LOCUS       sequence-310841-        5389 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Rescue mfn2 knockdown by tethering mitochondria and ER with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5389)
  AUTHORS   Zhou W, Hsu AY, Wang Y, Syahirah R, Wang T, Jeffries J, Wang X, 
            Mohammad H, Seleem MN, Umulis D, Deng Q
  TITLE     Mitofusin 2 regulates neutrophil adhesive migration and the actin 
            cytoskeleton.
  JOURNAL   J Cell Sci. 2020 Sep 4;133(17). pii: jcs.248880. doi: 
            10.1242/jcs.248880.
  PUBMED    32788232
REFERENCE   2  (bases 1 to 5389)
  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"; journalName: "J Cell 
            Sci."; date: "2020-09-4"; volume: "133(17). pii: jcs.248880. doi"; 
            pages: " 10.1242/jcs.248880"
COMMENT     Sequence Label: plko-shmfn2-mito-GFP-ER tethering
FEATURES             Location/Qualifiers
     source          1..5389
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        207..717
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     636..654
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             739..1338
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(739..758)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1235..1255
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1817..1942
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2435..2668
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2853..2897
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        3195..3443
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3195..3215
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3366..3385
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        3564..3867
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3868..4071
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4022..4042
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4110..4119
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4275..4277,4278..4280,4281..4991)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4275..4277,4278..4280,4281..4991)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4320..4341)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4581..4600)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4928..4949
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5091..5143
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    5267..5388
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5304..5323)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5358..5377
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggccgcgg ctcgaggggg ttggggttgc gccttttcca aggcagccct
      241 gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac
      301 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc
      361 cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg
      421 ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac
      481 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc
      541 tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt
      601 ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg
      661 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg
      721 gatccaccgg agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
      781 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
      841 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
      901 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
      961 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     1021 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     1081 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     1141 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     1201 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     1261 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     1321 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca
     1381 cgaccccatg catcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     1441 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     1501 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     1561 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     1621 gtgtgtgccc gtctgttgtg tgactcccac tgcttaagcc tcaataaagc ttgccttgag
     1681 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     1741 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
     1801 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
     1861 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
     1921 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
     1981 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
     2041 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
     2101 acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt
     2161 atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa
     2221 ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc
     2281 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     2341 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     2401 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     2461 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     2521 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     2581 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     2641 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     2701 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     2761 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     2821 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     2881 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     2941 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     3001 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     3061 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
     3121 gatccattcg attagtgaac ggatctcgac ggtatcgatc acgagactag cctcgagcgg
     3181 ccgccccctt caccgagggc ctatttccca tgattccttc atatttgcat atacgataca
     3241 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     3301 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     3361 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     3421 tcttgtggaa aggacgaaac accgggcagg tttactgcga ggaaatctcg agatttcctc
     3481 gcagtaaacc tgctttttga attcagttat taatagtaat caattacggg gtcattagtt
     3541 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     3601 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     3661 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaagctgc ccacttggca
     3721 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     3781 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     3841 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     3901 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     3961 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaagaactc cgccccattg
     4021 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg
     4081 aaccgtcaga tccgctagcg ctcccggtcg ccaccatggc aatccagttg cgttcgctct
     4141 tccccttggc attgcccgga atgctggccc tccttggctg gtggtggttt ttctctcgta
     4201 aaaaagacct ggagctgaag ctgcgcatcc tgcagagcac cgtgccccgc gccagagacc
     4261 cccctgtggc tacaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     4321 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     4381 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     4441 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     4501 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     4561 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     4621 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     4681 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     4741 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     4801 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     4861 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     4921 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     4981 agctgtacaa gtccggcctg agaagcagag ctcaggctag caacagcaga gtgatggttt
     5041 atattggcat cgctattttt ttgtttttgg tgggcctgtt tatgaaataa ctcgagctca
     5101 agcttcgaat tctgcagtcg acggtaccgc gggcccggga tccaccggat ctagataact
     5161 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     5221 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     5281 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5341 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaa
//
