LOCUS Exported 8605 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG305GAL-EYFP-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8605) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 8605) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8605 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" CDS complement(663..1757) /codon_start=1 /gene="S. cerevisiae LEU2" /product="3-isopropylmalate dehydrogenase, required for leucine biosynthesis" /label=LEU2 /note="yeast auxotrophic marker" /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG DLGGSNSTTEVGDAVAEEVKKILA" promoter complement(1770..2174) /gene="S. cerevisiae LEU2" /label=LEU2 promoter rep_origin complement(2474..2929) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2611..2632) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2823..2842 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3059..3081 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3073..3090 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3074..3090 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 3097..3116 /label=T7 /note="T7 promoter, forward primer" promoter 3097..3115 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 3135..3576 /gene="S. cerevisiae GAL1" /label=GAL1 promoter /note="inducible promoter, regulated by Gal4" protein_bind 3135..3252 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" primer_bind 3547..3570 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" primer_bind 3591..3607 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 3591..3607 /label=pBluescriptSK /note="For pBluescript vector" CDS 3620..4336 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3665..3686) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3926..3945) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4273..4294 /label=EGFP-C /note="EGFP, forward primer" protein_bind 4361..4485 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 4510..4540 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 4594..5253 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(4660..4679) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 5598..5903 /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 5782..5801 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(5944..6068) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" terminator 6075..6322 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(6091..6109) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(6345..6363) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(6384..6400) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6384..6400) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6397..6419) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6408..6424 /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)." promoter complement(6432..6462) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(6615..6632) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6786..7374) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7545..8405) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 8168..8187 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8406..8510) /gene="bla" /label=AmpR promoter primer_bind 8578..8596 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc 241 accattatgg gaaatggttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca 301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat 361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc 421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc 481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt 541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg 601 tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct 661 ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg 721 ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct 781 tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac 841 aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat 901 ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc 961 aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg 1021 ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca 1081 gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc 1141 acagtttttc tccataatct tgaagaggcc aaaacattag ctttatccaa ggaccaaata 1201 ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact 1261 tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc 1321 ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca 1381 aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt 1441 aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca 1501 ctaccggtac cccatttagg accacccaca gcacctaaca aaacggcatc aaccttcttg 1561 gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca 1621 attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga 1681 accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc 1741 ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata 1801 tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat 1861 tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat 1921 ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct 1981 ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca 2041 aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat 2101 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga 2161 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg 2221 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt 2281 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg 2341 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta 2401 gaagttctcc tcgagggtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa 2461 ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt 2521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 2581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 2641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 2701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 2761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 2821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 2881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg 2941 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 3001 cagctggcga aggggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 3061 cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa 3121 ttggagctct agtacggatt agaagccgcc gagcgggcga cagccctccg acggaagact 3181 ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc 3241 cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa 3301 aattggcagt aacctggccc cacaaacctt caaattaacg aatcaaatta acaaccatag 3361 gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg 3421 atttttgatc tattaacaga tatataaatg gaaaagctgc ataaccactt taactaatac 3481 tttcaacatt ttcagtttgt attacttctt attcaaatgt cataaaagta tcaacaaaaa 3541 attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat tctagaacta 3601 gtggatcccc cgggacaaaa tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc 3661 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg 3721 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct 3781 gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg 3841 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt 3901 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa 3961 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga 4021 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat 4081 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga 4141 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt 4201 gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga 4261 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat 4321 ggacgagctg tacaagggtg gaccaggtgg tggacatcaa acaagtttgt acaaaaaagc 4381 tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa 4441 acagactaca taatactgta aaacacaaca tatccagtca ctatggcggc cgcattaggc 4501 accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat 4561 ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc 4621 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct 4681 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag 4741 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct 4801 catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac 4861 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac 4921 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa 4981 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc 5041 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc 5101 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt 5161 caggttcatc atgccgtctg tgatggcttc catgtcggca gaatgcttaa tgaattacaa 5221 cagtactgcg atgagtggca gggcggggcg taaacgccgc gtggatccgg cttactaaaa 5281 gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat atatactgat 5341 atgtataccc gaagtatgtc aaaaagaggt atgctatgaa gcagcgtatt acagtgacag 5401 ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt 5461 aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa 5521 tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt ttgctgacga 5581 gaacaggggc tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc 5641 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc 5701 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc 5761 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg 5821 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta 5881 acctgatgtt ctggggaata taaatgtcag gctcccttat acacagccag tctgcaggtc 5941 gaccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa 6001 atctaattta atatattgat atttatatca ttttacgttt ctcgttcagc tttcttgtac 6061 aaagtggttc gatgtcatgt aattagttat gtcacgctta cattcacgcc ctccccccac 6121 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 6181 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 6241 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 6301 acgctcgaag gctttaattt gcggccggta cccagctttt gttcccttta gtgagggtta 6361 attccgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 6421 acaattccac acaacatagg agccggaagc ataaagtgta aagcctgggg tgcctaatga 6481 gtgaggtaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 6541 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 6601 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 6661 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 6721 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 6781 gcgtttttcc ataggctcgg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 6841 aggtggcgaa acccgacagg actataaaga taccaggcgt tcccccctgg aagctccctc 6901 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 6961 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 7021 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 7081 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 7141 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 7201 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 7261 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 7321 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 7381 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 7441 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 7501 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 7561 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactgccc 7621 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 7681 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 7741 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 7801 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 7861 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 7921 cgatcaaggc gagttacatg atcccccatg ttgtgaaaaa aagcggttag ctccttcggt 7981 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 8041 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 8101 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 8161 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 8221 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 8281 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 8341 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 8401 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 8461 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 8521 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 8581 aggcgtatca cgaggccctt tcgtc //