LOCUS Exported 8619 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG305GAL-ccdB-ECFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8619) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 8619) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8619 /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" protein_bind 3615..3739 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 3764..3794 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 3848..4507 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(3914..3933) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 4852..5157 /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 5036..5055 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(5198..5322) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 5354..6070 /codon_start=1 /product="enhanced CFP" /label=ECFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(5399..5420) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5660..5679) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 6007..6028 /label=EGFP-C /note="EGFP, forward primer" terminator 6089..6336 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(6105..6123) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(6359..6377) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(6398..6414) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6398..6414) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6411..6433) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6422..6438 /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(6446..6476) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(6629..6646) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6800..7388) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7559..8419) /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 8182..8201 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8420..8524) /gene="bla" /label=AmpR promoter primer_bind 8592..8610 /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 catcacaagt ttgtacaaaa aagctgaacg agaaacgtaa aatgatataa 3661 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac 3721 aacatatcca gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc 3781 cggctcgtat aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga 3841 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg 3901 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca 3961 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc 4021 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa 4081 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca 4141 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca 4201 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt 4261 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt 4321 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac 4381 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tctgtgatgg 4441 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg 4501 ggcgtaaacg ccgcgtggat ccggcttact aaaagccaga taacagtatg cgtatttgcg 4561 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag 4621 aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc 4681 aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc 4741 gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc 4801 ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt 4861 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat 4921 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca 4981 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg 5041 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc 5101 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg 5161 tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga tatgttgtgt 5221 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat 5281 atcattttac gtttctcgtt cagctttctt gtacaaagtg gtgatgggct gcaggaattc 5341 gatatcaagc ttaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 5401 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 5461 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 5521 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc 5581 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 5641 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 5701 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 5761 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga 5821 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag 5881 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 5941 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 6001 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 6061 gctgtacaag ctgtacaagt aactcgagtc atgtaattag ttatgtcacg cttacattca 6121 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag 6181 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt 6241 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt 6301 gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccagc ttttgttccc 6361 tttagtgagg gttaattccg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 6421 attgttatcc gctcacaatt ccacacaaca taggagccgg aagcataaag tgtaaagcct 6481 ggggtgccta atgagtgagg taactcacat taattgcgtt gcgctcactg cccgctttcc 6541 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 6601 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 6661 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 6721 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 6781 aggccgcgtt gctggcgttt ttccataggc tcggcccccc tgacgagcat cacaaaaatc 6841 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgttccccc 6901 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 6961 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt 7021 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 7081 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 7141 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 7201 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 7261 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 7321 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 7381 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 7441 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 7501 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 7561 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 7621 ttgcctgact gcccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 7681 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 7741 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 7801 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 7861 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 7921 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtga aaaaaagcgg 7981 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 8041 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 8101 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 8161 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 8221 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 8281 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 8341 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 8401 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 8461 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 8521 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat 8581 taacctataa aaataggcgt atcacgaggc cctttcgtc //