LOCUS Exported 8347 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION FX cloning Saccharomyces cerevisiae expression vector with GAL1 promoter and N-terminal 10x His tag, GFP and 3C protease cleavage site. ACCESSION . VERSION . KEYWORDS pYEXNHG3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8347) AUTHORS Geertsma ER, Dutzler R TITLE A versatile and efficient high-throughput cloning tool for structural biology. JOURNAL Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25. PUBMED 21410291 REFERENCE 2 (bases 1 to 8347) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8347 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 596..1476 /label=2u ori /note="yeast 2u plasmid origin of replication" rep_origin complement(1545..2000) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 1894..1913 /label=F1ori-R /note="F1 origin, reverse primer" promoter 2019..2460 /gene="S. cerevisiae GAL1" /label=GAL1 promoter /note="inducible promoter, regulated by Gal4" protein_bind 2019..2136 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" primer_bind 2431..2454 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" primer_bind 2492..2511 /label=T7 /note="T7 promoter, forward primer" promoter 2492..2510 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 2533..2562 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 2563..3273 /codon_start=1 /product="yeast-enhanced green fluorescent protein" /label=yeGFP /note="codons optimized for translation in S. cerevisiae and C. albicans" /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITHGMDELYK" CDS 3280..3303 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS complement(3352..3657) /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 complement(3454..3473) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(3999..4658) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 4573..4592 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(4712..4742) /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind complement(4767..4891) /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS complement(4910..4930) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(4934..4954) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5008..5049 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 5059..5076 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" terminator 5110..5357 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(5126..5144) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(5434..5451) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5605..6193) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5685..5704) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6364..7224) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" CDS complement(7320..8123) /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(8124..8339) /gene="S. cerevisiae URA3" /label=URA3 promoter ORIGIN 1 ctagcttatc gatgataagc tgtcaaagat gagaattaat tccacggact atagactata 61 ctagatactc cgtctactgt acgatacact tccgctcagg tccttgtcct ttaacgaggc 121 cttaccactc ttttgttact ctattgatcc agctcagcaa aggcagtgtg atctaagatt 181 ctatcttcgc gatgtagtaa aactagctag accgagaaag agactagaaa tgcaaaaggc 241 acttctacaa tggctgccat cattattatc cgatgtgacg ctgcagcttc tcaatgatat 301 tcgaatacgc tttgaggaga tacagcctaa tatccgacaa actgttttac agatttacga 361 tcgtacttgt tacccatcat tgaattttga acatccgaac ctgggagttt tccctgaaac 421 agatagtata tttgaacctg tataataata tatagtctag cgctttacgg aagacaatgt 481 atgtatttcg gttcctggag aaactattgc atctattgca taggtaatct tgcacgtcgc 541 atccccggtt cattttctgc gtttccatct tgcacttcaa tagcatatct ttgttaacga 601 agcatctgtg cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac 661 aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca 721 acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca acgcgacgag agcgctaatt 781 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta 841 ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc 901 tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca 961 gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg 1021 tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc 1081 gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt 1141 ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa 1201 aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt 1261 ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga 1321 gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag 1381 cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag caaagagata 1441 cttttgagca atgtttgtgg aagcggtatt cgcaatggga agctccaccc cggttgataa 1501 tcagaaaagc cccaaaaaca ggaagattgt ataagcaaat atttaaattg taaacgttaa 1561 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta acgaatagcc 1621 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt 1681 tccagtttcc aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa 1741 aagggtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg 1801 gtcgaggtgc cgtaaagcag taaatcggaa gggtaaacgg atgcccccat ttagagcttg 1861 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggggc 1921 tagggcggtg ggaagtgtag gggtcacgct gggcgtaacc accacacccg ccgcgcttaa 1981 tggggcgcta cagggcgcgt ggggatgatc cactagtacg gattagaagc cgccgagcgg 2041 gtgacagccc tccgaaggaa gactctcctc cgtgcgtcct cgtcttcacc ggtcgcgttc 2101 ctgaaacgca gatgtgcctc gcgccgcact gctccgaaca ataaagattc tacaatacta 2161 gcttttatgg ttatgaagag gaaaaattgg cagtaacctg gccccacaaa ccttcaaatg 2221 aacgaatcaa attaacaacc ataggatgat aatgcgatta gttttttagc cttatttctg 2281 gggtaattaa tcagcgaagc gatgattttt gatctattaa cagatatata aatgcaaaaa 2341 ctgcataacc actttaacta atactttcaa cattttcggt ttgtattact tcttattcaa 2401 atgtaataaa agtatcaaca aaaaattgtt aatatacctc tatactttaa cgtcaaggag 2461 aaaaaacccc ggatcggact actagcagct gtaatacgac tcactatagg gaatattaag 2521 cttggtacca tgcaccatca tcatcaccat catcatcatc attctaaagg agaagaatta 2581 ttcactggtg ttgtcccaat tttggttgaa ttagatggtg atgttaatgg tcacaaattt 2641 tctgtctccg gtgaaggtga aggtgatgct acttacggta aattgacctt aaaatttatt 2701 tgtactactg gtaaattgcc agttccatgg ccaaccttag tcactacttt cggttatggt 2761 gttcaatgtt ttgcgagata cccagatcat atgaaacaac atgacttttt caagtctgcc 