LOCUS       sequence-403468-        8242 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 tagBFP 
            fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8242)
  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 8242)
  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: pBCC089
FEATURES             Location/Qualifiers
     source          1..8242
                     /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      2459..2553
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        join(2702..2707,2708..2725,2726..2732)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2786..3445
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2852..2871)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3787..4092
                     /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     3971..3990
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     mobile_element  4246..4411
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        4454..4899
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4900..4986,4987..6321)
                     /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      6404..6498
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(6619..6638)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6630..7445
                     /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            7612..7720
                     /note="incP origin of transfer"
     rep_origin      7731..8119
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(8150..8236)
                     /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 cgtsgtatta 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 atgtctgaat tgattaaaga gaatatgcat atgaagttgt atatggaagg tactgttgat
     1801 aaccaccatt ttaaatgtac atctgaggga gaaggtaagc cttacgaagg tactcaaaca
     1861 atgcgtatta aagtagttga gggaggtcct ttaccattcg cttttgacat attggcaact
     1921 tcattcttat atggatctaa gacatttatt aaccatactc aaggtatacc agatttcttt
     1981 aaacaatcat ttcctgaagg attcacatgg gagagagtaa caacttatga agatggtgga
     2041 gttttgactg ctactcagga tacatcttta caagacggtt gtttgattta taatgttaag
     2101 attcgtggag taaacttcac ttctaatggt ccagttatgc agaagaaaac attgggatgg
     2161 gaggcattca ctgagacatt ataccctgct gatggtggat tggaaggtcg taacgacatg
     2221 gcattgaaat tagtaggagg ttcacacttg atagctaata ttaagactac atatcgttct
     2281 aagaaaccag ctaaaaactt gaaaatgcct ggtgtttact atgttgatta tcgtttagaa
     2341 agaattaagg aagcaaataa cgaaacttac gttgagcaac atgaagtagc tgttgcacgt
     2401 tattgtgact tgccttctaa gttaggacac aaattgaacg cttaattagc tgagcttgga
     2461 ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc
     2521 ggttgccgcc gggcgttttt tattggtgag aatctcccgc tttacgaatt cccatgggga
     2581 gcatgcggag ttacgtcatc cccaccttcc gctgcgttct tcatcgatgc ccgcttagtg
     2641 atatgcttaa aactccaagc acagccttct ccccggggcg gccgcattag gcaccccagg
     2701 ctttacactt tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccgtcgag
     2761 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
     2821 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     2881 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
     2941 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
     3001 attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
     3061 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
     3121 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
     3181 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     3241 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     3301 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     3361 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     3421 cgatgagtgg cagggcgggg cgtaaacgcg tggatccggc ttactaaaag ccagataaca
     3481 gtatgcgtat ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg
     3541 aagtatgtca aaaagaggta tgctatgaag cagcgtatta cagtgacagt tgacagcgac
     3601 agctatcagt tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca
     3661 tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga
     3721 tggctgaggt cgcccggttt attgaaatga acggctcttt tgctgacgag aacaggggct
     3781 ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
     3841 gatgtacaga gtgatattat tgacacgccc ggacgacgga tggtgatccc cctggccagt
     3901 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
     3961 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtatccgt tatcggggaa
     4021 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     4081 tggggaatat aaatgtcagg ctcccttata cacagccccc ggggatgctg gttgtcattg
     4141 cccctccttc ctctaagtga taaggttact tcctctaaat gaccaagcag ggtatctaat
     4201 cctgttaacg ctcgctcaga gaattcgcat gcggagtcga gggagaacca gataagtgaa
     4261 atctagttcc aaactatttt gtcattttta attttcgtat tagcttacga cgctacaccc
     4321 agttcccatc tattttgtca ctcttcccta aataatcctt aaaaactcca tttccacccc
