LOCUS       Exported                9231 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  C. difficile transposon mutagenesis system. Shuttle vector 
            containing tet-inducible Himar1 transposase and custom ErmR 
            transposon.
ACCESSION   .
VERSION     .
KEYWORDS    pRPF215
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 May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9231
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(3293..3916)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     3824..3843
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    4026..4044
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             complement(6525..6896)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            6929..7038
                     /note="incP origin of transfer"
     primer_bind     complement(8229..8246)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8400..8988)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8480..8499)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtgcaccaat tttttacttt tctattttag aatctagcat ttccaatgct tactaatact
       61 atttttgatt tttaacttgt tttaaaaaag cacctactaa ttaggattct agctctatat
      121 tctagttata cattctctat attctagctc tatattctag ttatacattc tctatattct
      181 agctctatat tctagttata cattctctat attctagctc tatattctag ttatacattc
      241 tctatattct agctctatat tctagttata cattctctat attctagctc tatattctag
      301 ttatacattc tctatattct agctctatat tctagttata cattctctaa aaaacatata
      361 tatatatact acatatatat gtacaattat tttaccaaaa gaaattttta aaacggctta
      421 atttcaatac tttgatgcca tttttttttt gcaattctcg tacaattatt ttaccaaaag
      481 aaatttttat taattagttt ttttgcataa aaataggact agaaaatcaa ttctaatcct
      541 atcaaaaatc tacttgttta atctatatgt atagtacata aatcctctat catttatttt
      601 agtttcccat acatctaaaa ttttagtatc taataaagat tctatcatat ttgaaattct
      661 ttgctgtgtt actcctaaat cttttgctaa ttcctcttga ttaattctta atatatttcc
      721 ttttatttta tcatctttga ccaataaatc ttgcttatat ctcatatgag tatataatct
      781 taattcttca gttgaaatta ttccccaaat tactgccatg gtaacaaagt aactaatagt
      841 atattgttta tctatattac atctaacttc ttttacattg tatgtatctt ttattcctag
      901 ctttgtaaca ccttttgttt ttgtaatata atcattatgt tgaagttctt ttaatgtttc
      961 tcttaatgtc ttttcactca taacaatgtt tttaactttc ttcttacgtt tataggttat
     1021 cagctccatt atatcgtcta tgtttaattc tctatcttta ttgttaagta acacattata
     1081 gataaacaat tgattaccag tcattatttt aacccctttc ctattcttat atttcaaatc
     1141 ctttgagtgc ttatgtggca tattgaacaa agtatcttca tcactatata taaaatctga
     1201 ttctatttta ttccaacaat cactattaaa ctcacaacct gtacaaccaa aattaaccgt
     1261 tttaagcttc tcatacatat acttgacttg atattctagc tcactatttg aaagactatt
     1321 ttgtgatata tagttccatt ctctagcaac ggatattact tgacttaaac ttttattcct
     1381 caatcttaaa gtcacaacta ttttttgcaa atctaggttt ttatgtccat ctttactacc
     1441 attttttaaa atatctacta tacaatttgg tagcttacta tctaatatat attttgtatt
     1501 agtattacct actgttttta catctttgca atatttttta gctaactttt ctatgtcata
     1561 tctatatata tttttgtctt ggtgtattat ttttacttgt ttagtagtat ttttaatatt
     1621 atatgtataa ggtactctta atacttgagt agataaattt gcttgtttat ctgcacccaa