2821 atgccagaag gttatgttca agaaagaact atttttttca aagatgacgg taactacaag 2881 accagagctg aagtcaagtt tgaaggtgat accttagtta atagaatcga attaaaaggt 2941 attgatttta aagaagatgg taacatttta ggtcacaaat tggaatacaa ctataactct 3001 cacaatgttt acatcatggc tgacaaacaa aagaatggta tcaaagttaa cttcaaaatt 3061 agacacaaca ttgaagatgg ttctgttcaa ttagctgacc attatcaaca aaatactcca 3121 attggtgatg gtccagtctt gttaccagac aaccattact tatccactca atctgcctta 3181 tccaaagatc caaacgaaaa gagagaccac atggtcttgt tagaatttgt tactgctgct 3241 ggtattaccc atggtatgga tgaattgtac aaaactagtt tagaagtttt gtttcaaggt 3301 ccaagtagaa gagcgagctg cagactggct gtgtataagg gagcctgaca tttatattcc 3361 ccagaacatc aggttaatgg cgtttttgat gtcattttcg cggtggctga gatcagccac 3421 ttcttccccg ataacggaga ccggcacact ggccatatcg gtggtcatca tgcgccagct 3481 ttcatccccg atatgcacca ccgggtaaag ttcacgggag actttatctg acagcagacg 3541 tgcactggcc agggggatca ccatccgtcg cccgggcgtg tcaataatat cactctgtac 3601 atccacaaac agacgataac ggctctctct tttataggtg taaaccttaa actgcatttc 3661 accagcccct gttctcgtca gcaaaagagc cgttcatttc aataaaccgg gcgacctcag 3721 ccatcccttc ctgattttcc gctttccagc gttcggcacg cagacgacgg gcttcattct 3781 gcatggttgt gcttaccaga ccggagatat tgacatcata tatgccttga gcaactgata 3841 gctgtcgctg tcaactgtca ctgtaatacg ctgcttcata gcatacctct ttttgacata 3901 cttcgggtat acatatcagt atatattctt ataccgcaaa aatcagcgcg caaatacgca 3961 tactgttatc tggcttttag taagccggat ccacgcgttt acgccccgcc ctgccactca 4021 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca 4081 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc 4141 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg 4201 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa 4261 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg 4321 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg 4381 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg 4441 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg 4501 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta 4561 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat 4621 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa 4681 aatctcgacg gatcctaact caaaatccac acattatacg agccggaagc ataaagtgta 4741 aagcctgggg tgcctaatgc ggccgccata gtgactggat atgttgtgtt ttacagtatt 4801 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg 4861 tttctcgttc agcttttttg tacaaacttg tgatatcgaa ttcgctagct acctttctct 4921 tcttttttgg atctaccttt ctcttctttt ttggggatcc actagttcag ctcttctgca 4981 taataactat ctagagggcc cttcgaaggt aagcctatcc ctaaccctct cctcggtctc 5041 gattctacgc gtaccggtca tcatcaccat caccattgag tttaaacccg ctgatcctag 5101 agggccgcat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 5161 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 5221 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 5281 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 5341 cgaaggcttt aatttgcaag ctgcggccct gcattaatga atcggccaac gcgcggggag 5401 aggcggtttg cgtattgggc gctattccgc ttcctcgctc actgactcgc tgcgctcggt 5461 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 5521 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagc ccaggaaccg 5581 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 5641 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 5701 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 5761 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 5821 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 5881 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 5941 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 6001 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 6061 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 6121 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 6181 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 6241 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 6301 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 6361 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 6421 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagcgct taccatctgg 6481 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 6541 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 6601 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 6661 caacgttgtt ggcattgcta caggcatcgt ggtgtcactc tcgtcgtttg gtatggcttc 6721 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 6781 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 6841 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 6901 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 6961 ttgctcttgc ccggcgtcaa tacgggataa tagtgtatca catagcagaa ctttaaaagt 7021 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 7081 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 7141 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 7201 gacacggaaa tgttgaatac tcatactctt cctttttcaa tgggtaataa ctgatataat 7261 taaattgaag ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt 7321 tagttttgct ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca 7381 ccctctacct tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca 7441 gatcctgtag agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg 7501 tcatctaaac ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc 7561 atgtctcttt gagcaataaa gccgataaca aaatctttgt cgcttttcgc aatgtcaaca 7621 gtacccttag tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc 7681 aaaaggcctc taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata 7741 cctgggccca ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca 7801 cccgcagagt actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag 7861 agtaaaaaat tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa 7921 aaatcagtca agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca 7981 actaactcca gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc 8041 ttttcgtgca tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt 8101 tccttatatg tagctttcga catgatttat cttcgtttcc tgcaggtttt tgttctgtgc 8161 agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat 8221 accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca 8281 aaaaaatttc aaagaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaat 8341 tgaaaag //