LOCUS       sequence-205135-        9231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C. difficile transposon mutagenesis system. Shuttle vector 
            containing tet-inducible Himar1 transposase and custom ErmR 
            transposon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9231)
  AUTHORS   Dembek M, Barquist L, Boinett CJ, Cain AK, Mayho M, Lawley TD, 
            Fairweather NF, Fagan RP
  TITLE     High-throughput analysis of gene essentiality and sporulation in 
            Clostridium difficile.
  JOURNAL   MBio. 2015 Feb 24;6(2):e02383. doi: 10.1128/mBio.02383-14.
  PUBMED    25714712
REFERENCE   2  (bases 1 to 9231)
  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"; journalName: "MBio."; 
            date: "2015-02-24"; volume: "6(2):e02383. doi"; pages: " 
            10.1128/mBio.02383-14"
COMMENT     Sequence Label: pRPF215
FEATURES             Location/Qualifiers
     source          1..9231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(2710..3336)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     3241..3260
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    3443..3461
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             complement(5942..6313)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            6346..6455
                     /note="incP origin of transfer"
     primer_bind     complement(7646..7663)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7817..8405)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7897..7916)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctctatcat ttattttagt ttcccataca tctaaaattt tagtatctaa taaagattct
       61 atcatatttg aaattctttg ctgtgttact cctaaatctt ttgctaattc ctcttgatta
      121 attcttaata tatttccttt tattttatca tctttgacca ataaatcttg cttatatctc
      181 atatgagtat ataatcttaa ttcttcagtt gaaattattc cccaaattac tgccatggta
      241 acaaagtaac taatagtata ttgtttatct atattacatc taacttcttt tacattgtat
      301 gtatctttta ttcctagctt tgtaacacct tttgtttttg taatataatc attatgttga
      361 agttctttta atgtttctct taatgtcttt tcactcataa caatgttttt aactttcttc
      421 ttacgtttat aggttatcag ctccattata tcgtctatgt ttaattctct atctttattg
      481 ttaagtaaca cattatagat aaacaattga ttaccagtca ttattttaac ccctttccta
      541 ttcttatatt tcaaatcctt tgagtgctta tgtggcatat tgaacaaagt atcttcatca
      601 ctatatataa aatctgattc tattttattc caacaatcac tattaaactc acaacctgta
      661 caaccaaaat taaccgtttt aagcttctca tacatatact tgacttgata ttctagctca
      721 ctatttgaaa gactattttg tgatatatag ttccattctc tagcaacgga tattacttga
      781 cttaaacttt tattcctcaa tcttaaagtc acaactattt tttgcaaatc taggttttta
      841 tgtccatctt tactaccatt ttttaaaata tctactatac aatttggtag cttactatct
      901 aatatatatt ttgtattagt attacctact gtttttacat ctttgcaata ttttttagct
      961 aacttttcta tgtcatatct atatatattt ttgtcttggt gtattatttt tacttgttta
     1021 gtagtatttt taatattata tgtataaggt actcttaata cttgagtaga taaatttgct
     1081 tgtttatctg cacccaattt atttattaat gtatttgtaa cttctgatac taacttaatg
     1141 ttattagttt tattaattag cacataaaca tggaatccat ttccactatc aactaggata
     1201 tgataatgta atcctatctt agtaaataaa ttaattatat ctttataatt aaaaccttgt
     1261 cctaattctt ttttgtcaaa atcaaatcct aatgcatatc tatttattaa gttttcattg
