LOCUS Exported 7549 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG303GAL-ccdB-Cerulean SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7549) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 7549) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7549 /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 315..502 /gene="S. cerevisiae HIS3" /label=HIS3 promoter CDS 503..1162 /codon_start=1 /gene="S. cerevisiae HIS3" /product="imidazoleglycerol-phosphate dehydratase, required for histidine biosynthesis" /label=HIS3 /note="yeast auxotrophic marker" /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM" rep_origin complement(1423..1878) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1560..1581) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1772..1791 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2008..2030 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2022..2039 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2023..2039 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 2046..2065 /label=T7 /note="T7 promoter, forward primer" promoter 2046..2064 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 2084..2525 /gene="S. cerevisiae GAL1" /label=GAL1 promoter /note="inducible promoter, regulated by Gal4" protein_bind 2084..2201 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" primer_bind 2496..2519 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" primer_bind 2540..2556 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 2540..2556 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 2564..2688 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 2713..2743 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 2797..3456 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(2863..2882) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 3801..4106 /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 3985..4004 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(4147..4271) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 4303..5019 /codon_start=1 /product="monomeric optimized variant of ECFP (Kremers et al., 2006)" /label=SCFP3A /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISDNVYITADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4348..4369) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4609..4628) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4956..4977 /label=EGFP-C /note="EGFP, forward primer" terminator 5029..5276 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(5045..5063) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(5299..5317) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5338..5354) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5338..5354) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5351..5373) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5362..5378 /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(5386..5416) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(5569..5586) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5740..6328) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6499..7359) /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 7122..7141 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7360..7464) /gene="bla" /label=AmpR promoter primer_bind 7522..7540 /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 accataattc cgttttaaga gcttggtgag cgctaggagt cactgccagg tatcgtttga 241 acacggcatt agtcagggaa gtcataacac agtcctttcc cgcaattttc tttttctatt 301 actcttggcc tcctctagta cactctatat ttttttatgc ctcggtaatg attttcattt 361 ttttttttcc acctagcgga tgactctttt tttttcttag cgattggcat tatcacataa 421 tgaattatac attatataaa gtaatgtgat ttcttcgaag aatatactaa aaaatgagca 481 ggcaagataa acgaaggcaa agatgacaga gcagaaagcc ctagtaaagc gtattacaaa 541 tgaaaccaag attcagattg cgatctcttt aaagggtggt cccctagcga tagagcactc 601 gatcttccca gaaaaagagg cagaagcagt agcagaacag gccacacaat cgcaagtgat 661 taacgtccac acaggtatag ggtttctgga ccatatgata catgctctgg ccaagcattc 721 cggctggtcg ctaatcgttg agtgcattgg tgacttacac atagacgacc atcacaccac 781 tgaagactgc gggattgctc tcggtcaagc ttttaaagag gccctactgg cgcgtggagt 841 aaaaaggttt ggatcaggat ttgcgccttt ggatgaggca ctttccagag cggtggtaga 901 tctttcgaac aggccgtacg cagttgtcga acttggtttg caaagggaga aagtaggaga 961 tctctcttgc gagatgatcc cgcattttct tgaaagcttt gcagaggcta gcagaattac 1021 cctccacgtt gattgtctgc gaggcaagaa tgatcatcac cgtagtgaga gtgcgttcaa 1081 ggctcttgcg gttgccataa gagaagccac ctcgcccaat ggtaccaacg atgttccctc 1141 caccaaaggt gttcttatgt agtgacaccg attatttaaa gctgcagcat acgatatata 1201 tacatgtgta tatatgtata cctatgaatg tcagtaagta tgtatacgaa cagtatgata 1261 ctgaagatga caaggtaatg catcattcta tacgtgtcat tctgaacgag gcgcgctttc 1321 cttttttctt tttgcttttt cttttttttt ctcttgaact cgacggatca tatgcggtgt 1381 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata 1441 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 1501 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 1561 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 1621 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 1681 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 1741 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 1801 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 1861 cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 1921 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa ggggggatgt gctgcaaggc 1981 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2041 aattgtaata cgactcacta tagggcgaat tggagctcta gtacggatta gaagccgccg 2101 agcgggcgac agccctccga cggaagactc tcctccgtgc gtcctcgtct tcaccggtcg 2161 cgttcctgaa acgcagatgt gcctcgcgcc gcactgctcc gaacaataaa gattctacaa 2221 tactagcttt tatggttatg aagaggaaaa attggcagta acctggcccc acaaaccttc 2281 aaattaacga atcaaattaa caaccatagg atgataatgc gattagtttt ttagccttat 2341 ttctggggta attaatcagc gaagcgatga tttttgatct attaacagat atataaatgg 2401 aaaagctgca taaccacttt aactaatact ttcaacattt tcagtttgta ttacttctta 2461 ttcaaatgtc ataaaagtat caacaaaaaa ttgttaatat acctctatac tttaacgtca 2521 aggagaaaaa accccggatt ctagaactag tggatccccc atcacaagtt tgtacaaaaa 2581 agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa 2641 aaaacagact acataatact gtaaaacaca acatatccag tcactatggc ggccgcatta 2701 ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag 2761 gatccgtcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat 2821 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt 2881 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta 2941 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat 3001 gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt 3061 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa 3121 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt 3181 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat 3241 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc 3301 cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg 3361 attcaggttc atcatgccgt ctgtgatggc ttccatgtcg gcagaatgct taatgaatta 3421 caacagtact gcgatgagtg gcagggcggg gcgtaaacgc cgcgtggatc cggcttacta 3481 aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag aatatatact 3541 gatatgtata cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt attacagtga 3601 cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata tctccggtct 3661 ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga 3721 aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct cttttgctga 3781 cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga gagagccgtt 3841 atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga 3901 tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg 3961 tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct 4021 ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca 4081 ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag 4141 gtcgaccata gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc 4201 aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc agctttcttg 4261 tacaaagtgg tgatgggctg caggaattcg atatcaagct taatggtgag caagggcgag 4321 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 4381 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag 4441 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc 4501 tggggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag 4561 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 4621 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 4681 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 4741 atcagcgaca acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc 4801 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 4861 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc 4921 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 4981 gccgccggga tcactctcgg catggacgag ctgtacaagt aactcgagtc atgtaattag 5041 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag 5101 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa 5161 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca 5221 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcggcc 5281 ggtacccagc ttttgttccc tttagtgagg gttaattccg agcttggcgt aatcatggtc 5341 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca taggagccgg 5401 aagcataaag tgtaaagcct ggggtgccta atgagtgagg taactcacat taattgcgtt 5461 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 5521 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 5581 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 5641 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5701 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tcggcccccc 5761 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 5821 aagataccag gcgttccccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 5881 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 5941 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 6001 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 6061 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 6121 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 6181 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 6241 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 6301 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 6361 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 6421 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 6481 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 6541 tctatttcgt tcatccatag ttgcctgact gcccgtcgtg tagataacta cgatacggga 6601 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 6661 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 6721 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 6781 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 6841 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 6901 catgttgtga aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 6961 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 7021 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 7081 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 7141 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 7201 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 7261 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 7321 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 7381 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 7441 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcttatt 7501 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc //