LOCUS       Exported                7950 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  E. coli-C. difficile shuttle vector: tetracycline-inducible promoter
            (Ptet) driving expression of red fluorescent protein (mCherryOpt), 
            multiple cloning site for fusion to N-terminus of target protein.
ACCESSION   .
VERSION     .
KEYWORDS    pRAN473
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7950)
  AUTHORS   Ransom EM, Ellermeier CD, Weiss DS
  TITLE     Use of mCherry Red fluorescent protein for studies of protein 
            localization and gene expression in Clostridium difficile.
  JOURNAL   Appl Environ Microbiol. 2015 Mar;81(5):1652-60. doi: 
            10.1128/AEM.03446-14. Epub 2014 Dec 19.
  PUBMED    25527559
REFERENCE   2  (bases 1 to 7950)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7950
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(136..759)
                     /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     667..686
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    869..887
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             complement(2047..2418)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            2451..2560
                     /note="incP origin of transfer"
     primer_bind     complement(3751..3768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3922..4510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4002..4021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttatgattat aacatcttct atttttccca aatccttaca tctcccctaa tttttagact
       61 taagggcgaa ttctgcatca agctagcata aaaataagaa gcctgcattt gcaggcttct
      121 tatttttatg gtaccttaag acccactttc acatttaagt tgtttttcta atccgcatat
      181 gatcaattca aggccgaata agaaggctgg ctctgcacct tggtgatcaa ataattcgat
      241 agcttgtcgt aataatggcg gcatactatc agtagtaggt gtttcccttt cttctttagc
      301 gacttgatgc tcttgatctt ccaatacgca acctaaagta aaatgcccca cagcgctgag
      361 tgcatataat gcattctcta gtgaaaaacc ttgttggcat aaaaaggcta attgattttc
      421 gagagtttca tactgttttt ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg
      481 atgacttagt aaagcacatc taaaactttt agcgttatta cgtaaaaaat cttgccagct
      541 ttccccttct aaagggcaaa agtgagtatg gtgcctatct aacatctcaa tggctaaggc
      601 gtcgagcaaa gcccgcttat tttttacatg ccaatacaat gtaggctgct ctacacctag
      661 cttctgggcg agtttacggg ttgttaaacc ttcgattccg acctcattaa gcagctctaa
      721 tgcgctgtta atcactttac ttttatctaa tctagacatc attaattcct cctttttgtt
      781 gacattatat cattgataga gttatttgtc aaactagttt tttatttcga tgccctggac
      841 ttcatgaaaa actaaaaaaa atattgacac tctatcattg atagagtata attaaaataa
      901 gcttgatcgt agcgttaaca gatctgagct cctgcagtaa aggagaaaat tttatggtat
      961 ctaaaggaga agaagataat atggctataa ttaaagaatt tatgagattt aaagttcata
     1021 tggaaggatc tgttaatgga catgaatttg aaatagaagg agaaggagaa ggaagacctt
     1081 atgaaggtac tcaaacagct aaattaaaag taactaaagg tggtccatta ccatttgcat
     1141 gggatatatt aagtcctcaa tttatgtatg gatctaaagc atatgttaaa catcctgctg
     1201 atatacctga ttatttaaaa ttatcatttc cagaaggttt taaatgggaa agagttatga
     1261 attttgaaga tggtggagta gttactgtaa cacaagatag ttcattacaa gatggagatt
     1321 ttatatataa agttaaatta agaggtacta attttccatc tgatggacca gtaatgcaaa
     1381 agaaaactat gggatgggaa gcatcatctg aaagaatgta tccagaagat ggagcattaa
     1441 aaggtgaaat taaacaaaga ttaaaattaa aagatggagg tcattatgat gctgaagtta
     1501 agactacata taaagctaag aaaccagttc aattaccagg tgcatataat gttaatatta
     1561 aattagatat aacttctcat aatgaagatt atactatagt tgaacaatat gaaagagcag
     1621 aaggaagaca ttcaacagga ggtatggatg aattatataa aatgaacgca tgcgtcgact
     1681 taattaaaca gctgaggcgc gccggatcct ataagtttta ataaaacttt aaatagaaaa
     1741 aggcttctct catgagaagt cttttttatt taaaataaat ataaaataaa atagaggcta