     1321 gttcctgctt ttccatcagt tgtacatagg ttaaaaaatg tattttcata ataaatattt
     1381 tttgcgaagg taaattctac tatatcatct atatttttaa aaaaagttgt tttactatat
     1441 tctttaaatt cttctccgag ttctaattgt ttactattac ttttattatt ttgaaatact
     1501 ctaacatatt cattatcaat aagattattt ccttgtccta taccatctct aaatactgcc
     1561 attagaaatt ttttcaactt atattttgaa tctaattgtt ccatatattt attatctcct
     1621 tatctagttt ttttaagaga taacactaca ttaaatatat acttattgac taatcaactt
     1681 ttacaactat tgaataattt tagcttagta tgtataataa tattgttaag atgctaaaat
     1741 tgatagcaat attattatat aactacagat acttaatcag ttagaccgat attggagttg
     1801 gtagctccat aaactcaatt ggtagttgag ttgtcattgt ctagtagtat tatctcattt
     1861 tgttgttatc ttaaaatatt attttaaaaa tactctgctt ggtcggctta gggtattttt
     1921 tttgtttaat tccaatattg gttcaaactc ctgtgtaata cttttgacac ttccatctgt
     1981 attgaattga cagttgatat ggtatctcat tattggcttg ttcttatgtt tttctatgta
     2041 tttatttttt actttcaaat aatttgtatt tccattttta gttatgttta acacactatc
     2101 cttattactc aataaaatta ttttttgttt taacatatga tttatagaat ctaaaaattg
     2161 atattgactt ataatagtac tttctacctc tctagctttt tctagtgttt caaattcttc
     2221 aaactctgct atattgctat agtatagctt atcattaata atattatctg acaactttaa
     2281 ttccatttta taaaacaatt ccaagttatg tataaaattt ttaatgttac aacatataga
     2341 cttattattt gttcttatat ggtatctcca tttactactt ctagctctca tgttatatgc
     2401 acaactactc ccaacataaa catatattgc ttgttctaac tcatattttt gtaaaatttc
     2461 aacatcatat ccagtcaaat cgtactctct cttatataca aatggttttt ctaatttttt
     2521 gttcaataaa tctcctccta tttcttactt aatttagcta aaaatctatt tatatttcac
     2581 ctcctttttg actttaagcc tacgaatacc atatttatga ttataacatc ttctattttt
     2641 cccaaatcct tacatctccc ctaattttta gacttaaggg cgaattctgc atcaagctag
     2701 cttggtacct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa
     2761 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg
     2821 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg
     2881 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata
     2941 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt
     3001 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact
     3061 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc
     3121 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag
     3181 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg
     3241 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct
     3301 gttaatcact ttacttttat ctaatctaga catcattaat tcctcctttt tgttgacatt
     3361 atatcattga tagagttatt tgtcaaacta gttttttatt tcgatgccct ggacttcatg
     3421 aaaaactaaa aaaaatattg acactctatc attgatagag tataattaaa ataagcttga
     3481 tcgtagcgtt aacagatctg agctcaataa tactaggagg tttttatgga aaaaaaagaa
     3541 tttagagtat taataaaata ttgtttttta aaaggaaaga atacagtaga agcaaaaaca
     3601 tggttagata atgaatttcc agatagtgca ccaggaaaaa gtacaataat agattggtat
     3661 gcaaaattta aaagaggtga aatgagtact gaagatggtg aaagaagtgg tagaccaaaa
     3721 gaagtagtaa ctgatgaaaa tataaaaaaa atacataaaa tgatacttaa tgatagaaaa
     3781 atgaaattaa tagaaatagc agaagcatta aaaataagta aagaaagagt aggacatata
     3841 atacatcaat atttagatat gagaaaatta tgtgcaaaat gggttccaag agaattaaca
     3901 tttgatcaaa aacaacaaag agtagatgat tctgaaagat gtttacaatt attaacaaga
