LOCUS Exported 6866 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG304GPD-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6866) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 6866) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6866 /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 1903..2547 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 2441..2464 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" primer_bind 2553..2569 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 2553..2569 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 2577..2701 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 2726..2756 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 2810..3469 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(2876..2895) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 3814..4119 /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 3998..4017 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(4160..4284) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(4317..4333) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4318..4334) /label=pBluescriptKS /note="For pBluescript vector" terminator 4336..4583 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(4352..4370) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(4606..4624) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4645..4661) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4645..4661) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4658..4680) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4669..4685 /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(4693..4723) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(4876..4893) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5047..5635) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5806..6666) /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 6429..6448 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6667..6771) /gene="bla" /label=AmpR promoter primer_bind 6839..6857 /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 tcttcgagtt tatcattatc aatactcgcc 1921 atttcaaaga atacgtaaat aattaatagt agtgattttc ctaactttat ttagtcaaaa 1981 aattagcctt ttaattctgc tgtaacccgt acatgcccaa aatagggggc gggttacaca 2041 gaatatataa caccgaaggt gcctgggtga acaggtttat tcctggcatc cactaaatat 2101 aatggagccc gctttttaag ctggcatcca gaaaaaaaaa gaatcccagc accaaaatat 2161 tgttttcttc accaaccatc agttcatagg tccattctct tagcgcaact acagagaaca 2221 ggggcacaaa caggcaaaaa acgggcacaa cctcaatgga gtgatgcaac ctgcctggag 2281 taaatgatga cacaaggcaa ttgacccacg catgtatcta tctcattttc ttacaccttc 2341 tattaccttc tgctctctct gatttggaaa aagctgaaaa aaaaggttga aaccagttcc 2401 ctgaaattat tcccctactt gactaataag tatataaaga cggtaggtat tgattgtaat 2461 tctgtaaatc tatttcttaa acttcttaaa ttctactttt atagttagtc ttttttttag 2521 ttttaaaaca ccagaactta gtttcgacgg attctagaac tagtggatcc cccatcacaa 2581 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt 2641 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat 2701 ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg 2761 gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa 2821 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc 2881 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt 2941 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc 3001 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat 3061 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc 3121 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt 3181 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt 3241 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga 3301 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct 3361 gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg tcggcagaat 3421 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa cgccgcgtgg 3481 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat 3541 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag 3601 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca 3661 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac 3721 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg 3781 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa 3841 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg 3901 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa 3961 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc 4021 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac 4081 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac 4141 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt 4201 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg 4261 ttcagctttc ttgtacaaag tggtgatggg ctgcaggaat tcgatatcaa gcttatcgat 4321 accgtcgacc tcgagtcatg taattagtta tgtcacgctt acattcacgc cctcccccca 4381 catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt 4441 tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc 4501 tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg 4561 gacgctcgaa ggctttaatt tgcggccggt acccagcttt tgttcccttt agtgagggtt 4621 aattccgagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 4681 cacaattcca cacaacatag gagccggaag cataaagtgt aaagcctggg gtgcctaatg 4741 agtgaggtaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 4801 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 4861 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 4921 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 4981 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 5041 ggcgtttttc cataggctcg gcccccctga cgagcatcac aaaaatcgac gctcaagtca 5101 gaggtggcga aacccgacag gactataaag ataccaggcg ttcccccctg gaagctccct 5161 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 5221 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 5281 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5341 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5401 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5461 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 5521 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 5581 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 5641 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 5701 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5761 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 5821 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactgcc 5881 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 5941 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 6001 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 6061 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 6121 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 6181 acgatcaagg cgagttacat gatcccccat gttgtgaaaa aaagcggtta gctccttcgg 6241 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 6301 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 6361 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 6421 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 6481 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 6541 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 6601 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 6661 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 6721 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6781 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 6841 taggcgtatc acgaggccct ttcgtc //