LOCUS       Exported                8795 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector pGWFDR196, complete sequence.
ACCESSION   EF515872
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8795)
  AUTHORS   Li X, Romanowsky SM, Harper JF, Cheung AY, Sze H.
  TITLE     Thapsigargin-Sensitive Ca2+/Mn2+ Pump AtECA3 Supports Pollen Tip 
            Growth and Promotes Male Fertility
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8795)
  AUTHORS   Li X, Sze H.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-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 8795)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8795
                     /organism="Shuttle vector pGWFDR196"
                     /lab_host="Escherichia coli DB3.1"
                     /mol_type="other DNA"
                     /note="yeast expression gateway destination vector; derived
                     from Saccharomyces cerevisae and Escherichia coli"
                     /db_xref="taxon:436865"
     promoter        complement(86..104)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      252..439
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(451..467)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      903..2249
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     gene            complement(2282..3389)
                     /gene="ura3"
                     /label=ura3
                     /note="yeast selective marker; uracil synthesis"
     CDS             complement(2360..3163)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(3164..3379)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        3490..3594
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3595..4455
                     /codon_start=1
                     /gene="ampR"
                     /product="AmpR"
                     /label=ampR
                     /note="ampicillin resistance protein"
                     /protein_id="ABP57392.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            3595..4455
                     /gene="ampR"
                     /label=ampR
     rep_origin      4626..5214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5502..5523
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5538..5568
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5576..5592
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5600..5616
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      5628..6401
                     /regulatory_class="promoter"
                     /note="PMA1 promoter; derived from yeast; causes 
                     constitutive overexpression in yeast"
     misc_recomb     6418..6542
                     /note="AttR1; gateway recombination site; derived from 
                     Escherichia coli"
     protein_bind    6418..6542
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        6567..6597
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             6651..7310
                     /codon_start=1
                     /gene="cmR"
                     /product="CmR"
                     /label=cmR
                     /note="chloramphenicol resistance protein"
                     /protein_id="ABP57391.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     gene            6651..7310
                     /gene="cmR"
                     /label=cmR
     CDS             7652..7957
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB"
                     /label=ccdB
                     /note="cytotoxic protein; derived from Escherichia coli"
                     /protein_id="ABP57390.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     gene            7652..7957
                     /gene="ccdB"
                     /label=ccdB
     misc_recomb     complement(7998..8122)
                     /note="AttR2; gateway recombination site; derived from 
                     Escherichia coli"
     protein_bind    complement(7998..8122)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             8130..54
                     /codon_start=1
                     /gene="eGFP"
                     /product="enhanced green fluorescent protein"
                     /label=eGFP
                     /note="fused to C-terminus of target protein"
                     /protein_id="ABP57393.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     gene            8130..54
                     /gene="eGFP"
                     /label=eGFP