     1801 taaatagcct ctattttatg tgagaaatcc ctaaataaaa agatgccagt gtgctggaat
     1861 tcgcccttag gctgcgctag ggacctcttt agctccttgg aagctgtcag tagtagggga
     1921 tcggtcttgc cttgctcgtc ggtgatgtac ttcaccagct ccgcgaagtc gctcttcttg
     1981 atggagcgca tggggacgtg cttggcaatc acgcgcaccc cccggccgtt ttagcggcta
     2041 aaaaagtcat ggctctgccc tcgggcggac cacgcccatc atgaccttgc caagctcgtc
     2101 ctgcttctct tcgatcttcg ccagcagggc gaggatcgtg gcatcaccga accgcgccgt
     2161 gcgcgggtcg tcggtgagcc agagtttcag caggccgccc aggcggccca ggtcgccatt
     2221 gatgcgggcc agctcgcgga cgtgctcata gtccacgacg cccgtgattt tgtagccctg
     2281 gccgacggcc agcaggtagg ccgacaggct catgccggcc gccgccgcct tttcctcaat
     2341 cgctcttcgt tcgtctggaa ggcagtacac cttgataggt gggctgccct tcctggttgg
     2401 cttggtttca tcagccatcc gcttgccctc atctgttacg ccggcggtag ccggccagcc
     2461 tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga
     2521 acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca
     2581 ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg
     2641 atataccgaa aaaatcgcta taatgacccc gaagcaggaa gggcgaattc tgcagatctg
     2701 accggtctct gaaaatataa aaaccacaga ttgatactaa aaccttggtt gtgttgcttt
     2761 tcggggctta aatcaaggaa aaatccttgt tttaagcctt tcaaaaagaa acacaaggtc
     2821 tttgtactaa cctgtggtta tgtataaaat tgtagatttt agggtaacaa aaaacaccgt
     2881 atttctacga tgtttttgct taaatacttg tttttagtta cagacaaacc tgaagttaac
     2941 tatttatcaa ttcctgcaat tcgtttacaa aacggcaaat gtgaaatccg tcacatactg
     3001 cgtgatgaac ttgaattgcc aaaggaagta taattttgtt atcttcttta taatatttcc
     3061 ccatagtaaa aataggaatc aaataatcat atcctttctg caaattcaga ttaaagccat
     3121 cgaaggttga ccacggtatc atagatacat taaaaatgtt ttccggagca tttggctttc
     3181 cttccattct atgattgttt ccataccgtt gcgtatcact ttcataatct gctaaaaatg
     3241 atttaaagtc agacttacac tcagtccaaa ggctggaaaa tgtttcagta tcattgtgaa
     3301 atattgtata gcttggtatc atctcatcat atatccccaa ttcaccatct tgattgattg
     3361 ccgtcctaaa ctctgaatgg cggtttacaa tcattgcaat ataataaagc attgcaggat
     3421 atagtttcat tcccttttcc tttatttgtg tgatatccac tttaacggtc atgctgtagg
     3481 tacaaggtac acttgcaaag tagtggtcaa aatactcttt tctgttccaa ctatttttat
     3541 caattttttc aaataccatc taagttccct ctcaaattca agtttatcgc tctaatgaac
     3601 aaagatatta taccacattt ttgtgaattt ttcaacttgc ccacttcgac tgcactcccg
     3661 acttaataac ttcttgaaca cttgccgaaa aagaaaaact gccgggtacg tacccgggga
     3721 tcgatccccg gccgagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3781 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3841 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3901 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3961 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4021 cccttgaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4081 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4141 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4201 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4261 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4321 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4381 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4441 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4501 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4561 ctcacgttaa gggattttgg tcatgaaatg caagtttcta actaacatac atcatagtta
     4621 ctaaactatg gtaactataa ttttattaac tatattaagc actgattagt actataactc
     4681 aatataagca tatcccctgt atcgtaacta gagaaccaaa cgacggaaaa agcgatatag
     4741 ataagtttag atagtgcacc aattttttac ttttctattt tagaatctag catttccaat
     4801 gcttactaat actatttttg atttttaact tgttttaaaa aagcacctac taattaggat
     4861 tctagctcta tattctagtt atacattctc tatattctag ctctatattc tagttataca
     4921 ttctctatat tctagctcta tattctagtt atacattctc tatattctag ctctatattc
