LOCUS       Exported                8419 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pKEK1140, complete sequence.
ACCESSION   EU499313
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8419)
  AUTHORS   Rodriguez SA, Yu JJ, Davis G, Arulanandam BP, Klose KE.
  TITLE     Targeted inactivation of francisella tularensis genes by group II 
            introns
  JOURNAL   Appl. Environ. Microbiol. 74 (9), 2619-2626 (2008)
  PUBMED    18310413
REFERENCE   2  (bases 1 to 8419)
  AUTHORS   Rodriguez SA, Petrosino JF, Klose KE.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-FEB-2008) Biology, South Texas Center for Emerging 
            Infectious Diseases, The University of Texas at San Antonio, One 
            UTSA Circle, San Antonio, TX 78249, USA
REFERENCE   3  (bases 1 to 8419)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8419
                     /organism="Cloning vector pKEK1140"
                     /lab_host="Francisella tularensis"
                     /mol_type="other DNA"
                     /note="temperature-sensitive cloning vector for targeted 
                     inactivation of genes in Francisella tularensis"
                     /db_xref="taxon:508959"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        142..172
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..195
                     /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)."
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             216..836
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYG
                     QDSRLPSEFDLSAFLRAGENVQSVGEPMGTKRMRCRESEFTKT"
     CDS             216..763
                     /codon_start=1
                     /gene="lacZ-alpha"
                     /product="LacZ-alpha"
                     /label=lacZ-alpha
                     /protein_id="ACA63830.1"
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYG
                     QDSRLPSEFDLSAFLRAGE"
     gene            216..763
                     /gene="lacZ-alpha"
                     /label=lacZ-alpha
     primer_bind     complement(236..252)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     intron          770..1372
                     /note="Lactococcus lactis LtrB group II intron"
     RBS             1405..1427
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1619..3418
                     /codon_start=1
                     /gene="ltrA"
                     /product="LtrA"
                     /label=ltrA
                     /note="maturase, endonuclease, reverse transcriptase; aids 
                     LtrB group II intron into its secondary structure, cleaves 
                     opposite strand DNA, reverse transcribes intron insertion"
                     /protein_id="ACA63831.1"
                     /translation="MKPTMAILERISKNSQENIDEVFTRLYRYLLRPDIYYVAYQNLYS
                     NKGASTKGILDDTADGFSEEKIKKIIQSLKDGTYYPQPVRRMYIAKKNSKKMRPLGIPT
                     FTDKLIQEAVRIILESIYEPVFEDVSHGFRPQRSCHTALKTIKREFGGARWFVEGDIKG
                     CFDNIDHVTLIGLINLKIKDMKMSQLIYKFLKAGYLENWQYHKTYSGTPQGGILSPLLA
                     NIYLHELDKFVLQLKMKFDRESPERITPEYRELHNEIKRISHRLKKLEGEEKAKVLLEY
                     QEKRKRLPTLPCTSQTNKVLKYVRYADDFIISVKGSKEDCQWIKEQLKLFIHNKLKMEL
                     SEEKTLITHSSQPARFLGYDIRVRRSGTIKRSGKVKKRTLNGSVELLIPLQDKIRQFIF
                     DKKIAIQKKDSSWFPVHRKYLIRSTDLEIITIYNSELRGICNYYGLASNFNQLNYFAYL
                     MEYSCLKTIASKHKGTLSKTISMFKDGSGSWGIPYEIKQGKQRRYFANFSECKSPYQFT
                     DEISQAPVLYGYARNTLENRLKAKCCELCGTSDENTSYEIHHVNKVKNLKGKEKWEMAM
                     IAKQRKTLVVCFHCHRHVIHKHK"
     gene            1619..3418
                     /gene="ltrA"
                     /label=ltrA
     terminator      3610..3696
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3788..3815
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     misc_feature    3999..4847
                     /note="encodes origin of replication of Escherichia coli 
                     p15A"
     rep_origin      4036..4581
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     misc_feature    complement(4912..6807)
                     /note="corresponds to region within Francisella tularensis 
                     subsp. novicida plasmid, pFNL10, which encodes origin of 
                     replication"
     CDS             complement(5273..6292)
                     /codon_start=1
                     /gene="repA"
                     /product="temperature-sensitive RepA"
                     /label=repA
                     /note="contains amino acid mutation, methionine to 
                     isoleucine M120I"
                     /protein_id="ACA63832.1"
                     /translation="MKEVKNVNAGKEIAMSNTLVAGKYSLTKEEQNLIFLVASMVKRED
                     KEFHRYKISLSDLEKATGVKHNRVRLKQLMHSIMSKPVWLNKEQTKIANWFAYIEADPK
                     SSALICEFHWSLMPHIIQLQEYFTKAERQLLFSFKSKYSSRLYLLLKSKLGEQAGYANI
                     VDCVLYVDDMINDFDLPKSYSNRYSNFKNKFLLPALEEINQLSDIFVTYDDKDHHRKHG
                     RKITNIKFTVSKVAETEEEFKQKLLDTKNKEDYIPIEASERLREVVLSDELDLDLYYVR
                     SIFRHHHLHDIERVCDDTWRNWDNKKIKDRKRVFIANIKKLEKKPEENHKLPFGFEEV"
     gene            complement(5273..6292)
                     /gene="repA"
                     /label=repA
     regulatory      7163..7322
                     /regulatory_class="promoter"
                     /note="region encodes promoter of FTN1451 of Francisella 
                     tularensis subsp. novicida"
     CDS             7326..8141
                     /codon_start=1
                     /gene="aph"
                     /product="aminoglycoside 3 phosphotransferase"
                     /label=aph
                     /protein_id="ACA63833.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAHAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            7326..8141
                     /gene="aph"
                     /label=aph
     regulatory      8181..8419
                     /regulatory_class="promoter"
                     /note="region encodes groELp promoter of Francisella 
                     tularensis subsp. holarctica strain, LVS"