LOCUS       Exported               11535 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway binary vector pKPWGF2, complete sequence.
ACCESSION   EF515875
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11535)
  AUTHORS   Wang C, Sze H.
  TITLE     Generating binary vectors for pollen-specific expression of proteins
            tagged to eGFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 11535)
  AUTHORS   Wang C, Sze H.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-MAR-2007) Cell Biology and Molecular Genetics, 
            University of Maryland, H J Patterson Hall Rm 3232, College Park, MD
            20742, USA
REFERENCE   3  (bases 1 to 11535)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..11535
                     /organism="Gateway binary vector pKPWGF2"
                     /lab_host="Escherichia coli DB3.1"
                     /mol_type="other DNA"
                     /note="gateway destination vector for GFP fusion expression
                     in mature pollen"
                     /db_xref="taxon:436868"
     regulatory      complement(7..720)
                     /regulatory_class="promoter"
                     /note="AtCHX08 promoter; promoter region of Arabidopsis 
                     thaliana CHX08; drives pollen-specific over-expression in 
                     Arabidopsis thaliana"
     misc_recomb     728..927
                     /note="RB; right border of Agrobacterium T-DNA"
     misc_feature    789..813
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2113..2742
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3176..4243
                     /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      4309..4503
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     misc_feature    4847..4987
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5173..5761)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6005..6937)
                     /codon_start=1
                     /gene="spR"
                     /product="SpR"
                     /label=spR
                     /note="spectinomycin resistance protein"
                     /protein_id="ABP57403.1"
                     /translation="MTCFFGVQSMPRASKQQARYAVGRCLMLWSSNDVTQQGSRPKTKL
                     NIMGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVR
                     LDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRND
                     ILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNS
                     PPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQ
                     EEDRLASRADQLEEFVHYVKGEITKVVGK"
     gene            complement(6005..6937)
                     /gene="spR"
                     /label=spR
     CDS             complement(6005..6796)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_recomb     complement(7086..7418)
                     /note="LB; left border of Agrobacterium T-DNA"
     misc_feature    7324..7348
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     terminator      7441..7693
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(7733..8527)
                     /codon_start=1
                     /gene="kan"
                     /product="Kan"
                     /label=kan
                     /note="kanamycin resistance protein"
                     /protein_id="ABP57404.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            complement(7733..8527)
                     /gene="kan"
                     /label=kan
     promoter        complement(8561..8740)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     regulatory      complement(8873..9098)
                     /regulatory_class="terminator"
                     /note="T35S; Cauliflower mosaic virus 35S terminator"
     misc_recomb     9108..9232
                     /note="AttR2; gateway recombination site; derived from 
                     Escherichia coli"
     protein_bind    9108..9232
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(9273..9578)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB"
                     /label=ccdB
                     /note="cell cycle death B; cytotoxic protein derived from 
                     Escherichia coli"
                     /protein_id="ABP57407.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     gene            complement(9273..9578)
                     /gene="ccdB"
                     /label=ccdB
     CDS             complement(9920..10579)
                     /codon_start=1
                     /gene="cmR"
                     /product="CmR"
                     /label=cmR
                     /note="chloramphenicol resistance protein"
                     /protein_id="ABP57406.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     gene            complement(9920..10579)
                     /gene="cmR"
                     /label=cmR
     promoter        complement(10633..10663)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_recomb     complement(10688..10812)
                     /note="AttR1; gateway recombination site; derived from 
                     Escherichia coli"
     protein_bind    complement(10688..10812)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(10777..11535)
                     /codon_start=1
                     /gene="eGFP"
                     /product="enhanced green fluorescent protein"
                     /label=eGFP
                     /note="fused to C-terminus of target protein"
                     /protein_id="ABP57405.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKDITSLYKKAERET"
     gene            complement(10777..11535)
                     /gene="eGFP"
                     /label=eGFP
     CDS             complement(10819..11535)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"