     3961 aatacaccag aattttttag aagatatgta acaatggatg aaacatggtt acatcattat
     4021 acaccagaaa gtaatagaca aagtgcagaa tggacagcaa caggtgaacc atctccaaaa
     4081 agaggaaaaa cacaaaaatc tgctggaaaa gtaatggcaa gtgtattttg ggatgcacat
     4141 ggaataatat ttatagatta tttagaaaaa ggtaaaacaa taaatagtga ttattatatg
     4201 gcattattag aaagattaaa agtagaaata gctgcaaaaa gaccacatat gaaaaaaaaa
     4261 aaagtattat ttcatcaaga taatgcacca tgtcataaaa gtttaagaac aatggcaaaa
     4321 atacatgaat taggatttga attattacca catccaccat atagtccaga tttagcacca
     4381 agtgattttt ttttatttag tgatttaaaa agaatgttag caggaaaaaa atttggttgt
     4441 aatgaagaag taatagcaga aacagaagca tattttgaag caaaaccaaa agaatattat
     4501 caaaatggaa taaaaaaatt agaaggtaga tataatagat gtatagcatt agaaggtaat
     4561 tatgtagaat aataaaggcc tcggatccta taagttttaa taaaacttta aatagaaaaa
     4621 ggcttctctc atgagaagtc ttttttattt aaaataaata taaaataaaa tagaggctat
     4681 aaatagcctc tattttatgt gagaaatccc taaataaaaa gatgccagtg tgctggtaac
     4741 aggttggctg ataagtcccc ggtctgacag aagcaaactt aagagtgtgt tgatagtgca
     4801 gtatcttaaa attttgtata ataggaattg aagttaaatt agatgctaaa aatttgtaat
     4861 taagaaggag tgattacatg aacaaaaata taaaatattc tcaaaacttt ttaacgagtg
     4921 aaaaagtact caaccaaata ataaaacaat tgaatttaaa agaaaccgat accgtttacg
     4981 aaattggaac aggtaaaggg catttaacga cgaaactggc taaaataagt aaacaggtaa
     5041 cgtctattga attagacagt catctattca acttatcgtc agaaaaatta aaactgaata
     5101 ctcgtgtcac tttaattcac caagatattc tacagtttca attccctaac aaacagaggt
     5161 ataaaattgt tgggagtatt ccttaccatt taagcacaca aattattaaa aaagtggttt
     5221 ttgaaagcca tgcgtctgac atctatctga ttgttgaaga aggattctac aagcgtacct
     5281 tggatattca ccgaacacta gggttgctct tgcacactca agtctcgatt cagcaattgc
     5341 ttaagctgcc agcggaatgc tttcatccta aaccaaaagt aaacagtgtc ttaataaaac
     5401 ttacccgcca taccacagat gttccagata aatattggaa gctatatacg tactttgttt
     5461 caaaatgggt caatcgagaa tatcgtcaac tgtttactaa aaatcagttt catcaagcaa
     5521 tgaaacacgc caaagtaaac aatttaagta ccgttactta tgagcaagta ttgtctattt
     5581 ttaatagtta tctattattt aacgggagga aataattcta tgagtcgctt ttgtaaattt
     5641 ggaaagttac acgttactaa aggcataaaa ataagaagcc tgcaaatgca ggcttcttat
     5701 ttttatgtgt tagaccgggg acttatcagc caacctgtta ccagtgtgct ggaattcgcc
     5761 cttaggctgc gctagggacc tctttagctc cttggaagct gtcagtagta ggggatcggt
     5821 cttgccttgc tcgtcggtga tgtacttcac cagctccgcg aagtcgctct tcttgatgga
     5881 gcgcatgggg acgtgcttgg caatcacgcg caccccccgg ccgttttagc ggctaaaaaa
     5941 gtcatggctc tgccctcggg cggaccacgc ccatcatgac cttgccaagc tcgtcctgct
     6001 tctcttcgat cttcgccagc agggcgagga tcgtggcatc accgaaccgc gccgtgcgcg
     6061 ggtcgtcggt gagccagagt ttcagcaggc cgcccaggcg gcccaggtcg ccattgatgc
     6121 gggccagctc gcggacgtgc tcatagtcca cgacgcccgt gattttgtag ccctggccga
     6181 cggccagcag gtaggccgac aggctcatgc cggccgccgc cgccttttcc tcaatcgctc
     6241 ttcgttcgtc tggaaggcag tacaccttga taggtgggct gcccttcctg gttggcttgg
     6301 tttcatcagc catccgcttg ccctcatctg ttacgccggc ggtagccggc cagcctcgca
     6361 gagcaggatt cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc
     6421 cgctcgcggg tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag
     6481 gaaagtctac acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata
