LOCUS       Exported               14508 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  URT cloning vector pKP77, complete sequence.
ACCESSION   JX181780
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14508)
  AUTHORS   Piatkov K, Graciet E, Varshavsky A.
  TITLE     Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking 
            Prokaryotes
  JOURNAL   PLoS ONE 8 (6), E67952 (2013)
  PUBMED    23825692
REFERENCE   2  (bases 1 to 14508)
  AUTHORS   Piatkov K, Graciet E, Varshavsky A.
  TITLE     Broad host range UPR vectors for Gram-negative Bacteria
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 14508)
  AUTHORS   Piatkov K, Graciet E, Varshavsky A.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-JUN-2012) Biology, California Institute of Technology,
            1200 E. California Blvd., 147-75, Pasadena, CA 91125, USA
REFERENCE   4  (bases 1 to 14508)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..14508
                     /organism="URT cloning vector pKP77"
                     /mol_type="other DNA"
                     /db_xref="taxon:1226529"
     CDS             complement(7..1092)
                     /codon_start=1
                     /product="URT reporter protein"
                     /label=URT reporter protein
                     /note="3xFLAG-DHFR-HA-Ub-cs-3xFLAG"
                     /protein_id="AFQ55276.1"
                     /translation="MGDYKDDDDKGSGSDYKDDDDKGSGSDYKDDDDKSGSHMVRPLNC
                     IVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGRKTWFSIPEKNR
                     PLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQPELASKVDMVWIVGGSSVYQEA
                     MNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEYPGVLSEVQEEKGIKYKFEVYE
                     KKDGTYPYDVPDYAAFLAGSGSTMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGI
                     PPDQQRLIFAGRQLEDGRTLSDYNIQKESTLHLVLRLRGGRDLGLSGSDYKDDDDKGSG
                     SDYKDDDDKGSGSDYKDDDDK"
     misc_feature    complement(7..114)
                     /label=Region: 3x FLAG tag
                     /note="Region: 3x FLAG tag"
     CDS             complement(10..33)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(46..69)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(82..105)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    complement(115..129)
                     /label=cloning site
                     /note="cloning site"
     misc_feature    complement(130..357)
                     /label=Region: ubiquitin
                     /note="Region: ubiquitin"
     CDS             complement(130..357)
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     misc_feature    complement(385..411)
                     /label=Region: HA tag
                     /note="Region: HA tag"
     CDS             complement(385..411)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    complement(412..978)
                     /label=Region: DHFR
                     /note="Region: DHFR"
     CDS             complement(418..978)
                     /codon_start=1
                     /gene="Mus musculus Dhfr"
                     /product="mouse dihydrofolate reductase"
                     /label=DHFR
                     /translation="MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVE
                     GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP
                     ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY
                     PGVLSEVQEEKGIKYKFEVYEKKD"
     misc_feature    complement(979..1092)
                     /label=Region: 3x FLAG tag
                     /note="Region: 3x FLAG tag"
     CDS             complement(991..1014)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1027..1050)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1063..1086)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    1571..1688
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     CDS             2387..4816
                     /codon_start=1
                     /product="Ubp1"
                     /label=Ubp1
                     /protein_id="AFQ55684.1"
                     /translation="MDLFIESKINSLLQFLFGSRQDFLRNFKTWSNNNNNLSIYLLIFG
                     IVVFFYKKPDHLNYIVESVSEMTTNFRNNNSLSRWLPRSKFTHLDEEILKRGGFIAGLV
                     NDGNTCFMNSVLQSLASSRELMEFLDNNVIRTYEEIEQNEHNEEGNGQESAQDEATHKK
                     NTRKGGKVYGKHKKKLNRKSSSKEDEEKSQEPDITFSVALRDLLSALNAKYYRDKPYFK
                     TNSLLKAMSKSPRKNILLGYDQEDAQEFFQNILAELESNVKSLNTEKLDTTPVAKSELP
                     DDALVGQLNLGEVGTVYIPTEQIDPNSILHDKSIQNFTPFKLMTPLDGITAERIGCLQC
                     GENGGIRYSVFSGLSLNLPNENIGSTLKLSQLLSDWSKPEIIEGVECNRCALTAAHSHL
                     FGQLKEFEKKPEGSIPEKLINAVKDRVHQIEEVLAKPVIDDEDYKKLHTANMVRKCSKS
                     KQILISRPPPLLSIHINRSVFDPRTYMIRKNNSKVLFKSRLNLAPWCCDINEINLDARL
                     PMSKKEKAAQQDSSEDENIGGEYYTKLHERFEQEFEDSEEEKEYDDAEGNYASHYNHTK
                     DISNYDPLNGEVDGVTSDDEDEYIEETDALGNTIKKRIIEHSDVENENVKDNEELQEID
                     NVSLDEPKINVEDQLETSSDEEDVIPAPPINYARSFSTVPATPLTYSLRSVIVHYGTHN
                     YGHYIAFRKYRGCWWRISDETVYVVDEAEVLSTPGVFMLFYEYDFDEETGKMKDDLEAI
                     QSNNEEDDEKEQEQKGVQEPKESQEQGEGEEQEEGQEQMKFERTEDHRDISGKDVN"
     promoter        5096..5124
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             5336..6139