     1681 tttatttatt aatgtatttg taacttctga tactaactta atgttattag ttttattaat
     1741 tagcacataa acatggaatc catttccact atcaactagg atatgataat gtaatcctat
     1801 cttagtaaat aaattaatta tatctttata attaaaacct tgtcctaatt cttttttgtc
     1861 aaaatcaaat cctaatgcat atctatttat taagttttca ttggttcctg cttttccatc
     1921 agttgtacat aggttaaaaa atgtattttc ataataaata ttttttgcga aggtaaattc
     1981 tactatatca tctatatttt taaaaaaagt tgttttacta tattctttaa attcttctcc
     2041 gagttctaat tgtttactat tacttttatt attttgaaat actctaacat attcattatc
     2101 aataagatta tttccttgtc ctataccatc tctaaatact gccattagaa attttttcaa
     2161 cttatatttt gaatctaatt gttccatata tttattatct ccttatctag tttttttaag
     2221 agataacact acattaaata tatacttatt gactaatcaa cttttacaac tattgaataa
     2281 ttttagctta gtatgtataa taatattgtt aagatgctaa aattgatagc aatattatta
     2341 tataactaca gatacttaat cagttagacc gatattggag ttggtagctc cataaactca
     2401 attggtagtt gagttgtcat tgtctagtag tattatctca ttttgttgtt atcttaaaat
     2461 attattttaa aaatactctg cttggtcggc ttagggtatt ttttttgttt aattccaata
     2521 ttggttcaaa ctcctgtgta atacttttga cacttccatc tgtattgaat tgacagttga
     2581 tatggtatct cattattggc ttgttcttat gtttttctat gtatttattt tttactttca
     2641 aataatttgt atttccattt ttagttatgt ttaacacact atccttatta ctcaataaaa
     2701 ttattttttg ttttaacata tgatttatag aatctaaaaa ttgatattga cttataatag
     2761 tactttctac ctctctagct ttttctagtg tttcaaattc ttcaaactct gctatattgc
     2821 tatagtatag cttatcatta ataatattat ctgacaactt taattccatt ttataaaaca
     2881 attccaagtt atgtataaaa tttttaatgt tacaacatat agacttatta tttgttctta
     2941 tatggtatct ccatttacta cttctagctc tcatgttata tgcacaacta ctcccaacat
     3001 aaacatatat tgcttgttct aactcatatt tttgtaaaat ttcaacatca tatccagtca
     3061 aatcgtactc tctcttatat acaaatggtt tttctaattt tttgttcaat aaatctcctc
     3121 ctatttctta cttaatttag ctaaaaatct atttatattt cacctccttt ttgactttaa
     3181 gcctacgaat accatattta tgattataac atcttctatt tttcccaaat ccttacatct
     3241 cccctaattt ttagacttaa gggcgaattc tgcatcaagc tagcttggta ccttaagacc
     3301 cactttcaca tttaagttgt ttttctaatc cgcatatgat caattcaagg ccgaataaga
     3361 aggctggctc tgcaccttgg tgatcaaata attcgatagc ttgtcgtaat aatggcggca
     3421 tactatcagt agtaggtgtt tccctttctt ctttagcgac ttgatgctct tgatcttcca
     3481 atacgcaacc taaagtaaaa tgccccacag cgctgagtgc atataatgca ttctctagtg
     3541 aaaaaccttg ttggcataaa aaggctaatt gattttcgag agtttcatac tgtttttctg
     3601 taggccgtgt acctaaatgt acttttgctc catcgcgatg acttagtaaa gcacatctaa
     3661 aacttttagc gttattacgt aaaaaatctt gccagctttc cccttctaaa gggcaaaagt
     3721 gagtatggtg cctatctaac atctcaatgg ctaaggcgtc gagcaaagcc cgcttatttt
     3781 ttacatgcca atacaatgta ggctgctcta cacctagctt ctgggcgagt ttacgggttg
     3841 ttaaaccttc gattccgacc tcattaagca gctctaatgc gctgttaatc actttacttt
     3901 tatctaatct agacatcatt aattcctcct ttttgttgac attatatcat tgatagagtt
     3961 atttgtcaaa ctagtttttt atttcgatgc cctggacttc atgaaaaact aaaaaaaata
     4021 ttgacactct atcattgata gagtataatt aaaataagct tgatcgtagc gttaacagat
     4081 ctgagctcaa taatactagg aggtttttat ggaaaaaaaa gaatttagag tattaataaa
