LOCUS Exported 8306 bp ds-DNA circular SYN 14-FEB-2022 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG426GPD-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8306) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 8306) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 14, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8306 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 246..293 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." promoter 1283..1387 /gene="bla" /label=AmpR promoter CDS 1388..2248 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1606..1625) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2419..3007 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2908..2927 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3161..3178 /label=L4440 /note="L4440 vector, forward primer" protein_bind 3295..3316 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3331..3361 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3369..3385 /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)." primer_bind 3374..3396 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3393..3409 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3393..3409 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 3428..3448 /label=T3 /note="T3 promoter, forward primer" promoter 3430..3448 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 3473..4116 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 4010..4033 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" primer_bind 4122..4138 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 4122..4138 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 4146..4270 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 4295..4325 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 4379..5038 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(4445..4464) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 5380..5685 /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 5564..5583 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(5726..5850) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(5883..5899) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5884..5900) /label=pBluescriptKS /note="For pBluescript vector" terminator 5902..6149 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(5918..5936) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(6167..6186) /label=T7 /note="T7 promoter, forward primer" promoter complement(6168..6186) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6196..6213) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6196..6212) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6205..6227) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 6353..6808 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6440..6459) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6650..6671 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6939..7742) /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" promoter complement(7743..7958) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(7982..8001) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8101..8123 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(8161..8179) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" rep_origin 8220..1256 /label=2u ori /note="yeast 2u plasmid origin of replication" ORIGIN 1 gctgaaggaa gcatacgata ccccgcatgg aatgggataa tatcacagga ggtactagac 61 tacctttcat cctacataaa tagacgcata taagtacgca tttaagcata aacacgcact 121 atgccgttct tctcatgtat atatatatac aggcaacacg cagatatagg tgcgacgtga 181 acagtgagct gtatgtgcgc agctcgcgtt gcattttcgg aagcgctcgt tttcggaaac 241 gctttgaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttcagagcgc 301 ttttgaaaac caaaagcgct ctgaagacgc actttcaaaa aaccaaaaac gcaccggact 361 gtaacgagct actaaaatat tgcgaatacc gcttccacaa acattgctca aaagtatctc 421 tttgctatat atctctgtgc tatatcccta tataacctac ccatccacct ttcgctcctt 481 gaacttgcat ctaaactcga cctctacatt ttttatgttt atctctagta ttactcttta 541 gacaaaaaaa ttgtagtaag aactattcat agagtgaatc gaaaacaata cgaaaatgta 601 aacatttcct atacgtagta tatagagaca aaatagaaga aaccgttcat aattttctga 661 ccaatgaaga atcatcaacg ctatcacttt ctgttcacaa agtatgcgca atccacatcg 721 gtatagaata taatcgggga tgcctttatc ttgaaaaaat gcacccgcag cttcgctagt 781 aatcagtaaa cgcgggaagt ggagtcaggc tttttttatg gaagagaaaa tagacaccaa 841 agtagccttc ttctaacctt aacggaccta cagtgcaaaa agttatcaag agactgcatt 901 atagagcgca caaaggagaa aaaaagtaat ctaagatgct ttgttagaaa aatagcgctc 961 tcgggatgca tttttgtaga acaaaaaaga agtatagatt ctttgttggt aaaatagcgc 1021 tctcgcgttg catttctgtt ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag 1081 cgctctcgcg ttgcattttt gttttacaaa aatgaagcac agattcttcg ttggtaaaat 1141 agcgctttcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa 1201 attagcgctc tcgcgttgca tttttgttct acaaaatgaa gcacagatgc ttcgttcagg 1261 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 1321 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 1381 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 1441 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 1501 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 1561 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 1621 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 1681 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 1741 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 1801 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 1861 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 1921 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 1981 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 2041 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 2101 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 2161 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 2221 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 2281 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 2341 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 2401 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 2461 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 2521 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc 2581 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 2641 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 