LOCUS Exported 5764 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses aggregation-prone FlucDM-HA-GFP11 in mammalian cells. ACCESSION . VERSION . KEYWORDS FlucDM-HA-GFP11-N1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5764) AUTHORS Ruan L, Zhou C, Jin E, Kucharavy A, Zhang Y, Wen Z, Florens L, Li R TITLE Cytosolic proteostasis through importing of misfolded proteins into mitochondria. JOURNAL Nature. 2017 Mar 16;543(7645):443-446. doi: 10.1038/nature21695. Epub 2017 Mar 1. PUBMED 28241148 REFERENCE 2 (bases 1 to 5764) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5764 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 152..455 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 456..659 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 610..630 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 719..2368 /codon_start=1 /gene="luc+" /product="firefly luciferase" /label=luciferase /note="enhanced luc+ version of the luciferase gene" /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS MYTFVTSHLPPGFNEYDFVPESFDQDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFQVVLMYRFEEELFLRSLQDYK IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV" primer_bind complement(785..803) /label=LucNrev /note="Firefly luciferase, reverse primer" CDS 2405..2431 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 2471..2518 /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" polyA_signal 2643..2764 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2680..2699) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2734..2753 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2771..3226) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2908..2929) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3120..3139 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3253..3357 /gene="bla" /label=AmpR promoter promoter 3359..3716 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3382..3402) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3567..3702 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3629..3648 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3751..4545 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3805..3824) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4415..4434 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4733..4752) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4777..4824 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5153..5741 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5642..5661 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 61 ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt 121 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 181 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 241 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 301 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 361 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 421 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 481 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 541 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 601 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg 661 gtttagtgaa ccgtcagatc cgctagcgct accggactca gatctcgagc tcaagcttat 721 ggaagacgcc aaaaacataa agaaaggccc ggcgccattc tatccgctgg aagatggaac 781 cgctggagag caactgcata aggctatgaa gagatacgcc ctggttcctg gaacaattgc 841 ttttacagat gcacatatcg aggtggacat cacttacgct gagtacttcg aaatgtccgt 901 tcggttggca gaagctatga aacgatatgg gctgaataca aatcacagaa tcgtcgtatg 961 cagtgaaaac tctcttcaat tctttatgcc ggtgttgggc gcgttattta tcggagttgc 1021 agttgcgccc gcgaacgaca tttataatga acgtgaattg ctcaacagta tgggcatttc 1081 gcagcctacc gtggtgttcg tttccaaaaa ggggttgcaa aaaattttga acgtgcaaaa 1141 aaagctccca atcatccaaa aaattattat catggattct aaaacggatt accagggatt 1201 tcagtcgatg tacacgttcg tcacatctca tctacctccc ggttttaatg aatacgattt 1261 tgtgccagag tccttcgatc aggacaagac aattgcactg atcatgaact cctctggatc 1321 tactggtctg cctaaaggtg tcgctctgcc tcatagaact gcctgcgtga gattctcgca 1381 tgccagagat cctatttttg gcaatcaaat cattccggat actgcgattt taagtgttgt 1441 tccattccat cacggttttg gaatgtttac tacactcgga tatttgatat gtggatttca 1501 agtcgtctta atgtatagat ttgaagaaga gctgtttctg aggagccttc aggattacaa 1561 gattcaaagt gcgctgctgg tgccaaccct attctccttc ttcgccaaaa gcactctgat 1621 tgacaaatac gatttatcta atttacacga aattgcttct ggtggcgctc ccctctctaa 1681 ggaagtcggg gaagcggttg ccaagaggtt ccatctgcca ggtatcaggc aaggatatgg 1741 gctcactgag actacatcag ctattctgat tacacccgag ggggatgata aaccgggcgc 1801 ggtcggtaaa gttgttccat tttttgaagc gaaggttgtg gatctggata ccgggaaaac 1861 gctgggcgtt aatcaaagag gcgaactgtg tgtgagaggt cctatgatta tgtccggtta 1921 tgtaaacaat ccggaagcga ccaacgcctt gattgacaag gatggatggc tacattctgg 1981 agacatagct tactgggacg aagacgaaca cttcttcatc gttgaccgcc tgaagtctct 2041 gattaagtac aaaggctatc aggtggctcc cgctgaattg gaatccatct tgctccaaca 2101 ccccaacatc ttcgacgcag gtgtcgcagg tcttcccgac gatgacgccg gtgaacttcc 2161 cgccgccgtt gttgttttgg agcacggaaa gacgatgacg gaaaaagaga tcgtggatta 2221 cgtcgccagt caagtaacaa ccgcgaaaaa gttgcgcgga ggagttgtgt ttgtggacga 2281 agtaccgaaa ggtcttaccg gaaaactcga cgcaagaaaa atcagagaga tcctcataaa 2341 ggccaagaag ggcggaaaga ttgccgtgaa acccggggga tccggtgacg gtccggtcgc 2401 cacctaccca tacgatgttc cagattacgc tggtcgacgg atcggtggtg gttctggagg 2461 cggttctatg agagatcata tggttttgca tgaatatgtt aatgctgctg gtattactta 2521 agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt 2581 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg 2641 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 2701 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 2761 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 2821 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 2881 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 2941 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 3001 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 3061 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 3121 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 3181 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt 3241 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 3301 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct 3361 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 3421 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 3481 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 3541 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 3601 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct 3661 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc 3721 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt 3781 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc 3841 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag 3901 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg 3961 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac 4021 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc 4081 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc 4141 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc 4201 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg 4261 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat 4321 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc 4381 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa 4441 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat 4501 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt 4561 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg 4621 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc 4681 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac 4741 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa 4801 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg 4861 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac 4921 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc 4981 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat 5041 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 5101 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 5161 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 5221 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 5281 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 5341 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 5401 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 5461 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 5521 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 5581 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 5641 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 5701 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 5761 tttt //