LOCUS       Exported                6778 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pMTL-SC7215, complete sequence.
ACCESSION   JQ040260
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6778)
  AUTHORS   Cartman ST, Kelly ML, Heeg D, Heap JT, Minton NP.
  TITLE     Precise Manipulation of the Clostridium difficile Chromosome Reveals
            a Lack of Association between the tcdC Genotype and Toxin Production
  JOURNAL   Appl. Environ. Microbiol. 78 (13), 4683-4690 (2012)
  PUBMED    22522680
REFERENCE   2  (bases 1 to 6778)
  AUTHORS   Cartman ST, Kelly ML, Heap JT, Minton NP.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-NOV-2011) Centre for Biomolecular Sciences, University
            of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom
REFERENCE   3  (bases 1 to 6778)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6778
                     /organism="Cloning vector pMTL-SC7215"
                     /mol_type="other DNA"
                     /label=allele exchange plasmid for Clostridium difficile
                     /note="allele exchange plasmid for Clostridium difficile"
                     /db_xref="taxon:1197975"
     regulatory      9..62
                     /regulatory_class="terminator"
                     /note="derived from CD0164 gene of Clostridium difficile 
                     630"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    254..278
                     /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)."
     RBS             297..305
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3 end of the 16S rRNA (Chen et al., 1994)"
     CDS             318..1601
                     /codon_start=1
                     /gene="codA"
                     /product="cytosine deaminase"
                     /label=codA
                     /protein_id="AFL93715.1"
                     /translation="MSNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITEN
                     SLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQR
                     AWQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSY
                     PNGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDE
                     QSRFVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNI
                     HLQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQ
                     LMGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSV
                     RGGKVIASTQPAQTTVYLEQPEAIDYKR"
     gene            318..1601
                     /gene="codA"
                     /label=codA
     CDS             321..1601
                     /codon_start=1
                     /gene="codA"
                     /product="E. coli cytosine deaminase"
                     /label=codA
                     /note="confers sensitivity to 5-fluorocytosine"
                     /translation="SNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITENS
                     LDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQRA
                     WQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSYP
                     NGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQ
                     SRFVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNIH
                     LQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQL
                     MGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSVR
                     GGKVIASTQPAQTTVYLEQPEAIDYKR"
     primer_bind     complement(1723..1739)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      1887..1928
                     /regulatory_class="terminator"
                     /note="derived from fdx gene of Clostridium pasteurianum"
     CDS             2449..3504
                     /codon_start=1
                     /gene="repA"
                     /product="plasmid replication protein"
                     /label=repA
                     /protein_id="AFL93716.1"
                     /translation="MTNYNQKNENKQEVKTALEKCTDKKRKNPRFTSYIAPLTTKKNIE
                     RTSTCGDYLFMLSDADLEHFKLHKGNFCGNRFCPMCSWRLACKDSLEISILMEHLRKEE
                     NKEFIFLTLTTPNVKSYDLNYSIKQYNKSFKKLMERKEVKDITKGYIRKLEVTYQKEKY
                     ITKDLWKIKKDYYQKKGLEIGDLEPNFDTYNPHFHVVIAVNKSYFTDKNYYINRERWLE
                     LWKFATKDDSITQVDVRKAKINDYKEVYELAKYSAKDTDYLISRPVFEIFYKALKGKQV
                     LVFSGFFKDAHKLYKQGKLDVYKKKDEIKYVYIVYYNWCKKQYEKTRIRELTEDEKEEL
                     NQDLIDEIEID"
     gene            2449..3504
                     /gene="repA"
                     /label=repA
     CDS             complement(3595..4098)
                     /codon_start=1
                     /product="putative plasmid replication protein"
                     /label=putative plasmid replication protein
                     /note="orf2"
                     /protein_id="AFL93717.1"
                     /translation="MKFKKIILYIFIVIIPLSITGCSINQITTPVPKNYLDNIVKKYVG
                     PEGVQPSFGGKTFYDYKIIDSEKSKNIIKIYLYYIGQEYYIEDDKLVEGTGSASFATVT
                     IKKENNNYKLVSYEVPKSPAQEDTVKIFPKSVRKKAIHANTPSLKNIEKEAETYFKKEL
                     KTKN"
     CDS             4422..5045
                     /codon_start=1
                     /gene="catP"
                     /product="chloramphenicol acetyltransferase"
                     /label=catP
                     /protein_id="AFL93718.1"
                     /translation="MVFEKIDKNSWNRKEYFDHYFASVPCTYSMTVKVDITQIKEKGMK
                     LYPAMLYYIAMIVNRHSEFRTAINQDGELGIYDEMIPSYTIFHNDTETFSSLWTECKSD
                     FKSFLADYESDTQRYGNNHRMEGKPNAPENIFNVSMIPWSTFDGFNLNLQKGYDYLIPI
                     FTMGKYYKEDNKIILPLAIQVHHAVCDGFHICRFVNELQELINS"
     gene            4422..5045
                     /gene="catP"
                     /label=catP
     rep_origin      5222..5810
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     oriT            6131..6240
                     /note="incP origin of transfer"
     CDS             6204..6644
                     /codon_start=1
                     /gene="traJ"
                     /product="plasmid conjugative transfer protein"
                     /label=traJ
                     /protein_id="AFL93719.1"
                     /translation="MLNGNPALRGWPATAGVTDEGKRMADETKPTRKGSPPIKVYCLPD
                     ERRAIEEKAAAAGMSLSAYLLAVGQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWL
                     TDDPRTARFGDATILALLAKIEEKQDELGKVMMGVVRPRAEP"
     gene            6204..6644
                     /gene="traJ"
                     /label=traJ
     CDS             6273..6644
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"