2701 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 2761 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 2821 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 2881 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 2941 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 3001 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 3061 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 3121 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 3181 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 3241 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt 3301 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 3361 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc 3421 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctcagtt tatcattatc 3481 aatactcgcc atttcaaaga atacgtaaat aattaatagt agtgattttc ctaactttat 3541 ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt acatgcccaa aatagggggc 3601 gggttacaca gaatatataa catcgtaggt gtctgggtga acagtttatt cctggcatcc 3661 actaaatata atggagcccg ctttttaagc tggcatccag aaaaaaaaag aatcccagca 3721 ccaaaatatt gttttcttca ccaaccatca gttcataggt ccattctctt agcgcaacta 3781 cagagaacag gggcacaaac aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc 3841 tgcctggagt aaatgatgac acaaggcaat tgacccacgc atgtatctat ctcattttct 3901 tacaccttct attaccttct gctctctctg atttggaaaa agctgaaaaa aaaggttgaa 3961 accagttccc tgaaattatt cccctacttg actaataagt atataaagac ggtaggtatt 4021 gattgtaatt ctgtaaatct atttcttaaa cttcttaaat tctactttta tagttagtct 4081 tttttttagt tttaaaacac cagaacttag tttcgacgga ttctagaact agtggatccc 4141 ccatcacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata aatatcaata 4201 tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca caacatatcc 4261 agtcactatg gcggccgcat taggcacccc aggctttaca ctttatgctt ccggctcgta 4321 taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg aagctaaaat 4381 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca 4441 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat 4501 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca 4561 cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga aagacggtga 4621 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac 4681 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc 4741 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa 4801 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc 4861 caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga 4921 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt 4981 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaaac 5041 gcgtggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg ctgatttttg 5101 cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag gtatgctatg 5161 aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa ggcatatatg 5221 atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg 5281 ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg tttattgaaa 5341 tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc 5401 tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat tattgacacg 5461 cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc 5521 cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat 5581 atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa 5641 aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc aggctccctt 5701 atacacagcc agtctgcagg tcgaccatag tgactggata tgttgtgttt tacagtatta 5761 tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat cattttacgt 5821 ttctcgttca gctttcttgt acaaagtggt gatgggctgc aggaattcga tatcaagctt 5881 atcgataccg tcgacctcga gtcatgtaat tagttatgtc acgcttacat tcacgccctc 5941 cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta 6001 tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 6061 tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 6121 ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc aattcgccct atagtgagtc 6181 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 6241 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 6301 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc 6361 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 6421 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 6481 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 6541 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 6601 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 6661 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 6721 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 6781 tttaacaaaa tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt 6841 gcggtatttc acaccgcata gggtaataac tgatataatt aaattgaagc tctaatttgt 6901 gagtttagta tacatgcatt tacttataat acagtttttt agttttgctg gccgcatctt 6961 ctcaaatatg cttcccagcc tgcttttctg taacgttcac cctctacctt agcatccctt 7021 ccctttgcaa atagtcctct tccaacaata ataatgtcag atcctgtaga gaccacatca 7081 tccacggttc tatactgttg acccaatgcg tctcccttgt catctaaacc cacaccgggt 7141 gtcataatca accaatcgta accttcatct cttccaccca tgtctctttg agcaataaag 7201 ccgataacaa aatctttgtc gctcttcgca atgtcaacag tacccttagt atattctcca 7261 gtagataggg agcccttgca tgacaattct gctaacatca aaaggcctct aggttccttt 7321 gttacttctt ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt 7381 gcattcgtaa tgtctgccca ttctgctatt ctgtatacac ccgcagagta ctgcaatttg 7441 actgtattac caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg 7501 gataatgcct ttagcggctt aactgtgccc tccatggaaa aatcagtcaa gatatccaca 7561 tgtgttttta gtaaacaaat tttgggacct aatgcttcaa ctaactccag taattccttg 7621 gtggtacgaa catccaatga agcacacaag tttgtttgct tttcgtgcat gatattaaat 7681 agcttggcag caacaggact aggatgagta gcagcacgtt ccttatatgt agctttcgac 7741 atgatttatc ttcgtttcct gcaggttttt gttctgtgca gttgggttaa gaatactggg 7801 caatttcatg tttcttcaac actacatatg cgtatatata ccaatctaag tctgtgctcc 7861 ttccttcgtt cttccttctg ttcggagatt accgaatcaa aaaaatttca aggaaaccga 7921 aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa ggtggtatgg tgcactctca 7981 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 8041 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 8101 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 8161 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagtatg 8221 atccaatatc aaaggaaatg atagcattga aggatgagac taatccaatt gaggagtggc 8281 agcatataga acagctaaag ggtagt //