     4381 tcccagttcc caactatttt gtccgcccac acgctgagcg aattccacgc ttgtggaatt
     4441 cctcgaggga gttcacatat acctgccgtt cactattatt tagtgaaatg agatattatg
     4501 atattttctg aattgtgatt aaaaaggcaa ctttatgccc atgcaacaga aactataaaa
     4561 aatacagaga atgaaaagaa acagatagat tttttagttc tttaggcccg tagtctgcaa
     4621 atccttttat gattttctat caaacaaaag aggaaaatag accagttgca atccaaacga
     4681 gagtctaata gaatgaggtc gaaaagtaaa tcgcgcgggt ttgttactga taaagcaggc
     4741 aagacctaaa atgtgtaaag ggcaaagtgt atactttggc gtcacccctt acatatttta
     4801 ggtctttttt tattgtgcgt aactaacttg ccatcttcaa acaggagggc tggaagaagc
     4861 agaccgctaa cacagtacat aaaaaaggag acatgaacga tgaacatcaa aaagtttgca
     4921 aaacaagcaa cagtattaac ctttactacc gcactgctgg caggaggcgc aactcaagcg
     4981 tttgcgaaag aaacgaacca aaagccatat aaggaaacat acggcatttc ccatattaca
     5041 cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca agttcctgaa
     5101 ttcgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt ttgggacagc
     5161 tggccattac aaaacgctga cggcactgtc gcaaactatc acggctacca catcgtcttt
     5221 gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt ctatcaaaaa
     5281 gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa agacagcgac
     5341 aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc aggttcagcc
     5401 acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg taaacattac
     5461 ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag catcagacag ctctttgaac
     5521 atcaacggtg tagaggatta taaatcaatc tttgacggtg acggaaaaac gtatcaaaat
     5581 gtacagcagt tcatcgatga aggcaactac agctcaggcg acaaccatac gctgagagat
     5641 cctcactacg tagaagataa aggccacaaa tacttagtat ttgaagcaaa cactggaact
     5701 gaagatggct accaaggcga agaatcttta tttaacaaag catactatgg caaaagcaca
     5761 tcattcttcc gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag
     5821 ttagcaaacg gcgctctcgg tatgattgag ctaaacgatg attacacact gaaaaaagtg
     5881 atgaaaccgc tgattgcatc taacacagta acagatgaaa ttgaacgcgc gaacgtcttt
     5941 aaaatgaacg gcaaatggta cctgttcact gactcccgcg gatcaaaaat gacgattgac
     6001 ggcattacgt ctaacgatat ttacatgctt ggttatgttt ctaattcttt aactggccca
     6061 tacaagccgc tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc taacgatgta
     6121 acctttactt actcacactt cgctgtacct caagcgaaag gaaacaatgt cgtgattaca
     6181 agctatatga caaacagagg attctacgca gacaaacaat caacgtttgc gccaagcttc
     6241 ctgctgaaca tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct tgaacaagga
     6301 caattaacag ttaacaaata aattcgctga gcgaggatgc acatgtgacc gagggaagct
     6361 tgcggccgcg tcgtgactgg gaaaaccctg gcgactagtc ttggactcct gttgatagat
     6421 ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg
     6481 ttttttattg gtgagaatcc aggggtcccc aataattacg atttaaattt gtgtctcaaa
     6541 atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg
     6601 cttacataaa cagtaataca aggggtgtta tgagccatat tcagcgtgaa acgagctgta
     6661 gccgtccgcg tctgaacagc aacatggatg cggatctgta tggctataaa tgggcgcgtg
     6721 ataacgtggg tcagagcggc gcgaccattt atcgtctgta tggcaaaccg gatgcgccgg
     6781 aactgtttct gaaacatggc aaaggcagcg tggcgaacga tgtgaccgat gaaatggtgc
     6841 gtctgaactg gctgaccgaa tttatgccgc tgccgaccat taaacatttt attcgcaccc
     6901 cggatgatgc gtggctgctg accaccgcga ttccgggcaa aaccgcgttt caggtgctgg
     6961 aagaatatcc ggatagcggc gaaaacattg tggatgcgct ggccgtgttt ctgcgtcgtc
     7021 tgcatagcat tccggtgtgc aactgcccgt ttaacagcga tcgtgtgttt cgtctggccc
     7081 aggcgcagag ccgtatgaac aacggcctgg tggatgcgag cgattttgat gatgaacgta
     7141 acggctggcc ggtggaacag gtgtggaaag aaatgcataa actgctgccg tttagcccgg
     7201 atagcgtggt gacccacggc gattttagcc tggataacct gattttcgat gaaggcaaac
     7261 tgattggctg cattgatgtg ggccgtgtgg gcattgcgga tcgttatcag gatctggcca
     7321 ttctgtggaa ctgcctgggc gaatttagcc cgagcctgca aaaacgtctg tttcagaaat
     7381 atggcattga taatccggat atgaacaaac tgcaatttca tctgatgctg gatgaatttt
     7441 tctaataatt aattggaccg cggtccgcgc gttgtccttt tccgctgcat aaccctgctt
     7501 cggggtcatt atagcgattt tttcggtata tccatccttt ttcgcacgat atacaggatt
     7561 ttgccaaagg gttcgtgtag actttccttg gtgtatccaa cggcgtcagc cgggcaggat
     7621 aggtgaagta ggcccacccg cgagcgggtg ttccttcttc actgtccctt attcgcacct
     7681 ggcggtgctc aacgggaatc ctgctctgcg aggctggccg taggccggcc gatctgaaga
     7741 tcagcagttc aacctgttga tagtacgtac taagctctca tgtttcacgt actaagctct
     7801 catgtttaac gtactaagct ctcatgttta acgaactaaa ccctcatggc taacgtacta
     7861 agctctcatg gctaacgtac taagctctca tgtttcacgt actaagctct catgtttgaa
     7921 caataaaatt aatataaatc agcaacttaa atagcctcta aggttttaag ttttataaga
     7981 aaaaaaagaa tatataaggc ttttaaagcc tttaaggttt aacggttgtg gacaacaagc
     8041 cagggatgta acgcactgag aagcccttag agcctctcaa agcaattttg agtgacacag
     8101 gaacacttaa cggctgacat ggggcgcgcc cagctgtcta gggcggcgga tttgtcctac
     8161 tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag
     8221 cctttcgttt tatttgatgc ct
//
