LOCUS Exported 8396 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG423GPD-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8396) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 8396) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8396 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" rep_origin 68..1410 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind 400..447 /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 1437..1541 /gene="bla" /label=AmpR promoter CDS 1542..2402 /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(1760..1779) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2573..3161 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3062..3081 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3315..3332 /label=L4440 /note="L4440 vector, forward primer" promoter 3485..3515 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3523..3539 /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 3528..3550 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3547..3563 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3547..3563 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 3582..3602 /label=T3 /note="T3 promoter, forward primer" promoter 3584..3602 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 3631..4275 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 4169..4192 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" primer_bind 4281..4297 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 4281..4297 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 4305..4429 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 4454..4484 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 4538..5197 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(4604..4623) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 5542..5847 /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 5726..5745 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(5888..6012) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(6045..6061) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6046..6062) /label=pBluescriptKS /note="For pBluescript vector" terminator 6064..6311 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(6080..6098) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(6329..6348) /label=T7 /note="T7 promoter, forward primer" promoter complement(6330..6348) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6358..6375) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6358..6374) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6367..6389) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 6519..6974 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6606..6625) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6816..6837 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(7234..7893) /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" promoter complement(7894..8080) /gene="S. cerevisiae HIS3" /label=HIS3 promoter primer_bind complement(8227..8246) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8346..8368 /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttagatgat ccaatatcaa aggaaatgat agcattgaag gatgagacta atccaattga 121 ggagtggcag catatagaac agctaaaggg tagtgctgaa ggaagcatac gataccccgc 181 atggaatggg ataatatcac aggaggtact agactacctt tcatcctaca taaatagacg 241 catataagta cgcatttaag cataaacacg cactatgccg ttcttctcat gtatatatat 301 atacaggcaa cacgcagata taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg 361 cgttgcattt tcggaagcgc tcgttttcgg aaacgctttg aagttcctat tccgaagttc 421 ctattctcta gaaagtatag gaacttcaga gcgcttttga aaaccaaaag cgctctgaag 481 acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa atattgcgaa 541 taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct gtgctatatc 601 cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac tcgacctcta 661 cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag taagaactat 721 tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt agtatataga 781 gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc aacgctatca 841 ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg gggatgcctt 901 tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg aagtggagtc 961 aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa ccttaacgga 1021 cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg agaaaaaaag 1081 taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg tagaacaaaa 1141 aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa 1201 aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta 1261 caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc atttctgttc 1321 tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg 1381 ttctacaaaa tgaagcacag atgcttcgtt caggtggcac ttttcgggga aatgtgcgcg 1441 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 1501 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 1561 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 1621 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 1681 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 1741 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 1801 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 1861 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 1921 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 1981 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 2041 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 2101 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 2161 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 2221 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 2281 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 2341 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 2401 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 2461 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 2521 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 2581 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 2641 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 2701 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 2761 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 2821 