     4141 atattgtttt ttaaaaggaa agaatacagt agaagcaaaa acatggttag ataatgaatt
     4201 tccagatagt gcaccaggaa aaagtacaat aatagattgg tatgcaaaat ttaaaagagg
     4261 tgaaatgagt actgaagatg gtgaaagaag tggtagacca aaagaagtag taactgatga
     4321 aaatataaaa aaaatacata aaatgatact taatgataga aaaatgaaat taatagaaat
     4381 agcagaagca ttaaaaataa gtaaagaaag agtaggacat ataatacatc aatatttaga
     4441 tatgagaaaa ttatgtgcaa aatgggttcc aagagaatta acatttgatc aaaaacaaca
     4501 aagagtagat gattctgaaa gatgtttaca attattaaca agaaatacac cagaattttt
     4561 tagaagatat gtaacaatgg atgaaacatg gttacatcat tatacaccag aaagtaatag
     4621 acaaagtgca gaatggacag caacaggtga accatctcca aaaagaggaa aaacacaaaa
     4681 atctgctgga aaagtaatgg caagtgtatt ttgggatgca catggaataa tatttataga
     4741 ttatttagaa aaaggtaaaa caataaatag tgattattat atggcattat tagaaagatt
     4801 aaaagtagaa atagctgcaa aaagaccaca tatgaaaaaa aaaaaagtat tatttcatca
     4861 agataatgca ccatgtcata aaagtttaag aacaatggca aaaatacatg aattaggatt
     4921 tgaattatta ccacatccac catatagtcc agatttagca ccaagtgatt tttttttatt
     4981 tagtgattta aaaagaatgt tagcaggaaa aaaatttggt tgtaatgaag aagtaatagc
     5041 agaaacagaa gcatattttg aagcaaaacc aaaagaatat tatcaaaatg gaataaaaaa
     5101 attagaaggt agatataata gatgtatagc attagaaggt aattatgtag aataataaag
     5161 gcctcggatc ctataagttt taataaaact ttaaatagaa aaaggcttct ctcatgagaa
     5221 gtctttttta tttaaaataa atataaaata aaatagaggc tataaatagc ctctatttta
     5281 tgtgagaaat ccctaaataa aaagatgcca gtgtgctggt aacaggttgg ctgataagtc
     5341 cccggtctga cagaagcaaa cttaagagtg tgttgatagt gcagtatctt aaaattttgt
     5401 ataataggaa ttgaagttaa attagatgct aaaaatttgt aattaagaag gagtgattac
     5461 atgaacaaaa atataaaata ttctcaaaac tttttaacga gtgaaaaagt actcaaccaa
     5521 ataataaaac aattgaattt aaaagaaacc gataccgttt acgaaattgg aacaggtaaa
     5581 gggcatttaa cgacgaaact ggctaaaata agtaaacagg taacgtctat tgaattagac
     5641 agtcatctat tcaacttatc gtcagaaaaa ttaaaactga atactcgtgt cactttaatt
     5701 caccaagata ttctacagtt tcaattccct aacaaacaga ggtataaaat tgttgggagt
     5761 attccttacc atttaagcac acaaattatt aaaaaagtgg tttttgaaag ccatgcgtct
     5821 gacatctatc tgattgttga agaaggattc tacaagcgta ccttggatat tcaccgaaca
     5881 ctagggttgc tcttgcacac tcaagtctcg attcagcaat tgcttaagct gccagcggaa
     5941 tgctttcatc ctaaaccaaa agtaaacagt gtcttaataa aacttacccg ccataccaca
     6001 gatgttccag ataaatattg gaagctatat acgtactttg tttcaaaatg ggtcaatcga
     6061 gaatatcgtc aactgtttac taaaaatcag tttcatcaag caatgaaaca cgccaaagta
     6121 aacaatttaa gtaccgttac ttatgagcaa gtattgtcta tttttaatag ttatctatta
     6181 tttaacggga ggaaataatt ctatgagtcg cttttgtaaa tttggaaagt tacacgttac
     6241 taaaggcata aaaataagaa gcctgcaaat gcaggcttct tatttttatg tgttagaccg
     6301 gggacttatc agccaacctg ttaccagtgt gctggaattc gcccttaggc tgcgctaggg
     6361 acctctttag ctccttggaa gctgtcagta gtaggggatc ggtcttgcct tgctcgtcgg
     6421 tgatgtactt caccagctcc gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct
     6481 tggcaatcac gcgcaccccc cggccgtttt agcggctaaa aaagtcatgg ctctgccctc
     6541 gggcggacca cgcccatcat gaccttgcca agctcgtcct gcttctcttc gatcttcgcc
     6601 agcagggcga ggatcgtggc atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag
