LOCUS       40924_23949        8795 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector pGWFDR196, complete sequence.
ACCESSION   .
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  JOURNAL   Unpublished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)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8795)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (21-MAR-2007) Cell Biology and Molecular Genetics, University of 
            Maryland, H J Patterson Hall Rm 3232, College Park, MD 20742, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(86..104)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      252..439
                     /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      complement(903..2249)
                     /direction=LEFT
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(2363..3163)
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(3164..3379)
                     /label=URA3 promoter
     promoter        3490..3594
                     /label=AmpR promoter
     CDS             3595..4452
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4626..5214
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5502..5523
                     /label=CAP binding site
                     /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
                     /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"
     protein_bind    6418..6542
                     /label=attR1
                     /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..7307
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             7652..7954
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(7998..8122)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(8130..8795,1..51)
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
ORIGIN
        1 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaccctcg
       61 agggggggcc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg gatccagctt
      121 tggacttctt cgccagaggt ttggtcaagt ctccaatcaa ggttgtcggc ttgtctacct
      181 tgccagaaat ttacgaaaag atggaaaagg gtcaaatcgt tggtagatac gttgttgaca
      241 cttctaaata agcgaatttc ttatgattta tgatttttat tattaaataa gttataaaaa
      301 aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaraa ttcttattct
      361 tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagcatgag gtcgctctta
      421 ttgaccacac ctctaccggc atgccaattc actggccgtc gttttacaac gtcgtgactg
      481 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
      541 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
      601 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
      661 aatcggatcg tacttgttac ccatcattga attttgaaca tccgaacctg ggagttttcc
      721 ctgaaacaga tagtatattt gaacctgtat aataatatat agtctagcgc tttacggaag
      781 acaatgtatg tatttcggtt cctggagaaa ctattgcatc tattgcatag gtaatcttgc
      841 acgtcgcatc cccggttcat tttctgcgtt tccatcttgc acttcaatag catatctttg
      901 ttaacgaagc atctgtgctt cattttgtag aacaaaaatg caacgcgaga gcgctaattt
      961 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg aaagcgctat
     1021 tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa aaatgcaacg cgagagcgct
     1081 aatttttcaa acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgagagc
     1141 gctattttac caacaaagaa tctatacttc ttttttgttc tacaaaaatg catcccgaga
     1201 gcgctatttt tctaacaaag catcttagat tacttttttt ctcctttgtg cgctctataa
     1261 tgcagtctct tgataacttt ttgcactgta ggtccgttaa ggttagaaga aggctacttt
     1321 ggtgtctatt ttctcttcca taaaaaaagc ctgactccac ttcccgcgtt tactgattac
     1381 tagcgaagct gcgggtgcat tttttcaaga taaaggcatc cccgattata ttctataccg
     1441 atgtggattg cgcatacttt gtgaacagaa agtgatagcg ttgatgattc ttcattggtc
     1501 agaaaattat gaacggtttc ttctattttg tctctatata ctacgtatag gaaatgttta
     1561 cattttcgta ttgttttcga ttcactctat gaatagttct tactacaatt tttttgtcta
     1621 aagagtaata ctagagataa acataaaaaa tgtagaggtc gagtttagat gcaagttcaa
     1681 ggagcgaaag gtggatgggt aggttatata gggatatagc acagagatat atagcaaaga
     1741 gatacttttg agcaatgttt gtggaagcgg tattcgcaat attttagtag ctcgttacag
     1801 tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag cgcttttggt tttcaaaagc
     1861 gctctgaagt tcctatactt tctagctaga gaataggaac ttcggaatag gaacttcaaa
     1921 gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat acagctcact
     1981 gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga gaagaacggc
     2041 atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta ggatgaaagg
     2101 tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat gcttccttca
     2161 gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt ctcatccttc
     2221 aatgctatca tttcctttga tattggatcg atccgatgat aagctgtcaa acatgagaat
     2281 tgggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat
     2341 ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc
     2401 ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca aatagtcctc
     2461 ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt
     2521 gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt
     2581 aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt
     2641 cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc
     2701 atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct
     2761 gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta atgtctgccc
     2821 attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag
     2881 caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct
     2941 taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt agtaaacaaa
     3001 ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg
     3061 aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac
     3121 taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc
     3181 tgcatgtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa
     3241 cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct
     3301 gttcggagat taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa aaagaataaa
     3361 aaaaaaatga tgaattgaaa agctaattct tgaagacgaa agggcctcgt gatacgccta
     3421 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     3481 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3541 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     3601 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     3661 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3721 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     3781 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     3841 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     3901 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     3961 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4021 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4081 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4141 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4201 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4261 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4321 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4381 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4441 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     4501 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4561 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4621 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4681 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4741 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4801 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4861 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4921 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4981 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5041 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5101 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5161 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5221 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5281 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5341 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     5401 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     5461 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     5521 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     5581 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcttcctgaa
     5641 acggagaaac ataaacaggc attgctggga tcacccatac atcactctgt tttgcctgac
     5701 cttttccggt aatttgaaaa caaacccggt ctcgaagcgg agatccggcg ataattaccg
     5761 cagaaataaa cccatacacg agacgtagaa ccagccgcac atggccggag aaactcctgc
     5821 gagaatttcg taaactcgcg cgcattgcat ctgtatttcc taatgcggca cttccaggcc
     5881 tcgatcgaga ccgtttatcc attgcttttt tgttgtcttt ttccctcgtt cacagaaagt
     5941 ctgaagaagc tatagtagaa ctatgagctt tttttgtttc tgttttcctt tttttttttt
     6001 ttacctctgt ggaaattgtt actctcacac tctttagttc gtttgtttgt tttgtttatt
     6061 ccaattatga ccggtgacga aacgtggtcg atggtgggta ccgcttatgc tcccctccat
     6121 tagtttcgat tatataaaaa ggccaaatat tgtattattt tcaaatgtcc tatcattatc
     6181 gtctaacatc taatttctct taaatttttt ctctttcttt cctataacac caatagtgaa
     6241 aatctttttt tcttctatat ctacaaaaac tttttttttc tatcaacctc gttgataaat
     6301 tttttcttta acaatcgtta ataattaatt aattggaaaa taaccatttt ttctctcttt
     6361 tatacacaca ttcaaaagaa agaaaaaaaa tataccccag cctcgactag tgatatcaca
     6421 agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa tatattaaat
     6481 tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta
     6541 tggcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt
     6601 ggattttgag ttaggatccg gcgagatttt caggagctaa ggaagctaaa atggagaaaa
     6661 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg
     6721 catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct
     6781 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg
     6841 cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga
     6901 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat
     6961 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg
     7021 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt
     7081 tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg
     7141 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc
     7201 tgatgccgct ggcgattcag gttcatcatg ccgtctgtga tggcttccat gtcggcagaa
     7261 tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa acgcgtggat
     7321 ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa
     7381 gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtgtgcta tgaagcagcg
     7441 tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat
     7501 atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc
     7561 tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc
     7621 tcttttgctg acgagaacag ggactggtga aatgcagttt aaggtttaca cctataaaag
     7681 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg
     7741 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     7801 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     7861 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     7921 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag
     7981 ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct
     8041 gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt
     8101 cagctttctt gtacaaagtg gtgatatcaa tggtgagcaa gggcgaggag ctgttcaccg
     8161 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     8221 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     8281 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     8341 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     8401 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     8461 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     8521 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     8581 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     8641 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     8701 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     8761 accccaacga gaagcgcgat cacatggtcc tgctg
//
