LOCUS       Exported               11845 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector pMTL007, complete sequence.
ACCESSION   EF525477
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11845)
  AUTHORS   Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP.
  TITLE     The ClosTron: a universal gene knock-out system for the genus 
            Clostridium
  JOURNAL   J. Microbiol. Methods 70 (3), 452-464 (2007)
  PUBMED    17658189
REFERENCE   2  (bases 1 to 11845)
  AUTHORS   Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-MAR-2007) Centre for Biomolecular Sciences, The 
            University of Nottingham, University Park, Nottingham NG7 2RD, 
            United Kingdom
REFERENCE   3  (bases 1 to 11845)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..11845
                     /organism="Shuttle vector pMTL007"
                     /mol_type="other DNA"
                     /db_xref="taxon:459063"
     misc_feature    1..338
                     /note="group II intron IBS and EBS; replaced to alter the 
                     intron target specificity"
     intron          31..2204
                     /note="group II intron"
     CDS             complement(join(740..1509,1903..1906))
                     /codon_start=1
                     /gene="ErmBtdRAM2"
                     /product="rRNA adenine N-6-methyltransferase"
                     /label=ErmBtdRAM2
                     /note="erythromycin resistance protein"
                     /protein_id="ABU90832.1"
                     /translation="MDPRDAGASGAGMNKNIKYSQNFLTSEKVLNQIIKQLNLKETDTV
                     YEIGTGKGHLTTKLAKISKQVTSIELDSHLFNLSSEKLKLNTRVTLIHQDILQFQFPNK
                     QRYKIVGSIPYHLSTQIIKKVVFESHASDIYLIVEEGFYKRTLDIHRTLGLLLHTQVSI
                     QQLLKLPAECFHPKPKVNSVLIKLTRHTTDVPDKYWKLYTYFVSKWVNREYRQLFTKNQ
                     FHQAMKHAKVNNLSTVTYEQVLSIFNSYLLFNGRK"
     gene            complement(740..1906)
                     /gene="ErmBtdRAM2"
                     /label=ErmBtdRAM2
     intron          1510..1902
                     /gene="ErmBtdRAM2"
                     /note="group I intron from td gene of phage T4 with 
                     intron-encoded protein deletion; inserted in reverse 
                     orientation to confer RAM functionality"
     RBS             2237..2259
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2451..4250
                     /codon_start=1
                     /gene="ltrA"
                     /product="LtrA intron-encoded protein"
                     /label=ltrA
                     /note="maturase, endonuclease and reverse-transcriptase 
                     activities"
                     /protein_id="ABU90833.1"
                     /translation="MKPTMAILERISKNSQENIDEVFTRLYRYLLRPDIYYVAYQNLYS
                     NKGASTKGILDDTADGFSEEKIKKIIQSLKDGTYYPQPVRRMYIAKKNSKKMRPLGIPT
                     FTDKLIQEAVRIILESIYEPVFEDVSHGFRPQRSCHTALKTIKREFGGARWFVEGDIKG
                     CFDNIDHVTLIGLINLKIKDMKMSQLIYKFLKAGYLENWQYHKTYSGTPQGGILSPLLA
                     NIYLHELDKFVLQLKMKFDRESPERITPEYRELHNEIKRISHRLKKLEGEEKAKVLLEY
                     QEKRKRLPTLPCTSQTNKVLKYVRYADDFIISVKGSKEDCQWIKEQLKLFIHNKLKMEL
                     SEEKTLITHSSQPARFLGYDIRVRRSGTIKRSGKVKKRTLNGSVELLIPLQDKIRQFIF
                     DKKIAIQKKDSSWFPVHRKYLIRSTDLEIITIYNSELRGICNYYGLASNFNQLNYFAYL
                     MEYSCLKTIASKHKGTLSKTISMFKDGSGSWGIPYEIKQGKQRRYFANFSECKSPYQFT
                     DEISQAPVLYGYARNTLENRLKAKCCELCGTSDENTSYEIHHVNKVKNLKGKEKWEMAM
                     IAKQRKTLVVCFHCHRHVIHKHK"
     gene            2451..4250
                     /gene="ltrA"
                     /label=ltrA
     terminator      4436..4522
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4614..4641
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4888..4904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5784..6101
                     /codon_start=1
                     /product="putative plasmid replication protein"
                     /label=putative plasmid replication protein
                     /note="ORFH"
                     /protein_id="ABU90834.1"
                     /translation="MKRRAYMKTCKNCKEFIKDTEICKIHSLMIHDKTVATYCSKYNES
                     RCLHKGKVQCINCSKMNRYGWCAIKMRCFTEEEQKKERTCIKYYARSFKKAHVKKSKKK
                     K"
     CDS             6893..7516
                     /codon_start=1
                     /gene="catP"
                     /product="chloramphenicol acetyltransferase"
                     /label=catP
                     /note="chloramphenicol resistance protein"
                     /protein_id="ABU90835.1"
                     /translation="MVFEKIDKNSWNRKEYFDHYFASVPCTYSMTVKVDITQIKEKGMK
                     LYPAMLYYIAMIVNRHSEFRTAINQDGELGIYDEMIPSYTIFHNDTETFSSLWTECKSD
                     FKSFLADYESDTQRYGNNHRMEGKPNAPENIFNVSMIPWSTFDGFNLNLQKGYDYLIPI
                     FTMGKYYKEDNKIILPLAIQVHHAVCDGFHICRFVNELQELINS"
     gene            6893..7516
                     /gene="catP"
                     /label=catP
     rep_origin      7998..8586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8877..9968)
                     /codon_start=1
                     /gene="lacI"
                     /product="Lac repressor"
                     /label=lacI
                     /protein_id="ABU90836.1"
                     /translation="MQHMKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     gene            complement(8877..9968)
                     /gene="lacI"
                     /label=lacI
     CDS             complement(8877..9959)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     oriT            10671..10780
                     /note="incP origin of transfer"
     CDS             10813..11184
                     /codon_start=1
                     /gene="traJ"
                     /product="conjugal transfer protein TraJ"
                     /label=traJ
                     /protein_id="ABU90837.1"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     gene            10813..11184
                     /gene="traJ"
                     /label=traJ
     regulatory      11585..11686
                     /regulatory_class="promoter"
                     /note="IPTG-inducible promoter fac"
     protein_bind    11692..11716
                     /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             11735..11743
                     /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)"