     6661 agtttcagca ggccgcccag gcggcccagg tcgccattga tgcgggccag ctcgcggacg
     6721 tgctcatagt ccacgacgcc cgtgattttg tagccctggc cgacggccag caggtaggcc
     6781 gacaggctca tgccggccgc cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg
     6841 cagtacacct tgataggtgg gctgcccttc ctggttggct tggtttcatc agccatccgc
     6901 ttgccctcat ctgttacgcc ggcggtagcc ggccagcctc gcagagcagg attcccgttg
     6961 agcaccgcca ggtgcgaata agggacagtg aagaaggaac acccgctcgc gggtgggcct
     7021 acttcaccta tcctgcccgg ctgacgccgt tggatacacc aaggaaagtc tacacgaacc
     7081 ctttggcaaa atcctgtata tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata
     7141 atgaccccga agcaggaagg gcgaattctg cagatctgac cggtctctga aaatataaaa
     7201 accacagatt gatactaaaa ccttggttgt gttgcttttc ggggcttaaa tcaaggaaaa
     7261 atccttgttt taagcctttc aaaaagaaac acaaggtctt tgtactaacc tgtggttatg
     7321 tataaaattg tagattttag ggtaacaaaa aacaccgtat ttctacgatg tttttgctta
     7381 aatacttgtt tttagttaca gacaaacctg aagttaacta tttatcaatt cctgcaattc
     7441 gtttacaaaa cggcaaatgt gaaatccgtc acatactgcg tgatgaactt gaattgccaa
     7501 aggaagtata attttgttat cttctttata atatttcccc atagtaaaaa taggaatcaa
     7561 ataatcatat cctttctgca aattcagatt aaagccatcg aaggttgacc acggtatcat
     7621 agatacatta aaaatgtttt ccggagcatt tggctttcct tccattctat gattgtttcc
     7681 ataccgttgc gtatcacttt cataatctgc taaaaatgat ttaaagtcag acttacactc
     7741 agtccaaagg ctggaaaatg tttcagtatc attgtgaaat attgtatagc ttggtatcat
     7801 ctcatcatat atccccaatt caccatcttg attgattgcc gtcctaaact ctgaatggcg
     7861 gtttacaatc attgcaatat aataaagcat tgcaggatat agtttcattc ccttttcctt
     7921 tatttgtgtg atatccactt taacggtcat gctgtaggta caaggtacac ttgcaaagta
     7981 gtggtcaaaa tactcttttc tgttccaact atttttatca attttttcaa ataccatcta
     8041 agttccctct caaattcaag tttatcgctc taatgaacaa agatattata ccacattttt
     8101 gtgaattttt caacttgccc acttcgactg cactcccgac ttaataactt cttgaacact
     8161 tgccgaaaaa gaaaaactgc cgggtacgta cccggggatc gatccccggc cgagcgctct
     8221 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     8281 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     8341 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     8401 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     8461 cgaaacccga caggactata aagataccag gcgtttcccc cttgaagctc cctcgtgcgc
     8521 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     8581 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     8641 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     8701 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     8761 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     8821 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     8881 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     8941 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     9001 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     9061 atgaaatgca agtttctaac taacatacat catagttact aaactatggt aactataatt
     9121 ttattaacta tattaagcac tgattagtac tataactcaa tataagcata tcccctgtat
     9181 cgtaactaga gaaccaaacg acggaaaaag cgatatagat aagtttagat a
//
