LOCUS Exported 7479 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG306GAL-ccdB-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7479) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 7479) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7479 /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" promoter 195..410 /gene="S. cerevisiae URA3" /label=URA3 promoter CDS 411..1214 /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" rep_origin complement(1343..1798) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1480..1501) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1692..1711 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1928..1950 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 1942..1959 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 1943..1959 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 1966..1985 /label=T7 /note="T7 promoter, forward primer" promoter 1966..1984 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 2004..2445 /gene="S. cerevisiae GAL1" /label=GAL1 promoter /note="inducible promoter, regulated by Gal4" protein_bind 2004..2121 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" primer_bind 2416..2439 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" primer_bind 2460..2476 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 2460..2476 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 2484..2608 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 2633..2663 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 2717..3376 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(2783..2802) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 3721..4026 /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 3905..3924 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(4067..4191) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 4223..4942 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 4223..4939 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4268..4289) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4529..4548) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4876..4897 /label=EGFP-C /note="EGFP, forward primer" terminator 4949..5196 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(4965..4983) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(5219..5237) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5258..5274) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5258..5274) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5271..5293) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5282..5298 /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(5306..5336) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(5489..5506) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5660..6248) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6419..7279) /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 7042..7061 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7280..7384) /gene="bla" /label=AmpR promoter primer_bind 7452..7470 /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 accacgcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc 241 tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca 301 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat 361 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag 421 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata 481 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat 541 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata 601 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt 661 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc 721 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg 781 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac 841 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat 901 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg 961 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg 1021 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg 1081 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg 1141 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg 1201 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta 1261 gagcttcaat ttaattatat cagttattac cctgcggtgt gaaataccgc acagatgcgt 1321 aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa attcgcgtta 1381 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1441 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1501 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1561 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 1621 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 1681 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 1741 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg 1801 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 1861 ctattacgcc agctggcgaa ggggggatgt gctgcaaggc gattaagttg ggtaacgcca 1921 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta 1981 tagggcgaat tggagctcta gtacggatta gaagccgccg agcgggcgac agccctccga 2041 cggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa acgcagatgt 2101 gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt tatggttatg 2161 aagaggaaaa attggcagta acctggcccc acaaaccttc aaattaacga atcaaattaa 2221 caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta attaatcagc 2281 gaagcgatga tttttgatct attaacagat atataaatgg aaaagctgca taaccacttt 2341 aactaatact ttcaacattt tcagtttgta ttacttctta ttcaaatgtc ataaaagtat 2401 caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa accccggatt 2461 ctagaactag tggatccccc atcacaagtt tgtacaaaaa agctgaacga gaaacgtaaa 2521 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact 2581 gtaaaacaca acatatccag tcactatggc ggccgcatta ggcaccccag gctttacact 2641 ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccgtcga gattttcagg 2701 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca 2761 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca 2821 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt 2881 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat 2941 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt 3001 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca 3061 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc 3121 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag 3181 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa 3241 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt 3301 ctgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg 3361 gcagggcggg gcgtaaacgc cgcgtggatc cggcttacta aaagccagat aacagtatgc 3421 gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat 3481 gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat 3541 cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga 3601 atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg 3661 aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa 3721 atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta 3781 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt 3841 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc 3901 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg 3961 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga 4021 atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata gtgactggat 4081 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt 4141 gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg tgatgggctg 4201 caggaattcg atatcaagct taatggtgag caagggcgag gagctgttca ccggggtggt 4261 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga 4321 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa 4381 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag 4441 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta 4501 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt 4561 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga 4621 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat 4681 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga 4741 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc 4801 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa 4861 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg 4921 catggacgag ctgtacaagt aactcgagtc atgtaattag ttatgtcacg cttacattca 4981 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag 5041 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt 5101 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt 5161 gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccagc ttttgttccc 5221 tttagtgagg gttaattccg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 5281 attgttatcc gctcacaatt ccacacaaca taggagccgg aagcataaag tgtaaagcct 5341 ggggtgccta atgagtgagg taactcacat taattgcgtt gcgctcactg cccgctttcc 5401 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 5461 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 5521 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 5581 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 5641 aggccgcgtt gctggcgttt ttccataggc tcggcccccc tgacgagcat cacaaaaatc 5701 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgttccccc 5761 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 5821 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt 5881 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 5941 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6001 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6061 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 6121 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6181 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 6241 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 6301 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 6361 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 6421 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 6481 ttgcctgact gcccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 6541 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 6601 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 6661 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 6721 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 6781 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtga aaaaaagcgg 6841 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 6901 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 6961 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 7021 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 7081 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 7141 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 7201 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 7261 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7321 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 7381 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat 7441 taacctataa aaataggcgt atcacgaggc cctttcgtc //