LOCUS Exported 7474 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG306GAL-ECFP-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7474) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 7474) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7474 /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 195..410 /gene="S. cerevisiae URA3" /label=URA3 promoter CDS 411..1214 /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" rep_origin complement(1343..1798) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1480..1501) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1692..1711 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1928..1950 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 1942..1959 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 1943..1959 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 1966..1985 /label=T7 /note="T7 promoter, forward primer" promoter 1966..1984 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 2004..2445 /gene="S. cerevisiae GAL1" /label=GAL1 promoter /note="inducible promoter, regulated by Gal4" protein_bind 2004..2121 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" primer_bind 2416..2439 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" primer_bind 2460..2476 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 2460..2476 /label=pBluescriptSK /note="For pBluescript vector" CDS 2489..3205 /codon_start=1 /product="enhanced CFP" /label=ECFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2534..2555) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2795..2814) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3142..3163 /label=EGFP-C /note="EGFP, forward primer" protein_bind 3230..3354 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 3379..3409 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 3463..4122 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(3529..3548) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 4467..4772 /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 4651..4670 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(4813..4937) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" terminator 4944..5191 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(4960..4978) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(5214..5232) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5253..5269) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5253..5269) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5266..5288) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5277..5293 /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(5301..5331) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(5484..5501) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5655..6243) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6414..7274) /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 7037..7056 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7275..7379) /gene="bla" /label=AmpR promoter primer_bind 7447..7465 /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 accacgcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc 241 tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca 301 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat 361 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag 421 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata 481 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat 541 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata 601 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt 661 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc 721 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg 781 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac 841 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat 901 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg 961 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg 1021 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg 1081 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg 1141 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg 1201 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta 1261 gagcttcaat ttaattatat cagttattac cctgcggtgt gaaataccgc acagatgcgt 1321 aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa attcgcgtta 1381 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1441 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1501 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1561 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 1621 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 1681 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 1741 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg 1801 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 1861 ctattacgcc agctggcgaa ggggggatgt gctgcaaggc gattaagttg ggtaacgcca 1921 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta 1981 tagggcgaat tggagctcta gtacggatta gaagccgccg agcgggcgac agccctccga 2041 cggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa acgcagatgt 2101 gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt tatggttatg 2161 aagaggaaaa attggcagta acctggcccc acaaaccttc aaattaacga atcaaattaa 2221 caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta attaatcagc 2281 gaagcgatga tttttgatct attaacagat atataaatgg aaaagctgca taaccacttt 2341 aactaatact ttcaacattt tcagtttgta ttacttctta ttcaaatgtc ataaaagtat 2401 caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa accccggatt 2461 ctagaactag tggatccccc gggacaaaat ggtgagcaag ggcgaggagc tgttcaccgg 2521 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 2581 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 2641 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg 2701 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 2761 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 2821 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 2881 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacatca gccacaacgt 2941 ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa 3001 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 3061 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga 3121 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 3181 tctcggcatg gacgagctgt acaagggtgg accaggtggt ggacatcaaa caagtttgta 3241 caaaaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt 3301 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc 3361 gcattaggca ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg 3421 agttaggatc cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact 3481 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag 3541 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag 3601 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg 3661 atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat 3721 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg 3781 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt 3841 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca 3901 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc 3961 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg 4021 ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaat 4081 gaattacaac agtactgcga tgagtggcag ggcggggcgt aaacgccgcg tggatccggc 4141 ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg tataagaata 4201 tatactgata tgtatacccg aagtatgtca aaaagaggta tgctatgaag cagcgtatta 4261 cagtgacagt tgacagcgac agctatcagt tgctcaaggc atatatgatg tcaatatctc 4321 cggtctggta agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa 4381 agcggaaaat caggaaggga tggctgaggt cgcccggttt attgaaatga acggctcttt 4441 tgctgacgag aacaggggct ggtgaaatgc agtttaaggt ttacacctat aaaagagaga 4501 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga 4561 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc 4621 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc 4681 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa 4741 acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata cacagccagt 4801 ctgcaggtcg accatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt 4861 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct 4921 ttcttgtaca aagtggttcg atgtcatgta attagttatg tcacgcttac attcacgccc 4981 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc 5041 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct 5101 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa 5161 ggttttggga cgctcgaagg ctttaatttg cggccggtac ccagcttttg ttccctttag 5221 tgagggttaa ttccgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 5281 tatccgctca caattccaca caacatagga gccggaagca taaagtgtaa agcctggggt 5341 gcctaatgag tgaggtaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 5401 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 5461 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 5521 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 5581 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 5641 gcgttgctgg cgtttttcca taggctcggc ccccctgacg agcatcacaa aaatcgacgc 5701 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt cccccctgga 5761 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 5821 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg 5881 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 5941 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 6001 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 6061 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 6121 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 6181 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 6241 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 6301 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 6361 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 6421 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 6481 tgactgcccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 6541 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 6601 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 6661 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 6721 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 6781 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgaaaaaa agcggttagc 6841 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 6901 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 6961 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 7021 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 7081 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 7141 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 7201 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 7261 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 7321 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 7381 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc 7441 tataaaaata ggcgtatcac gaggcccttt cgtc //