     4981 tagttataca ttctctatat tctagctcta tattctagtt atacattctc tatattctag
     5041 ctctatattc tagttataca ttctctatat tctagctcta tattctagtt atacattctc
     5101 taaaaaacat atatatatat actacatata tatgtacaat tattttacca aaagaaattt
     5161 ttaaaacggc ttaatttcaa tactttgatg ccattttttt tttgcaattc tcgtacaatt
     5221 attttaccaa aagaaatttt tattaattag tttttttgca taaaaatagg actagaaaat
     5281 caattctaat cctatcaaaa atctacttgt ttaatctata tgtatagtac ataaatcctc
     5341 tatcatttat tttagtttcc catacatcta aaattttagt atctaataaa gattctatca
     5401 tatttgaaat tctttgctgt gttactccta aatcttttgc taattcctct tgattaattc
     5461 ttaatatatt tccttttatt ttatcatctt tgaccaataa atcttgctta tatctcatat
     5521 gagtatataa tcttaattct tcagttgaaa ttattcccca aattactgcc atggtaacaa
     5581 agtaactaat agtatattgt ttatctatat tacatctaac ttcttttaca ttgtatgtat
     5641 cttttattcc tagctttgta acaccttttg tttttgtaat ataatcatta tgttgaagtt
     5701 cttttaatgt ttctcttaat gtcttttcac tcataacaat gtttttaact ttcttcttac
     5761 gtttataggt tatcagctcc attatatcgt ctatgtttaa ttctctatct ttattgttaa
     5821 gtaacacatt atagataaac aattgattac cagtcattat tttaacccct ttcctattct
     5881 tatatttcaa atcctttgag tgcttatgtg gcatattgaa caaagtatct tcatcactat
     5941 atataaaatc tgattctatt ttattccaac aatcactatt aaactcacaa cctgtacaac
     6001 caaaattaac cgttttaagc ttctcataca tatacttgac ttgatattct agctcactat
     6061 ttgaaagact attttgtgat atatagttcc attctctagc aacggatatt acttgactta
     6121 aacttttatt cctcaatctt aaagtcacaa ctattttttg caaatctagg tttttatgtc
     6181 catctttact accatttttt aaaatatcta ctatacaatt tggtagctta ctatctaata
     6241 tatattttgt attagtatta cctactgttt ttacatcttt gcaatatttt ttagctaact
     6301 tttctatgtc atatctatat atatttttgt cttggtgtat tatttttact tgtttagtag
     6361 tatttttaat attatatgta taaggtactc ttaatacttg agtagataaa tttgcttgtt
     6421 tatctgcacc caatttattt attaatgtat ttgtaacttc tgatactaac ttaatgttat
     6481 tagttttatt aattagcaca taaacatgga atccatttcc actatcaact aggatatgat
     6541 aatgtaatcc tatcttagta aataaattaa ttatatcttt ataattaaaa ccttgtccta
     6601 attctttttt gtcaaaatca aatcctaatg catatctatt tattaagttt tcattggttc
     6661 ctgcttttcc atcagttgta cataggttaa aaaatgtatt ttcataataa atattttttg
     6721 cgaaggtaaa ttctactata tcatctatat ttttaaaaaa agttgtttta ctatattctt
     6781 taaattcttc tccgagttct aattgtttac tattactttt attattttga aatactctaa
     6841 catattcatt atcaataaga ttatttcctt gtcctatacc atctctaaat actgccatta
     6901 gaaatttttt caacttatat tttgaatcta attgttccat atatttatta tctccttatc
     6961 tagttttttt aagagataac actacattaa atatatactt attgactaat caacttttac
     7021 aactattgaa taattttagc ttagtatgta taataatatt gttaagatgc taaaattgat
     7081 agcaatatta ttatataact acagatactt aatcagttag accgatattg gagttggtag
     7141 ctccataaac tcaattggta gttgagttgt cattgtctag tagtattatc tcattttgtt
     7201 gttatcttaa aatattattt taaaaatact ctgcttggtc ggcttagggt attttttttg
     7261 tttaattcca atattggttc aaactcctgt gtaatacttt tgacacttcc atctgtattg
     7321 aattgacagt tgatatggta tctcattatt ggcttgttct tatgtttttc tatgtattta
     7381 ttttttactt tcaaataatt tgtatttcca tttttagtta tgtttaacac actatcctta
     7441 ttactcaata aaattatttt ttgttttaac atatgattta tagaatctaa aaattgatat
     7501 tgacttataa tagtactttc tacctctcta gctttttcta gtgtttcaaa ttcttcaaac
     7561 tctgctatat tgctatagta tagcttatca ttaataatat tatctgacaa ctttaattcc
     7621 attttataaa acaattccaa gttatgtata aaatttttaa tgttacaaca tatagactta
     7681 ttatttgttc ttatatggta tctccattta ctacttctag ctctcatgtt atatgcacaa
     7741 ctactcccaa cataaacata tattgcttgt tctaactcat atttttgtaa aatttcaaca
     7801 tcatatccag tcaaatcgta ctctctctta tatacaaatg gtttttctaa ttttttgttc
     7861 aataaatctc ctcctatttc ttacttaatt tagctaaaaa tctatttata tttcacctcc
     7921 tttttgactt taagcctacg aataccatat
//
