LOCUS       Exported               17681 bp ds-DNA    circular SYN 29-5-2014
DEFINITION  High-throughput binary Gateway(R) destination vector with two 
            introns, for silencing genes in plants using intron-containing 
            hairpin RNA.
ACCESSION   .
VERSION     .
KEYWORDS    pHELLSGATE 12
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17681)
  AUTHORS   Helliwell CA, Waterhouse PM.
  TITLE     Constructs and methods for hairpin RNA-mediated gene silencing in 
            plants.
  JOURNAL   Meth. Enzymol. 2005;392:24-35.
  PUBMED    15644173
REFERENCE   2  (bases 1 to 17681)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..17681
                     /organism="synthetic DNA construct"
                     /lab_host="Plant Cells"
                     /mol_type="other DNA"
     promoter        264..447
                     /function="NOS promoter"
                     /note="NOS promoter"
                     /note="nopaline synthase promoter"
     CDS             448..1269
                     /codon_start=1
                     /gene="nos-nptII"
                     /product="fusion between an N-terminal peptide of nopaline 
                     synthase and Tn5 aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin"
                     /protein_id="
                     "
                     /translation="MAITLSATSLPISARIRAGSPAAWVERLFGYDWAQQTIGCSDAAV
                     FRLSAQGRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLG
                     EVPGQDLLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVD
                     QDDLDEEHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLG
                     VADRYQDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     terminator      1894..2146
                     /note="NOS terminator"
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    2268..2292
                     /note="LB T-DNA repeat"
                     /note="left border repeat from nopaline C58 T-DNA"
     oriT            2846..2955
                     /note="incP origin of transfer"
     mobile_element  3015..3782
                     /mobile_element_type="insertion sequence:IS1"
                     /note="IS1"
                     /note="prokaryotic transposable element"
     CDS             4407..5555
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /note="trfA"
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(6915..7503)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             8600..9388
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase"
                     /note="SmR"
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      10071..10684
                     /note="oriV"
                     /note="origin of replication for the bacterial F plasmid"
     misc_feature    11182..11206
                     /note="RB T-DNA repeat"
                     /note="right border repeat from nopaline C58 T-DNA"
     promoter        12672..13017
                     /note="CaMV 35S promoter"
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     protein_bind    13026..13150
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR1"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(13579..13884)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /note="ccdB"
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(14324..14448)
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR2"
                     /note="recombination site for the Gateway(R) LR reaction"
     intron          14475..15241
                     /gene="pdk"
                     /number="3"
                     /note="PDK intron"
                     /note="pyruvate orthophosphate dikinase intron from 
                     Flaveria trinervia"
     intron          15276..15465
                     /note="cat1 intron"
                     /note="castor bean catalase intron, modified"
     protein_bind    15489..15613
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR2"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(15942..16247)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /note="ccdB"
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(16779..16903)
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR1"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      16912..17618
                     /note="OCS terminator"
                     /note="octopine synthase terminator"