LOCUS Exported 5584 bp ds-DNA circular SYN 21-JAN-2022 DEFINITION Eleventh beta-strand of GFP (GFP11) fused to a mitochondria signal sequence for mitochondrial targeting, HA tagged in yeast plasmid with URA marker. ACCESSION . VERSION . KEYWORDS p406 – TDH3-MTS-GFP11-Ura SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5584) TITLE ER-misfolded proteins become sequestered with mitochondria and impair mitochondrial function JOURNAL Unpublished REFERENCE 2 (bases 1 to 5584) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 21, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5584 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 128..147 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 381..398 /label=L4440 /note="L4440 vector, forward primer" protein_bind 515..536 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 551..581 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 589..605 /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 594..616 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 613..629 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 613..629 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 648..668 /label=T3 /note="T3 promoter, forward primer" promoter 650..668 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 693..1336 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 1230..1253 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" CDS 1617..1643 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1683..1730 /codon_start=1 /product="11th beta-strand of superfolder GFP" /label=GFP11 /note="associates with GFP(1-10), consisting of the first 10 beta-strands, to reconstitute fluorescent GFP (Cabantous et al., 2005; Kamiyama et al., 2016)" /translation="RDHMVLHEYVNAAGIT" terminator 1742..1989 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(1758..1776) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(2007..2026) /label=T7 /note="T7 promoter, forward primer" promoter complement(2008..2026) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2036..2053) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2036..2052) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2045..2067) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2193..2648 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2280..2299) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2490..2511 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2779..3582) /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(3583..3798) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(3822..3841) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3941..3963 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4001..4019) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4087..4191 /gene="bla" /label=AmpR promoter CDS 4192..5052 /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(4410..4429) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5223..227 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" ORIGIN 1 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 61 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 121 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 181 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 241 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 301 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 361 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 421 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 481 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 541 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 601 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc 661 actaaaggga acaaaagctg gagctcagtt tatcattatc aatactcgcc atttcaaaga 721 atacgtaaat aattaatagt agtgattttc ctaactttat ttagtcaaaa aattagcctt 781 ttaattctgc tgtaacccgt acatgcccaa aatagggggc gggttacaca gaatatataa 841 catcgtaggt gtctgggtga acagtttatt cctggcatcc actaaatata atggagcccg 901 ctttttaagc tggcatccag aaaaaaaaag aatcccagca ccaaaatatt gttttcttca 961 ccaaccatca gttcataggt ccattctctt agcgcaacta cagagaacag gggcacaaac 1021 aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc tgcctggagt aaatgatgac 1081 acaaggcaat tgacccacgc atgtatctat ctcattttct tacaccttct attaccttct 1141 gctctctctg atttggaaaa agctgaaaaa aaaggttgaa accagttccc tgaaattatt 1201 cccctacttg actaataagt atataaagac ggtaggtatt gattgtaatt ctgtaaatct 1261 atttcttaaa cttcttaaat tctactttta tagttagtct tttttttagt tttaaaacac 1321 cagaacttag tttcgacgga ttctagaact agtgagctca agcttcgaat tctgcagtcg 1381 acatggcctc cactcgtgtc ctcgcctctc gcctggcctc ccggatggct gcttccgcca 1441 aggttgcccg ccctgctgtc cgcgttgctc aggtcagcaa gcgcaccatc cagactggct 1501 cccccctcca gaccctcaag cgcacccaga tgacctccat cgtcaacgcc accacccgcc 1561 aggctttcca gaagcgcgcc tactcttccg gatccggtga cggtccggtc gccacctacc 1621 catacgatgt tccagattac gctggtcgac ggatcggtgg tggttctgga ggcggttcta 1681 tgagagatca tatggttttg catgaatatg ttaatgctgc tggtattact taagcctcga 1741 gtcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 1801 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 1861 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg 1921 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct 1981 ttaatttgcg gccggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg 2041 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 2101 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 2161 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc 2221 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 2281 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 2341 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 2401 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 2461 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 2521 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 2581 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 2641 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 2701 gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt 2761 tacttataat acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc 2821 tgcttttctg taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct 2881 tccaacaata ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg 2941 acccaatgcg tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta 3001 accttcatct cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc 3061 gctcttcgca atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca 3121 tgacaattct gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg 3181 cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca 3241 ttctgctatt ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc 3301 aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt 3361 aactgtgccc tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat 3421 tttgggacct aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga 3481 agcacacaag tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact 3541 aggatgagta gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct 3601 gcaggttttt gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac 3661 actacatatg cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg 3721 ttcggagatt accgaatcaa aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa 3781 aaaaaatgat gaattgaaaa ggtggtatgg tgcactctca gtacaatctg ctctgatgcc 3841 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 3901 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 3961 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 4021 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 4081 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 4141 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 4201 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 4261 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 4321 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 4381 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 4441 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 4501 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 4561 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 4621 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 4681 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 4741 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 4801 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 4861 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 4921 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 4981 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 5041 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 5101 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 5161 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 5221 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 5281 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 5341 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 5401 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 5461 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 5521 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 5581 acct //