LOCUS Exported 6800 bp ds-DNA circular SYN 25-JAN-2023 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG303GPD-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6800) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 6800) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 25, 2023 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6800 /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 317..504 /gene="S. cerevisiae HIS3" /label=HIS3 promoter CDS 505..1167 /codon_start=1 /gene="S. cerevisiae HIS3" /product="imidazoleglycerol-phosphate dehydratase, required for histidine biosynthesis" /label=HIS3 /note="yeast auxotrophic marker" /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM" rep_origin complement(1427..1882) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1564..1585) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1776..1795 /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 2085..2728 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 2622..2645 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" primer_bind 2734..2750 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 2734..2750 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 2758..2882 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 2907..2937 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 2991..3650 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(3057..3076) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 3992..4297 /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 4176..4195 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(4338..4462) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(4495..4511) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4496..4512) /label=pBluescriptKS /note="For pBluescript vector" promoter complement(4540..4558) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4579..4595) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4579..4595) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4592..4614) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4603..4619 /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(4627..4657) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4672..4693 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4810..4827) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4981..5569) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5061..5080) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5740..6600) /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 6363..6382 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6601..6705) /gene="bla" /label=AmpR promoter primer_bind 6773..6791 /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 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 241 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 301 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 361 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat 421 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag 481 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca 541 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac 601 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg 661 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat 721 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc 781 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt 841 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg 901 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta 961 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga 1021 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg 1081 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt 1141 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat 1201 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta 1261 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg 1321 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg 1381 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 1441 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1501 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1561 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1621 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 1681 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 1741 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 1801 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 1861 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc 1921 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 1981 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2041 aattgtaata cgactcacta tagggcgaat tggagctcag tttatcatta tcaatactcg 2101 ccatttcaaa gaatacgtaa ataattaata gtagtgattt tcctaacttt atttagtcaa 2161 aaaattagcc ttttaattct gctgtaaccc gtacatgccc aaaatagggg gcgggttaca 2221 cagaatatat aacatcgtag gtgtctgggt gaacagttta ttcctggcat ccactaaata 2281 taatggagcc cgctttttaa gctggcatcc agaaaaaaaa agaatcccag caccaaaata 2341 ttgttttctt caccaaccat cagttcatag gtccattctc ttagcgcaac tacagagaac 2401 aggggcacaa acaggcaaaa aacgggcaca acctcaatgg agtgatgcaa cctgcctgga 2461 gtaaatgatg acacaaggca attgacccac gcatgtatct atctcatttt cttacacctt 2521 ctattacctt ctgctctctc tgatttggaa aaagctgaaa aaaaaggttg aaaccagttc 2581 cctgaaatta ttcccctact tgactaataa gtatataaag acggtaggta ttgattgtaa 2641 ttctgtaaat ctatttctta aacttcttaa attctacttt tatagttagt ctttttttta 2701 gttttaaaac accagaactt agtttcgacg gattctagaa ctagtggatc ccccatcaca 2761 agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa tatattaaat 2821 tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta 2881 tggcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt 2941 ggattttgag ttaggatccg tcgagatttt caggagctaa ggaagctaaa atggagaaaa 3001 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg 3061 catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct 3121 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg 3181 cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga 3241 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat 3301 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg 3361 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt 3421 tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg 3481 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc 3541 tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa 3601 tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa acgcgtggat 3661 ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa 3721 gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg 3781 tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat 3841 atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc 3901 tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc 3961 tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag 4021 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg 4081 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact 4141 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag 4201 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat 4261 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag 4321 ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct 4381 gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt 4441 cagctttctt gtacaaagtg gtgatgggct gcaggaattc gatatcaagc ttatcgatac 4501 cgtcgacctc gagggggggc ccggtaccag cttttgttcc ctttagtgag ggttaatttc 4561 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 4621 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 4681 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 4741 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 4801 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 4861 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 4921 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 4981 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 5041 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 5101 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 5161 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 5221 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 5281 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 5341 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 5401 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 5461 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 5521 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 5581 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 5641 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 5701 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 5761 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 5821 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 5881 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 5941 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 6001 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 6061 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 6121 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 6181 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 6241 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 6301 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 6361 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 6421 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 6481 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 6541 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 6601 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 6661 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 6721 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg 6781 tatcacgagg ccctttcgtc //