LOCUS       sequence-403466-        8254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 empty backbone for strain-specific barcode DNA tag through 
            Tn7 bacterial insertion, Gentamicin selectable marker and tagRFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8254)
  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 8254)
  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: pBCC088
FEATURES             Location/Qualifiers
     source          1..8254
                     /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)."
     terminator      2471..2565
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        join(2714..2719,2720..2737,2738..2744)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2798..3457
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2864..2883)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3799..4104
                     /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     3983..4002
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     mobile_element  4258..4423
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        4466..4911
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4912..4998,4999..6333)
                     /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      6416..6510
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(6631..6650)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6642..7457
                     /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            7624..7732
                     /note="incP origin of transfer"
     rep_origin      7743..8131
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(8162..8248)
                     /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 aatttttgac aattaatcat
     1681 cggctcgtat aatgtgtgga attgtgagcg gataacaatt ttcacacagg aaacagctaa
     1741 atggtgagta aaggtgaaga gttgattaaa gagaacatgc atatgaagtt atacatggag
     1801 ggaactgtca ataatcacca ctttaagtgt acatcagagg gtgagggaaa gccatacgag
     1861 ggaacccaaa ctatgagaat caaagtagtg gagggaggtc ctcttccatt tgcttttgat
     1921 atactagcaa caagtttcat gtatggttcc aggaccttca ttaaccatac tcagggaatc
     1981 cctgacttct ttaaacagtc ttttcctgaa ggttttacat gggagagggt taccacttac
     2041 gaggacggtg gagtcttgac agcaacccag gacacttcat tacaagatgg atgcctaata
     2101 tacaatgtga aaattagggg tgtgaatttc cctagtaacg gaccagttat gcagaagaaa
     2161 acactaggtt gggaagctaa tactgaaatg ttgtaccctg ccgacggagg tttagaaggt
     2221 agatccgaca tggcccttaa gctagtcgga ggtggacact tgatctgtaa ctttaaaacc
     2281 acatataggt ctaagaagcc agcaaagaat ctaaaaatgc ctggtgttta ctatgtggac
     2341 catagactag aaaggataaa agaagcagac aaagaaactt acgtggagca acatgaggtc
     2401 gccgtcgcta ggtattgcga cttaccttcc aagctaggtc acaaattgaa cgcttaatta
     2461 gctgagcttg gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg
     2521 ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatctccc gctttacgaa
     2581 ttcccatggg gagcatgcgg agttacgtca tccccacctt ccgctgcgtt cttcatcgat
     2641 gcccgcttag tgatatgctt aaaactccaa gcacagcctt ctccccgggg cggccgcatt
     2701 aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga ttttgagtta
     2761 ggatccgtcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     2821 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     2881 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     2941 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     3001 tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     3061 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     3121 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     3181 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     3241 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     3301 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     3361 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     3421 acaacagtac tgcgatgagt ggcagggcgg ggcgtaaacg cgtggatccg gcttactaaa
     3481 agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa tatatactga
     3541 tatgtatacc cgaagtatgt caaaaagagg tatgctatga agcagcgtat tacagtgaca
     3601 gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc tccggtctgg
     3661 taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa
     3721 atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct tttgctgacg
     3781 agaacagggg ctggtgaaat gcagtttaag gtttacacct ataaaagaga gagccgttat
     3841 cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccggacgacg gatggtgatc
     3901 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
     3961 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtatcc
     4021 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     4081 aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagccc ccggggatgc
     4141 tggttgtcat tgcccctcct tcctctaagt gataaggtta cttcctctaa atgaccaagc
     4201 agggtatcta atcctgttaa cgctcgctca gagaattcgc atgcggagtc gagggagaac
     4261 cagataagtg aaatctagtt ccaaactatt ttgtcatttt taattttcgt attagcttac
