LOCUS Exported 6764 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG304GAL-ccdB-HA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6764) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 6764) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6764 /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" CDS 4126..4215 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" terminator 4234..4481 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(4250..4268) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(4504..4522) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4543..4559) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4543..4559) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4556..4578) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4567..4583 /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(4591..4621) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(4774..4791) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4945..5533) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5704..6564) /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 6327..6346 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6565..6669) /gene="bla" /label=AmpR promoter primer_bind 6737..6755 /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 agcttaggtg gaatgtaccc atacgatgtt 4141 cctgactatg cgggctatcc ctatgacgtc ccggactatg caggatccta tccatatgac 4201 gttccagatt acgctgctca gtgctaactc gagtcatgta attagttatg tcacgcttac 4261 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag 4321 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc 4381 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct 4441 tgcttgagaa ggttttggga cgctcgaagg ctttaatttg cggccggtac ccagcttttg 4501 ttccctttag tgagggttaa ttccgagctt ggcgtaatca tggtcatagc tgtttcctgt 4561 gtgaaattgt tatccgctca caattccaca caacatagga gccggaagca taaagtgtaa 4621 agcctggggt gcctaatgag tgaggtaact cacattaatt gcgttgcgct cactgcccgc 4681 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 4741 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 4801 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4861 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4921 taaaaaggcc gcgttgctgg cgtttttcca taggctcggc ccccctgacg agcatcacaa 4981 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 5041 cccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 5101 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct 5161 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 5221 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 5281 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 5341 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 5401 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 5461 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 5521 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 5581 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 5641 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 5701 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 5761 catagttgcc tgactgcccg tcgtgtagat aactacgata cgggagggct taccatctgg 5821 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5881 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5941 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 6001 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 6061 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgaaaaaa 6121 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 6181 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 6241 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 6301 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 6361 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 6421 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 6481 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 6541 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 6601 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 6661 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 6721 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc //