LOCUS Exported 8422 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG426GPD-ccdB-HA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8422) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 8422) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8422 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" rep_origin 69..1411 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind 401..448 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." promoter 1438..1542 /gene="bla" /label=AmpR promoter CDS 1543..2403 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1761..1780) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2574..3162 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3063..3082 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3316..3333 /label=L4440 /note="L4440 vector, forward primer" promoter 3486..3516 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3524..3540 /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 3529..3551 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3548..3564 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3548..3564 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 3583..3603 /label=T3 /note="T3 promoter, forward primer" promoter 3585..3603 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 3632..4276 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 4170..4193 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" primer_bind 4282..4298 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 4282..4298 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 4306..4430 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 4455..4485 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 4539..5198 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(4605..4624) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 5543..5848 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 5727..5746 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(5889..6013) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 6054..6143 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" terminator 6162..6409 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(6178..6196) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(6427..6446) /label=T7 /note="T7 promoter, forward primer" promoter complement(6428..6446) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6456..6473) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6456..6472) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6465..6487) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 6617..7072 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6704..6723) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6914..6935 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(7203..8006) /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(8007..8222) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(8253..8272) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8372..8394 /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg 121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg 181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac 241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata 301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc 361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt 421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa 481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga 541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat 601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct 661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta 721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag 781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc 841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct 901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt 961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg 1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa 1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa 1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta 1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt 1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt 1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt 1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc 1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc 3481 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa 3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 3601 agggaacaaa agctggagct cttcgagttt atcattatca atactcgcca tttcaaagaa 3661 tacgtaaata attaatagta gtgattttcc taactttatt tagtcaaaaa attagccttt 3721 taattctgct gtaacccgta catgcccaaa atagggggcg ggttacacag aatatataac 3781 accgaaggtg cctgggtgaa caggtttatt cctggcatcc actaaatata atggagcccg 3841 ctttttaagc tggcatccag aaaaaaaaag aatcccagca ccaaaatatt gttttcttca 3901 ccaaccatca gttcataggt ccattctctt agcgcaacta cagagaacag gggcacaaac 3961 aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc tgcctggagt aaatgatgac 4021 acaaggcaat tgacccacgc atgtatctat ctcattttct tacaccttct attaccttct 4081 gctctctctg atttggaaaa agctgaaaaa aaaggttgaa accagttccc tgaaattatt 4141 cccctacttg actaataagt atataaagac ggtaggtatt gattgtaatt ctgtaaatct 4201 atttcttaaa cttcttaaat tctactttta tagttagtct tttttttagt tttaaaacac 4261 cagaacttag tttcgacgga ttctagaact agtggatccc ccatcacaag tttgtacaaa 4321 aaagctgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat 4381 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat 4441 taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt 4501 aggatccgtc gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat 4561 ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag 4621 ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg 4681 taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga 4741 atgctcatcc ggaattccgt atggcaatga aagacggtga gctggtgata tgggatagtg 4801 ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg 4861 aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg 4921 gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca 4981 atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg 5041 cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg 5101 cgattcaggt tcatcatgcc gtctgtgatg gcttccatgt cggcagaatg cttaatgaat 5161 tacaacagta ctgcgatgag tggcagggcg gggcgtaaac gccgcgtgga tccggcttac 5221 taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata 5281 ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc gtattacagt 5341 gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt 5401 ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg 5461 gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct 5521 gacgagaaca ggggctggtg aaatgcagtt taaggtttac acctataaaa gagagagccg 5581 ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt 5641 gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt 5701 ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt 5761 ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc 5821 cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc 5881 aggtcgacca tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat 5941 gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcagctttct 6001 tgtacaaagt ggtgatgggc tgcaggaatt cgatatcaag cttaggtgga atgtacccat 6061 acgatgttcc tgactatgcg ggctatccct atgacgtccc ggactatgca ggatcctatc 6121 catatgacgt tccagattac gctgctcagt gctaactcga gtcatgtaat tagttatgtc 6181 acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca 6241 acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt 6301 tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact 6361 gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc 6421 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt 6481 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 6541 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 6601 aatggcgaat ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 6661 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 6721 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 6781 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 6841 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 6901 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 6961 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 7021 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcctgatgc 7081 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catagggtaa taactgatat 7141 aattaaattg aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt 7201 ttttagtttt gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt 7261 tcaccctcta ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg 7321 tcagatcctg tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc 7381 ttgtcatcta aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca 7441 cccatgtctc tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca 7501 acagtaccct tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac 7561 atcaaaaggc ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca 7621 atacctgggc ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat 7681 acacccgcag agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg 7741 aagagtaaaa aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg 7801 gaaaaatcag tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct 7861 tcaactaact ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt 7921 tgcttttcgt gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca 7981 cgttccttat atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg 8041 tgcagttggg ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata 8101 tataccaatc taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa 8161 tcaaaaaaat ttcaaagaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg 8221 aattgaaaag ctgtggtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 8281 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 8341 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 8401 ccgtcatcac cgaaacgcgc ga //