LOCUS Exported 6667 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG304GAL-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6667) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 6667) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6667 /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 185..465 /gene="S. cerevisiae TRP1" /label=TRP1 promoter CDS 466..1140 /codon_start=1 /gene="S. cerevisiae TRP1" /product="phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis" /label=TRP1 /note="yeast auxotrophic marker" /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA KK" rep_origin complement(1237..1692) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1374..1395) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1586..1605 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1822..1844 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 1836..1853 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 1837..1853 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 1860..1879 /label=T7 /note="T7 promoter, forward primer" promoter 1860..1878 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 1898..2339 /gene="S. cerevisiae GAL1" /label=GAL1 promoter /note="inducible promoter, regulated by Gal4" protein_bind 1898..2015 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" primer_bind 2310..2333 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" primer_bind 2354..2370 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 2354..2370 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 2378..2502 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 2527..2557 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 2611..3270 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(2677..2696) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 3615..3920 /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 3799..3818 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(3961..4085) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(4118..4134) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4119..4135) /label=pBluescriptKS /note="For pBluescript vector" terminator 4137..4384 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(4153..4171) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(4407..4425) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4446..4462) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4446..4462) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4459..4481) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4470..4486 /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(4494..4524) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(4677..4694) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4848..5436) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5607..6467) /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 6230..6249 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6468..6572) /gene="bla" /label=AmpR promoter primer_bind 6640..6658 /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 accaaacgac attactatat atataatata ggaagcattt aatagacagc atcgtaatat 241 atgtgtactt tgcagttatg acgccagatg gcagtagtgg aagatattct ttattgaaaa 301 atagcttgtc accttacgta caatcttgat ccggagcttt tctttttttg ccgattaaga 361 attaattcgg tcgaaaaaag aaaaggagag ggccaagagg gagggcattg gtgactattg 421 agcacgtgag tatacgtgat taagcacaca aaggcagctt ggagtatgtc tgttattaat 481 ttcacaggta gttctggtcc attggtgaaa gtttgcggct tgcagagcac agaggccgca 541 gaatgtgctc tagattccga tgctgacttg ctgggtatta tatgtgtgcc caatagaaag 601 agaacaattg acccggttat tgcaaggaaa atttcaagtc ttgtaaaagc atataaaaat 661 agttcaggca ctccgaaata cttggttggc gtgtttcgta atcaacctaa ggaggatgtt 721 ttggctctgg tcaatgatta cggcattgat atcgtccaac tgcatggaga tgagtcgtgg 781 caagaatacc aagagttcct cggtttgcca gttattaaaa gactcgtatt tccaaaagac 841 tgcaacatac tactcagtgc agcttcacag aaacctcatt cgtttattcc cttgtttgat 901 tcagaagcag gtgggacagg tgaacttttg gattggaact cgatttctga ctgggttgga 961 aggcaagaga gccccgaaag cttacatttt atgttagctg gtggactgac gccagaaaat 1021 gttggtgatg cgcttagatt aaatggcgtt attggtgttg atgtaagcgg aggtgtggag 1081 acaaatggtg taaaagactc taacaaaata gcaaatttcg tcaaaaatgc taagaaatag 1141 gttattactg agtagtattt atttaagtat tgtttgtgca cttgcctgcg gtgtgaaata 1201 ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaaacgtt aatattttgt 1261 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg 1321 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt 1381 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct 1441 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt 1501 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa 1561 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc 1621 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc 1681 tacagggcgc gtcgcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 1741 tgcgggcctc ttcgctatta cgccagctgg cgaagggggg atgtgctgca aggcgattaa 1801 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt 1861 aatacgactc actatagggc gaattggagc tctagtacgg attagaagcc gccgagcggg 1921 cgacagccct ccgacggaag actctcctcc gtgcgtcctc gtcttcaccg gtcgcgttcc 1981 tgaaacgcag atgtgcctcg cgccgcactg ctccgaacaa taaagattct acaatactag 2041 cttttatggt tatgaagagg aaaaattggc agtaacctgg ccccacaaac cttcaaatta 2101 acgaatcaaa ttaacaacca taggatgata atgcgattag ttttttagcc ttatttctgg 2161 ggtaattaat cagcgaagcg atgatttttg atctattaac agatatataa atggaaaagc 2221 tgcataacca ctttaactaa tactttcaac attttcagtt tgtattactt cttattcaaa 2281 tgtcataaaa gtatcaacaa aaaattgtta atatacctct atactttaac gtcaaggaga 2341 aaaaaccccg gattctagaa ctagtggatc ccccatcaca agtttgtaca aaaaagctga 2401 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca 2461 gactacataa tactgtaaaa cacaacatat ccagtcacta tggcggccgc attaggcacc 2521 ccaggcttta cactttatgc ttccggctcg tataatgtgt ggattttgag ttaggatccg 2581 tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc 2641 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa 2701 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa 2761 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat 2821 ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct 2881 tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac 2941 gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac 3001 ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg 3061 gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt 3121 ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag 3181 gttcatcatg ccgtctgtga tggcttccat gtcggcagaa tgcttaatga attacaacag 3241 tactgcgatg agtggcaggg cggggcgtaa acgccgcgtg gatccggctt actaaaagcc 3301 agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg 3361 tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca gtgacagttg 3421 acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag 3481 cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca 3541 ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa 3601 caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc 3661 tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc 3721 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata 3781 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta 3841 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc 3901 tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgac 3961 catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc 4021 taatttaata tattgatatt tatatcattt tacgtttctc gttcagcttt cttgtacaaa 4081 gtggtgatgg gctgcaggaa ttcgatatca agcttatcga taccgtcgac ctcgagtcat 4141 gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa 4201 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt 4261 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc 4321 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat 4381 ttgcggccgg tacccagctt ttgttccctt tagtgagggt taattccgag cttggcgtaa 4441 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 4501 ggagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgaggta actcacatta 4561 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 4621 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 4681 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 4741 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4801 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 4861 ggcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 4921 ggactataaa gataccaggc gttcccccct ggaagctccc tcgtgcgctc tcctgttccg 4981 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 5041 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 5101 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 5161 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 5221 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 5281 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 5341 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 5401 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 5461 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 5521 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 5581 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 5641 gcgatctgtc tatttcgttc atccatagtt gcctgactgc ccgtcgtgta gataactacg 5701 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 5761 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 5821 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 5881 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 5941 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 6001 tgatccccca tgttgtgaaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 6061 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 6121 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 6181 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 6241 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 6301 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 6361 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 6421 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 6481 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 6541 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 6601 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 6661 tttcgtc //