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 2881 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 2941 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 3001 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 3061 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 3121 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 3181 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 3241 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 3301 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 3361 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 3421 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta cctcactcat taggcacccc 3481 aggctttaca ctttatgctt ccggctccta tgttgtgtgg aattgtgagc ggataacaat 3541 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa 3601 gggaacaaaa gctggagctc ttcgagttta tcattatcaa tactcgccat ttcaaagaat 3661 acgtaaataa ttaatagtag tgattttcct aactttattt agtcaaaaaa ttagcctttt 3721 aattctgctg taacccgtac atgcccaaaa tagggggcgg gttacacaga atatataaca 3781 ccgaaggtgc ctgggtgaac aggtttattc ctggcatcca ctaaatataa tggagcccgc 3841 tttttaagct ggcatccaga aaaaaaaaga atcccagcac caaaatattg ttttcttcac 3901 caaccatcag ttcataggtc cattctctta gcgcaactac agagaacagg ggcacaaaca 3961 ggcaaaaaac gggcacaacc tcaatggagt gatgcaacct gcctggagta aatgatgaca 4021 caaggcaatt gacccacgca tgtatctatc tcattttctt acaccttcta ttaccttctg 4081 ctctctctga tttggaaaaa gctgaaaaaa aaggttgaaa ccagttccct gaaattattc 4141 ccctacttga ctaataagta tataaagacg gtaggtattg attgtaattc tgtaaatcta 4201 tttcttaaac ttcttaaatt ctacttttat agttagtctt ttttttagtt ttaaaacacc 4261 agaacttagt ttcgacggat tctagaacta gtggatcccc catcacaagt ttgtacaaaa 4321 aagctgaacg agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata 4381 aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgg cggccgcatt 4441 aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga ttttgagtta 4501 ggatccgtcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata 4561 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt 4621 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt 4681 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa 4741 tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt 4801 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga 4861 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg 4921 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa 4981 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc 5041 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc 5101 gattcaggtt catcatgccg tctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt 5161 acaacagtac tgcgatgagt ggcagggcgg ggcgtaaacg ccgcgtggat ccggcttact 5221 aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac 5281 tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg 5341 acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc 5401 tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg 5461 aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg 5521 acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt 5581 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg 5641 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg 5701 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc 5761 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc 5821 attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag ccagtctgca 5881 ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg 5941 caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt cagctttctt 6001 gtacaaagtg gtgatgggct gcaggaattc gatatcaagc ttatcgatac cgtcgacctc 6061 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa 6121 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta 6181 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg 6241 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg 6301 ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact 6361 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 6421 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 6481 ttcccaacag ttgcgcagcc tgaatggcga atggcgcgac gcgccctgta gcggcgcatt 6541 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 6601 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 6661 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 6721 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 6781 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 6841 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc 6901 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt 6961 aacgtttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 7021 cgcatagatc cgtcgagttc aagagaaaaa aaaagaaaaa gcaaaaagaa aaaaggaaag 7081 cgcgcctcgt tcagaatgac acgtatagaa tgatgcatta ccttgtcatc ttcagtatca 7141 tactgttcgt atacatactt actgacattc ataggtatac atatatacac atgtatatat 7201 atcgtatgct gcagctttaa ataatcggtg tcactacata agaacacctt tggtggaggg 7261 aacatcgttg gtaccattgg gcgaggtggc ttctcttatg gcaaccgcaa gagccttgaa 7321 cgcactctca ctacggtgat gatcattctt gcctcgcaga caatcaacgt ggagggtaat 7381 tctgctagcc tctgcaaagc tttcaagaaa atgcgggatc atctcgcaag agagatctcc 7441 tactttctcc ctttgcaaac caagttcgac aactgcgtac ggcctgttcg aaagatctac 7501 caccgctctg gaaagtgcct catccaaagg cgcaaatcct gatccaaacc tttttactcc 7561 acgcgccagt agggcctctt taaaagcttg accgagagca atcccgcagt cttcagtggt 7621 gtgatggtcg tctatgtgta agtcaccaat gcactcaacg attagcgacc agccggaatg 7681 cttggccaga gcatgtatca tatggtccag aaaccctata cctgtgtgga cgttaatcac 7741 ttgcgattgt gtggcctgtt ctgctactgc ttctgcctct ttttctggga agatcgagtg 7801 ctctatcgct aggggaccac cctttaaaga gatcgcaatc tgaatcttgg tttcatttgt 7861 aatacgcttt actagggctt tctgctctgt catctttgcc ttcgtttatc ttgcctgctc 7921 attttttagt atattcttcg aagaaatcac attactttat ataatgtata attcattatg 7981 tgataatgcc aatcgctaag aaaaaaaaag agtcatccgc taggggaaaa aaaaaaatga 8041 aaatcattac cgaggcataa aaaaatatag agtgtactag aggaggccaa gagtaataga 8101 aaaagaaaat tgcgggaaag gactgtgtta tgacttccct gactaatgcc gtgttcaaac 8161 gatacctggc agtgactcct agcgctcacc aagctcttaa aacgggaatt tatggtgcac 8221 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 8281 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 8341 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcga //