtt cactgactcc cgcggatcaa aaatgacgat tgacggcatt acgtctaacg
     6241 atatttacat gcttggttat gtttctaatt ctttaactgg cccatacaag ccgctgaaca
     6301 aaactggcct tgtgttaaaa atggatcttg atcctaacga tgtaaccttt acttactcac
     6361 acttcgctgt acctcaagcg aaaggaaaca atgtcgtgat tacaagctat atgacaaaca
     6421 gaggattcta cgcagacaaa caatcaacgt ttgcgccaag cttcctgctg aacatcaaag
     6481 gcaagaaaac atctgttgtc aaagacagca tccttgaaca aggacaatta acagttaaca
     6541 aataaattcg ctgagcgagg atgcacatgt gaccgaggga agcttgcggc cgcgtcgtga
     6601 ctgggaaaac cctggcgact agtcttggac tcctgttgat agatccagta atgacctcag
     6661 aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga
     6721 atccaggggt ccccaataat tacgatttaa atttgtgtct caaaatctct gatgttacat
     6781 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     6841 tacaaggggt gttatgagcc atattcagcg tgaaacgagc tgtagccgtc cgcgtctgaa
     6901 cagcaacatg gatgcggatc tgtatggcta taaatgggcg cgtgataacg tgggtcagag
     6961 cggcgcgacc atttatcgtc tgtatggcaa accggatgcg ccggaactgt ttctgaaaca
     7021 tggcaaaggc agcgtggcga acgatgtgac cgatgaaatg gtgcgtctga actggctgac
     7081 cgaatttatg ccgctgccga ccattaaaca ttttattcgc accccggatg atgcgtggct
     7141 gctgaccacc gcgattccgg gcaaaaccgc gtttcaggtg ctggaagaat atccggatag
     7201 cggcgaaaac attgtggatg cgctggccgt gtttctgcgt cgtctgcata gcattccggt
     7261 gtgcaactgc ccgtttaaca gcgatcgtgt gtttcgtctg gcccaggcgc agagccgtat
     7321 gaacaacggc ctggtggatg cgagcgattt tgatgatgaa cgtaacggct ggccggtgga
     7381 acaggtgtgg aaagaaatgc ataaactgct gccgtttagc ccggatagcg tggtgaccca
     7441 cggcgatttt agcctggata acctgatttt cgatgaaggc aaactgattg gctgcattga
     7501 tgtgggccgt gtgggcattg cggatcgtta tcaggatctg gccattctgt ggaactgcct
     7561 gggcgaattt agcccgagcc tgcaaaaacg tctgtttcag aaatatggca ttgataatcc
     7621 ggatatgaac aaactgcaat ttcatctgat gctggatgaa tttttctaat aattaattgg
     7681 accgcggtcc gcgcgttgtc cttttccgct gcataaccct gcttcggggt cattatagcg
     7741 attttttcgg tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt
     7801 gtagactttc cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca
     7861 cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg
     7921 aatcctgctc tgcgaggctg gccgtaggcc ggccgatctg aagatcagca gttcaacctg
     7981 ttgatagtac gtactaagct ctcatgtttc acgtactaag ctctcatgtt taacgtacta
     8041 agctctcatg tttaacgaac taaaccctca tggctaacgt actaagctct catggctaac
     8101 gtactaagct ctcatgtttc acgtactaag ctctcatgtt tgaacaataa aattaatata
     8161 aatcagcaac ttaaatagcc tctaaggttt taagttttat aagaaaaaaa agaatatata
     8221 aggcttttaa agcctttaag gtttaacggt tgtggacaac aagccaggga tgtaacgcac
     8281 tgagaagccc ttagagcctc tcaaagcaat tttgagtgac acaggaacac ttaacggctg
     8341 acatggggcg cgcccagctg tctagggcgg cggatttgtc ctactcagga gagcgttcac
     8401 cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg
     8461 atgcct
//