     6541 ccgaaaaaat cgctataatg accccgaagc aggaagggcg aattctgcag atctgaccgg
     6601 tctctgaaaa tataaaaacc acagattgat actaaaacct tggttgtgtt gcttttcggg
     6661 gcttaaatca aggaaaaatc cttgttttaa gcctttcaaa aagaaacaca aggtctttgt
     6721 actaacctgt ggttatgtat aaaattgtag attttagggt aacaaaaaac accgtatttc
     6781 tacgatgttt ttgcttaaat acttgttttt agttacagac aaacctgaag ttaactattt
     6841 atcaattcct gcaattcgtt tacaaaacgg caaatgtgaa atccgtcaca tactgcgtga
     6901 tgaacttgaa ttgccaaagg aagtataatt ttgttatctt ctttataata tttccccata
     6961 gtaaaaatag gaatcaaata atcatatcct ttctgcaaat tcagattaaa gccatcgaag
     7021 gttgaccacg gtatcataga tacattaaaa atgttttccg gagcatttgg ctttccttcc
     7081 attctatgat tgtttccata ccgttgcgta tcactttcat aatctgctaa aaatgattta
     7141 aagtcagact tacactcagt ccaaaggctg gaaaatgttt cagtatcatt gtgaaatatt
     7201 gtatagcttg gtatcatctc atcatatatc cccaattcac catcttgatt gattgccgtc
     7261 ctaaactctg aatggcggtt tacaatcatt gcaatataat aaagcattgc aggatatagt
     7321 ttcattccct tttcctttat ttgtgtgata tccactttaa cggtcatgct gtaggtacaa
     7381 ggtacacttg caaagtagtg gtcaaaatac tcttttctgt tccaactatt tttatcaatt
     7441 ttttcaaata ccatctaagt tccctctcaa attcaagttt atcgctctaa tgaacaaaga
     7501 tattatacca catttttgtg aatttttcaa cttgcccact tcgactgcac tcccgactta
     7561 ataacttctt gaacacttgc cgaaaaagaa aaactgccgg gtacgtaccc ggggatcgat
     7621 ccccggccga gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     7681 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     7741 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     7801 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     7861 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctt
     7921 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     7981 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     8041 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     8101 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     8161 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     8221 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     8281 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     8341 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     8401 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     8461 gttaagggat tttggtcatg aaatgcaagt ttctaactaa catacatcat agttactaaa
     8521 ctatggtaac tataatttta ttaactatat taagcactga ttagtactat aactcaatat
     8581 aagcatatcc cctgtatcgt aactagagaa ccaaacgacg gaaaaagcga tatagataag
     8641 tttagatagt gcaccaattt tttacttttc tattttagaa tctagcattt ccaatgctta
     8701 ctaatactat ttttgatttt taacttgttt taaaaaagca cctactaatt aggattctag
     8761 ctctatattc tagttataca ttctctatat tctagctcta tattctagtt atacattctc
     8821 tatattctag ctctatattc tagttataca ttctctatat tctagctcta tattctagtt
     8881 atacattctc tatattctag ctctatattc tagttataca ttctctatat tctagctcta
     8941 tattctagtt atacattctc tatattctag ctctatattc tagttataca ttctctaaaa
     9001 aacatatata tatatactac atatatatgt acaattattt taccaaaaga aatttttaaa
     9061 acggcttaat ttcaatactt tgatgccatt ttttttttgc aattctcgta caattatttt
     9121 accaaaagaa atttttatta attagttttt ttgcataaaa ataggactag aaaatcaatt
     9181 ctaatcctat caaaaatcta cttgtttaat ctatatgtat agtacataaa t
//
