LOCUS       Exported               11746 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRGK374, complete sequence.
ACCESSION   FJ268733
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11746)
  AUTHORS   Fitzsimmons K, Zhu H, Kranz R.
  TITLE     Direct Submission
  JOURNAL   Submitted (01-OCT-2008) Biology, Washington University in St Louis, 
            1 Brookings Dr. Campus Box #1137, St Louis, MO 63146, USA
REFERENCE   2  (bases 1 to 11746)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..11746
                     /organism="Cloning vector pRGK374"
                     /mol_type="other DNA"
                     /db_xref="taxon:571943"
     misc_feature    547..2385
                     /label=Region: GUS
                     /note="Region: GUS"
     CDS             553..2358
                     /codon_start=1
                     /gene="uidA (or gusA) from E. coli"
                     /product="beta-glucuronidase"
                     /label=GUS
                     /translation="LRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESRAIA
                     VPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGKVWVNNQEVM
                     EHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGKKKQSYFHDF
                     FNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQV
                     VATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGQQFL
                     INHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYPYAEEMLDWA
                     DEHGIVVIDETAAVGFQLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQAIKELIARDK
                     NHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCDAHTDTISDL
                     FDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMYT
                     DMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIFTRDRKP
                     KSAAFLLQKRWTGMNFGEKPQQGGKQ"
     CDS             2365..2382
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2414..2666
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     3UTR           2418..2670
                     /label=NOS
                     /note="NOS"
     misc_feature    2688..2712
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     misc_feature    2710..2733
                     /note="T-DNA; right border"
     CDS             4012..4641
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             5075..6142
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     rep_origin      6208..6402
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     misc_feature    6746..6886
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(7072..7660)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    7747..8541
                     /label=derived from pBR322
                     /note="derived from pBR322"
     CDS             complement(7747..8541)
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /label=kanamycin resistance protein
                     /protein_id="ACJ06732.1"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    8966..8990
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     misc_feature    8968..8994
                     /note="T-DNA; left border"
     primer_bind     complement(9170..9186)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        9415..10091
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             10154..10951
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /label=kanamycin resistance protein
                     /protein_id="ACJ06733.1"
                     /translation="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    11008..11182
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     protein_bind    11278..11372
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     misc_feature    11279..11373
                     /note="5X UAS; upstream activating sequence"
     regulatory      11381..11477
                     /regulatory_class="promoter"
                     /note="m35S; minimal 35S CaMV"
     promoter        11433..11479
                     /label=minimal CaMV 35S promoter
                     /note="minimal 35S promoter from cauliflower mosaic virus"
     CDS             11516..2388
                     /codon_start=1
                     /product="mGFP/GUS"
                     /label=mGFP/GUS
                     /protein_id="ACJ06731.1"
                     /translation="MKTNLFLFLIFSLLLSLSSAEFSKGEELFTGVVPILVELDGDVNG
                     HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDF
                     FKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLE
                     YNYNSHNVYIMADKQKNGIKANFKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYL
                     STQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKASLRPVETPTREIKKLDGLWAF
                     SLDRENCGIDQRWWESALQESRAIAVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWA
                     GQRIVLRFDAVTHYGKVWVNNQEVMEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELN
                     WQTIPPGMVITDENGKKKQSYFHDFFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCN
                     HASVDWQVVANGDVSVELRDADQQVVATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAK
                     SQTECDIYPLRVGIRSVAVKGQQFLINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHA
                     LMDWIGANSYRTSHYPYAEEMLDWADEHGIVVIDETAAVGFQLSLGIGFEAGNKPKELY
                     SEEAVNGETQQAHLQAIKELIARDKNHPSVVMWSIANEPDTRPQGAREYFAPLAEATRK
                     LDPTRPITCVNVMFCDAHTDTISDLFDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQ
                     EKLHQPIIITEYGVDTLAGLHSMYTDMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFA
                     DFATSQGILRVGGNKKGIFTRDRKPKSAAFLLQKRWTGMNFGEKPQQGGKQASHHHHHH
                     V"
     misc_feature    11516..546
                     /label=Region: endoplasmic reticulum signal + mGFP
                     /note="Region: endoplasmic reticulum signal + mGFP"
     CDS             11582..546
                     /codon_start=1
                     /gene="mgfp5"
                     /product="GFP with folding enhancement mutations"
                     /label=mgfp5
                     /note="suitable for expression in plants due to removal of 
                     a cryptic intron"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"