LOCUS Exported 5599 bp ds-DNA circular SYN 07-OCT-2021 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1 to 5599) TITLE ER-misfolded proteins become sequestered with mitochondria and impair mitochondrial function JOURNAL Unpublished REFERENCE 2 (bases 1 to 5599) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 7, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5599 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 96..200 /gene="bla" /label=AmpR promoter CDS 201..1061 /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(419..438) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1232..1820 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1721..1740 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1974..1991 /label=L4440 /note="L4440 vector, forward primer" promoter 2144..2174 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2182..2198 /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 2187..2209 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2206..2222 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2206..2222 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 2241..2261 /label=T3 /note="T3 promoter, forward primer" promoter 2243..2261 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 2286..2929 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 2823..2846 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" CDS 3193..3219 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 3259..3306 /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" primer_bind complement(3320..3336) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3321..3337) /label=pBluescriptKS /note="For pBluescript vector" terminator 3339..3586 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(3355..3373) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(3604..3623) /label=T7 /note="T7 promoter, forward primer" promoter complement(3605..3623) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3633..3650) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3633..3649) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3642..3664) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3794..4249 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3881..3900) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4091..4112 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(4380..5183) /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(5184..5399) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(5430..5449) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5549..5571 /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2101 acgcaattaa tgtgagttac ctcactcatt aggcacccca ggctttacac tttatgcttc 2161 cggctcctat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2221 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctca 2281 gtttatcatt atcaatactc gccatttcaa agaatacgta aataattaat agtagtgatt 2341 ttcctaactt tatttagtca aaaaattagc cttttaattc tgctgtaacc cgtacatgcc 2401 caaaataggg ggcgggttac acagaatata taacatcgta ggtgtctggg tgaacagttt 2461 attcctggca tccactaaat ataatggagc ccgcttttta agctggcatc cagaaaaaaa 2521 aagaatccca gcaccaaaat attgttttct tcaccaacca tcagttcata ggtccattct 2581 cttagcgcaa ctacagagaa caggggcaca aacaggcaaa aaacgggcac aacctcaatg 2641 gagtgatgca acctgcctgg agtaaatgat gacacaaggc aattgaccca cgcatgtatc 2701 tatctcattt tcttacacct tctattacct tctgctctct ctgatttgga aaaagctgaa 2761 aaaaaaggtt gaaaccagtt ccctgaaatt attcccctac ttgactaata agtatataaa 2821 gacggtaggt attgattgta attctgtaaa tctatttctt aaacttctta aattctactt 2881 ttatagttag tctttttttt agttttaaaa caccagaact tagtttcgac ggattctaga 2941 actagtatgg cctccactcg tgtcctcgcc tctcgcctgg cctcccggat ggctgcttcc 3001 gccaaggttg cccgccctgc tgtccgcgtt gctcaggtca gcaagcgcac catccagact 3061 ggctcccccc tccagaccct caagcgcacc cagatgacct ccatcgtcaa cgccaccacc 3121 cgccaggctt tccagaagcg cgcctactct tccggatccc ccgggggatc gggtgacggt 3181 ccggtcgcca cctacccata cgatgttcca gattacgctg gtcgacggat cggtggtggt 3241 tctggaggcg gttctatgag agatcatatg gttttgcatg aatatgttaa tgctgctggt 3301 attacttagt aaagcttatc gataccgtcg acctcgagtc atgtaattag ttatgtcacg 3361 cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc 3421 tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat 3481 atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa 3541 aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccaat 3601 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac 3661 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 3721 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 3781 ggcgaatggc gcgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 3841 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 3901 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 3961 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 4021 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 4081 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 4141 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 4201 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc ctgatgcggt 4261 attttctcct tacgcatctg tgcggtattt cacaccgcat agggtaataa ctgatataat 4321 taaattgaag ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt 4381 tagttttgct ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca 4441 ccctctacct tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca 4501 gatcctgtag agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg 4561 tcatctaaac ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc 4621 atgtctcttt gagcaataaa gccgataaca aaatctttgt cgctcttcgc aatgtcaaca 4681 gtacccttag tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc 4741 aaaaggcctc taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata 4801 cctgggccca ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca 4861 cccgcagagt actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag 4921 agtaaaaaat tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa 4981 aaatcagtca agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca 5041 actaactcca gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc 5101 ttttcgtgca tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt 5161 tccttatatg tagctttcga catgatttat cttcgtttcc tgcaggtttt tgttctgtgc 5221 agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat 5281 accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca 5341 aaaaaatttc aaagaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaat 5401 tgaaaagctg tggtatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 5461 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 5521 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 5581 tcatcaccga aacgcgcga //