                     /codon_start=1
                     /product="Sm resistance protein A"
                     /label=Sm resistance protein A
                     /protein_id="AFQ55265.1"
                     /translation="MNRTNIFFGESHSDWLPVRGGESGDFVFRRGDGHAFAKIAPASRR
                     GELAGERDRLIWLKGRGVACPEVINWQEEQEGACLVITAIPGVPAADLSGADLLKAWPS
                     MGQQLGAVHSLSVDQCPFERRLSRMFGRAVDVVSRNAVNPDFLPDEDKSTPLHDLLARV
                     ERELPVRLDQERTDMVVCHGDPCMPNFMVDPKTLQCTGLIDLGRLGTADRYADLALMIA
                     NAEENWAAPDEAERAFAVLFNVLGIEAPDRERLAFYLRLDPLTWG"
     CDS             6139..6975
                     /codon_start=1
                     /product="Sm resistance protein B"
                     /label=Sm resistance protein B
                     /protein_id="AFQ55266.1"
                     /translation="MFMPPVFPAHWHVSQPVLIADTFSSLVWKVSLPDGTPAIVKGLKP
                     IEDIADELRGADYLVWRNGRGAVRLLGRENNLMLLEYAGERMLSHIVAEHGDYQATEIA
                     AELMAKLYAASEEPLPSALLPIRDRFAALFQRARDDQNAGCQTDYVHAAIIADQMMSNA
                     SELRGLHGDLHHENIMFSSRGWLVIDPVGLVGEVGFGAANMFYDPADRDDLCLDPRRIA
                     QMADAFSRALDVDPRRLLDQAYAYGCLSAAWNADGEEEQRDLAIAAAIKQVRQTSY"
     rep_origin      7619..8013
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     CDS             complement(8040..8324)
                     /codon_start=1
                     /product="mobilization protein C"
                     /label=mobilization protein C
                     /protein_id="AFQ55267.1"
                     /translation="MVKGSNKAADRLAKLEEQRARINAEIQRVRAREQQQERKNETRRK
                     VLVGAMILAKVNSSEWPEDRLMAAMDAYLERDHDRALFGLPPRQKDEPG"
     oriT            8355..8442
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     CDS             8523..10652
                     /codon_start=1
                     /product="mobilization protein A"
                     /label=mobilization protein A
                     /protein_id="AFQ55268.1"
                     /translation="MAIYHLTAKTGSRSGGQSARAKADYIQREGKYARDMDEVLHAESG
                     HMPEFVERPADYWDAADLYERANGRLFKEVEFALPVELTLDQQKALASEFAQHLTGAER
                     LPYTLAIHAGGGENPHCHLMISERINDGIERPAAQWFKRYNGKTPEKGGAQKTEALKPK
                     AWLEQTREAWADHANRALERAGHDARIDHRTLEAQGIERLPGVHLGPNVVEMEGRGIRT
                     DRADVALNIDTANAQIIDLQEYREAIDHERNRQSEEIQRHQRVSGADRTAGPEHGDTGR
                     RSPAGHEPDPAGQRGAGGGVAESPAPDRGGMGGAGQRVAGGSRRGEQRRAERPERVAGV
                     ALEAMANRDAGFHDAYGGAADRIVALARPDATDNRGRLDLAALGGPMKNDRTLQAIGRQ
                     LKAMGCERFDIGVRDATTGQMMNREWSAAEVLQNTPWLKRMNAQGNDVYIRPAEQERHG
                     LVLVDDLSEFDLDDMKAEGREPALVVETSPKNYQAWVKVADAAGGELRGQIARTLASEY
                     DADPASADSRHYGRLAGFTNRKDKHTTRAGYQPWVLLRESKGKTATAGPALVQQAGQQI
                     EQAQRQQEKARRLASLELPERQLSRHRRTALDEYRSEMAGLVKRFGDDLSKCDFIAAQK
                     LASRGRSAEEIGKAMAEASPALAERKPGHEADYIERTVSKVMGLPSVQLARAELARAPA
                     PRQRGMDRGGPDFSM"
     CDS             9271..9684
                     /codon_start=1
                     /product="mobilization protein B"
                     /label=mobilization protein B
                     /protein_id="AFQ55269.1"
                     /translation="MNAIDRVKKSRGINELAEQIEPLAQSMATLADEARQVMSQTQQAS
                     EAQAAEWLKAQRQTGAAWVELAKELREVAAEVSSAAQSARSASRGWHWKLWLTVMLASM
                     MPTVVLLIASLLLLDLTPLTTEDGSIWLRLVAR"
     CDS             9681..10652
                     /codon_start=1
                     /product="replication protein B"
                     /label=replication protein B
                     /protein_id="AFQ55270.1"
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     CDS             10713..10925
                     /codon_start=1
                     /product="unknown"
                     /label=unknown
                     /note="E"
                     /protein_id="AFQ55271.1"
                     /translation="MEYEKSASGSVYLIKSDKGYWLPGGFGYTSNKAEAGRFSVADMAS
                     LNLDGCTLSLFREDKPFGPGKFLGD"
     CDS             10927..11133
                     /codon_start=1
                     /product="repressor protein F"
                     /label=repressor protein F
                     /protein_id="AFQ55272.1"
                     /translation="MKDQKDKQTGDLLASPDAVRQARYAERMKAKGMRQRKFWLTDDEY
                     EALRECLEELRAAQGGGSDPASA"
     CDS             11163..12002
                     /codon_start=1
                     /product="replication protein A"
                     /label=replication protein A
                     /protein_id="AFQ55273.1"
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             11989..12840
                     /codon_start=1
                     /product="replication protein C"
                     /label=replication protein C
                     /protein_id="AFQ55274.1"
                     /translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
                     GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
                     TAECHVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     CDS             complement(13362..14156)
                     /codon_start=1
                     /product="Km resistance protein"
                     /label=Km resistance protein
                     /protein_id="AFQ55275.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"