LOCUS       sequence-180680-        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FlucWT-HA-GFP11 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature21695"; journalName: "Nature"; date: "2017-03-16- 
            16"; volume: "543"; issue: "7645"; pages: "443-446"
COMMENT     Sequence Label: FlucWT-HA-GFP11-N1
FEATURES             Location/Qualifiers
     source          1..5764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     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"
     enhancer        3359..3550
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     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"
     CDS             3751..4545
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     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 gggacaagac aattgcactg atcatgaact cctctggatc
     1321 tactggtctg cctaaaggtg tcgctctgcc tcatagaact gcctgcgtga gattctcgca
     1381 tgccagagat cctatttttg gcaatcaaat cattccggat actgcgattt taagtgttgt
     1441 tccattccat cacggttttg gaatgtttac tacactcgga tatttgatat gtggatttcg
     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
//