     4321 gacgctacac ccagttccca tctattttgt cactcttccc taaataatcc ttaaaaactc
     4381 catttccacc cctcccagtt cccaactatt ttgtccgccc acacgctgag cgaattccac
     4441 gcttgtggaa ttcctcgagg gagttcacat atacctgccg ttcactatta tttagtgaaa
     4501 tgagatatta tgatattttc tgaattgtga ttaaaaaggc aactttatgc ccatgcaaca
     4561 gaaactataa aaaatacaga gaatgaaaag aaacagatag attttttagt tctttaggcc
     4621 cgtagtctgc aaatcctttt atgattttct atcaaacaaa agaggaaaat agaccagttg
     4681 caatccaaac gagagtctaa tagaatgagg tcgaaaagta aatcgcgcgg gtttgttact
     4741 gataaagcag gcaagaccta aaatgtgtaa agggcaaagt gtatactttg gcgtcacccc
     4801 ttacatattt taggtctttt tttattgtgc gtaactaact tgccatcttc aaacaggagg
     4861 gctggaagaa gcagaccgct aacacagtac ataaaaaagg agacatgaac gatgaacatc
     4921 aaaaagtttg caaaacaagc aacagtatta acctttacta ccgcactgct ggcaggaggc
     4981 gcaactcaag cgtttgcgaa agaaacgaac caaaagccat ataaggaaac atacggcatt
     5041 tcccatatta cacgccatga tatgctgcaa atccctgaac agcaaaaaaa tgaaaaatat
     5101 caagttcctg aattcgattc gtccacaatt aaaaatatct cttctgcaaa aggcctggac
     5161 gtttgggaca gctggccatt acaaaacgct gacggcactg tcgcaaacta tcacggctac
     5221 cacatcgtct ttgcattagc cggagatcct aaaaatgcgg atgacacatc gatttacatg
     5281 ttctatcaaa aagtcggcga aacttctatt gacagctgga aaaacgctgg ccgcgtcttt
     5341 aaagacagcg acaaattcga tgcaaatgat tctatcctaa aagaccaaac acaagaatgg
     5401 tcaggttcag ccacatttac atctgacgga aaaatccgtt tattctacac tgatttctcc
     5461 ggtaaacatt acggcaaaca aacactgaca actgcacaag ttaacgtatc agcatcagac
     5521 agctctttga acatcaacgg tgtagaggat tataaatcaa tctttgacgg tgacggaaaa
     5581 acgtatcaaa atgtacagca gttcatcgat gaaggcaact acagctcagg cgacaaccat
     5641 acgctgagag atcctcacta cgtagaagat aaaggccaca aatacttagt atttgaagca
     5701 aacactggaa ctgaagatgg ctaccaaggc gaagaatctt tatttaacaa agcatactat
     5761 ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac ttctgcaaag cgataaaaaa
     5821 cgcacggctg agttagcaaa cggcgctctc ggtatgattg agctaaacga tgattacaca
     5881 ctgaaaaaag tgatgaaacc gctgattgca tctaacacag taacagatga aattgaacgc
     5941 gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca ctgactcccg cggatcaaaa
     6001 atgacgattg acggcattac gtctaacgat atttacatgc ttggttatgt ttctaattct
     6061 ttaactggcc catacaagcc gctgaacaaa actggccttg tgttaaaaat ggatcttgat
     6121 cctaacgatg taacctttac ttactcacac ttcgctgtac ctcaagcgaa aggaaacaat
     6181 gtcgtgatta caagctatat gacaaacaga ggattctacg cagacaaaca atcaacgttt
     6241 gcgccaagct tcctgctgaa catcaaaggc aagaaaacat ctgttgtcaa agacagcatc
     6301 cttgaacaag gacaattaac agttaacaaa taaattcgct gagcgaggat gcacatgtga
     6361 ccgagggaag cttgcggccg cgtcgtgact gggaaaaccc tggcgactag tcttggactc
     6421 ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt
     6481 tgccgccggg cgttttttat tggtgagaat ccaggggtcc ccaataatta cgatttaaat
     6541 ttgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     6601 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcagcgtg
     6661 aaacgagctg tagccgtccg cgtctgaaca gcaacatgga tgcggatctg tatggctata
     6721 aatgggcgcg tgataacgtg ggtcagagcg gcgcgaccat ttatcgtctg tatggcaaac
     6781 cggatgcgcc ggaactgttt ctgaaacatg gcaaaggcag cgtggcgaac gatgtgaccg
     6841 atgaaatggt gcgtctgaac tggctgaccg aatttatgcc gctgccgacc attaaacatt
     6901 ttattcgcac cccggatgat gcgtggctgc tgaccaccgc gattccgggc aaaaccgcgt
     6961 ttcaggtgct ggaagaatat ccggatagcg gcgaaaacat tgtggatgcg ctggccgtgt
     7021 ttctgcgtcg tctgcatagc attccggtgt gcaactgccc gtttaacagc gatcgtgtgt
     7081 ttcgtctggc ccaggcgcag agccgtatga acaacggcct ggtggatgcg agcgattttg
     7141 atgatgaacg taacggctgg ccggtggaac aggtgtggaa agaaatgcat aaactgctgc
     7201 cgtttagccc ggatagcgtg gtgacccacg gcgattttag cctggataac ctgattttcg
     7261 atgaaggcaa actgattggc tgcattgatg tgggccgtgt gggcattgcg gatcgttatc
     7321 aggatctggc cattctgtgg aactgcctgg gcgaatttag cccgagcctg caaaaacgtc
     7381 tgtttcagaa atatggcatt gataatccgg atatgaacaa actgcaattt catctgatgc
     7441 tggatgaatt tttctaataa ttaattggac cgcggtccgc gcgttgtcct tttccgctgc
     7501 ataaccctgc ttcggggtca ttatagcgat tttttcggta tatccatcct ttttcgcacg
     7561 atatacagga ttttgccaaa gggttcgtgt agactttcct tggtgtatcc aacggcgtca
     7621 gccgggcagg ataggtgaag taggcccacc cgcgagcggg tgttccttct tcactgtccc
     7681 ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg cgaggctggc cgtaggccgg
     7741 ccgatctgaa gatcagcagt tcaacctgtt gatagtacgt actaagctct catgtttcac
     7801 gtactaagct ctcatgttta acgtactaag ctctcatgtt taacgaacta aaccctcatg
     7861 gctaacgtac taagctctca tggctaacgt actaagctct catgtttcac gtactaagct
     7921 ctcatgtttg aacaataaaa ttaatataaa tcagcaactt aaatagcctc taaggtttta
     7981 agttttataa gaaaaaaaag aatatataag gcttttaaag cctttaaggt ttaacggttg
     8041 tggacaacaa gccagggatg taacgcactg agaagccctt agagcctctc aaagcaattt
     8101 tgagtgacac aggaacactt aacggctgac atggggcgcg cccagctgtc tagggcggcg
     8161 gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt
     8221 ctttcgactg agcctttcgt tttatttgat gcct
//
