LOCUS       Exported                7535 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pQF, complete sequence.
ACCESSION   KF536588
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7535)
  AUTHORS   Kaczmarczyk A, Vorholt JA, Francez-Charlot A.
  TITLE     Cumate-inducible gene expression system for sphingomonads and other 
            alphaproteobacteria
  JOURNAL   Appl. Environ. Microbiol. 79 (21), 6795-6802 (2013)
  PUBMED    23995928
REFERENCE   2  (bases 1 to 7535)
  AUTHORS   Kaczmarczyk A.
  TITLE     Direct Submission
  JOURNAL   Submitted (12-AUG-2013) Department of Biology, Institute of 
            Microbiology, ETH Zurich, Wolfgang-Pauli-Str. 10, Zurich 8093, 
            Switzerland
REFERENCE   3  (bases 1 to 7535)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7535
                     /organism="Expression vector pQF"
                     /mol_type="other DNA"
                     /db_xref="taxon:1394911"
     rep_origin      4..712
                     /label=IncP incompatibility group
                     /note="IncP incompatibility group"
     rep_origin      11..642
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      1105..1693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      1180..1735
                     /label=ColE1 origin of replication
                     /note="ColE1 origin of replication"
     CDS             complement(1755..2375)
                     /codon_start=1
                     /gene="cymR*"
                     /product="CymR"
                     /label=cymR*
                     /note="transcriptional repressor; high-GC codon-optimized"
                     /protein_id="AGU99854.1"
                     /translation="MVIMSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPG
                     AAGVSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEF
                     FLDDDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDIL
                     WLIFNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     gene            complement(1755..2375)
                     /gene="cymR*"
                     /label=cymR*
     CDS             complement(1755..2366)
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     regulatory      complement(2382..2390)
                     /regulatory_class="ribosome_binding_site"
     regulatory      complement(2406..2434)
                     /regulatory_class="promoter"
                     /gene="Pbla-mut1T"
                     /note="modified bla promoter"
     gene            complement(2406..2434)
                     /gene="Pbla-mut1T"
                     /label=Pbla-mut1T
     regulatory      complement(2406..2411)
                     /regulatory_class="minus_10_signal"
                     /gene="Pbla-mut1T"
     regulatory      complement(2429..2434)
                     /regulatory_class="minus_35_signal"
                     /gene="Pbla-mut1T"
     regulatory      2443..2540
                     /regulatory_class="promoter"
                     /gene="PQ5"
                     /note="cumate-inducible promoter PQ5"
     gene            2443..2540
                     /gene="PQ5"
                     /label=PQ5
     protein_bind    2443..2470
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     regulatory      2476..2481
                     /regulatory_class="minus_35_signal"
                     /gene="PQ5"
     regulatory      2499..2505
                     /regulatory_class="minus_10_signal"
                     /gene="PQ5"
     protein_bind    2513..2540
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     regulatory      2547..2554
                     /regulatory_class="ribosome_binding_site"
     CDS             2562..2753
                     /codon_start=1
                     /product="3xFLAG-MCS-3xFLAG"
                     /label=3xFLAG-MCS-3xFLAG
                     /note="open reading frame of multiple cloning sites 
                     including 3xFLAG tags"
                     /protein_id="AGU99855.1"
                     /translation="MDYKDHDGDYKDHDIDYKDDDDKLINMTLEDLEVPSSNWDPDYKD
                     HDGDYKDHDIDYKDDDDK"
     misc_feature    join(2565..2630,2685..2750)
                     /label=3xFLAG tag
                     /note="3xFLAG tag"
     CDS             2565..2630
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2685..2750
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      2775..2815
                     /regulatory_class="terminator"
                     /note="putative transcriptional terminator T193*"
     primer_bind     complement(2822..2838)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3259..3924)
                     /codon_start=1
                     /gene="tetR"
                     /product="TetR"
                     /label=tetR
                     /note="tetracycline resistance"
                     /protein_id="AGU99856.1"
                     /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
                     RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
                     PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
                     QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGAKKPLLS"
     gene            complement(3259..3924)
                     /gene="tetR"
                     /label=tetR
     CDS             4030..5229
                     /codon_start=1
                     /gene="tetA"
                     /product="TetA"
                     /label=tetA
                     /note="tetracycline resistance"
                     /protein_id="AGU99857.1"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "
     gene            4030..5229
                     /gene="tetA"
                     /label=tetA
     CDS             complement(5460..6608)
                     /codon_start=1
                     /gene="trfA"
                     /product="TrfA"
                     /label=trfA
                     /note="plasmid replication protein"
                     /protein_id="AGU99858.1"
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     gene            complement(5460..6608)
                     /gene="trfA"
                     /label=trfA
     misc_feature    6790..7528
                     /label=origin of transfer
                     /note="origin of transfer"
     CDS             complement(6880..7251)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7284..7393
                     /note="incP origin of transfer"