LOCUS       sequence-379626-        8493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To produce mutants mimicking dephosphorylated Mfn1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8493)
  AUTHORS   Pyakurel A, Savoia C, Hess D, Scorrano L
  TITLE     Extracellular regulated kinase phosphorylates mitofusin 1 to control
            mitochondrial morphology and apoptosis.
  JOURNAL   Mol Cell. 2015 Apr 16;58(2):244-54. doi: 
            10.1016/j.molcel.2015.02.021. Epub 2015 Mar 19.
  PUBMED    25801171
REFERENCE   2  (bases 1 to 8493)
  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: 
            "10.1016/j.molcel.2015.02.021"; journalName: "Mol Cell"; date: 
            "2015-04-16- 16"; volume: "58"; issue: "2"; pages: "244-54"
COMMENT     Sequence Label: p3xFLAG-CMV-14-Mfn1-T562A
FEATURES             Location/Qualifiers
     source          1..8493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..64)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(218..806)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(298..317)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(977..1768,1769..1837))
                     /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     1600..1619
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1838..1942)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1969..2424
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2056..2075)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2266..2287
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2550..2572
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2564..2581
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2565..2581
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2742..3121
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3122..3325
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3276..3296
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             5600..5665
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    5676..6298
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5875..5894)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(6322..6342)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6340..6637
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             6691..7485
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6745..6764)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7355..7374
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    8137..8271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8174..8193)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8228..8247
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8276..8295)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8277..8295)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8309..8325)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8309..8325)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8322..8344)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8333..8349
                     /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)."
     promoter        complement(join(8357..8363,8364..8381,8382..8387))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8402..8423
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
       61 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
      121 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
      181 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
      241 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
      301 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
      361 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
      421 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
      481 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
      541 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
      601 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
      661 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
      721 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
      781 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
      841 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
      901 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
      961 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     1021 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     1081 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     1141 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     1201 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     1261 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     1321 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     1381 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     1441 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     1501 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     1561 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     1621 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     1681 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     1741 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     1801 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     1861 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     1921 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta
     1981 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2041 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2101 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2161 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2221 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2281 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2341 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2401 acaaaatatt aacgcttaca atttccattc gccattcagg ctgcgcaact gttgggaagg
     2461 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     2521 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     2581 tgccaagctg atctatacat tgaatcaata ttggcaatta gccatattag tcattggtta
     2641 tatagcataa atcaatattg gctattggcc attgcatacg ttgtatctat atcataatat
     2701 gtacatttat attggctcat gtccaatatg accgccatgt tgacattgat tattgactag
     2761 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     2821 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     2881 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     2941 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     3001 tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     3061 gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     3121 ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt
     3181 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     3241 ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg
     3301 gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagaattaag cttccaccat
     3361 ggcagaaacg gtatctccac tgaagcactt cgtgctggca aagaaggcca tcactgcgat
     3421 cttcggccag ttactggagt ttgttactga gggctcacat tttgttgaag caacatacag
     3481 gaacccggaa cttgatcgga tagcaaccga ggatgatctg gtggagatac agggctacag
     3541 aaacaagctc gctgtcattg gggaggtgct gtctcggagg catatgaaag tggcattttt
     3601 tggcaggaca agtagtggca agagctctgt catcaacgca atgctgtggg ataaagtcct
     3661 ccccagcggg attggtcaca caaccaactg cttcctgagt gtggagggaa ctgatggaga
     3721 taaagcctac cttatgaccg aagggtcaga tgagaagaaa agcgtgaaga ctgttaatca
     3781 gctggcccat gccctccata tggataaaga cttgaaagct ggttgtcttg tgcacgtatt
     3841 ttggcccaaa gcaaaatgtg ccctcttgag agatgacctg gttttagtag acagcccagg
     3901 tacagatgtc accacagagc tggacatctg gattgataag ttctgccttg atgctgatgt
     3961 ttttgttttg gtggcaaact cggaatcaac gctgatgaac acggagaaac attttttcca
     4021 taaggtgaat gagcggctct ccaagccaaa catttttatc ctgaataatc gttgggatgc
     4081 ttctgcttca gagccagaat atatggagga tgtgcgcaga cagcacatgg aaagatgtct
     4141 tcacttcttg gtagaagagc tcaaggttgt gagtccttta gaagctcgga atcggatctt
     4201 ttttgtttca gccaaggaag ttctcaactc cagaatgaat aaagcccagg ggatgccaga
     4261 aggtggaggg gcacttgcag aaggatttca ggcaagatta caggagtttc aaaattttga
     4321 acaaactttt gaggagtgta tctcgcagtc agcagtgaaa acaaagtttg aacagcacac
     4381 tatcagagct aaacagatac tagacactgt gaaaaacata ctggactcag taaacgtggc
     4441 agcagcagag aagagggttt attcaatgga agagagggaa gaccaaatcg acagactgga
     4501 ctttattcga aaccagatga accttttaac aatggatgtt aagaagaaga tcaaggaggt
     4561 cacggaggag gtggcaaaca aggtttcttg tgcaatgaca gatgagattt gtcgcctgtc
     4621 tgttttggtt gatgagtttt gttctgagtt tcatcctacc cccagtgtgc tgaaagtgta
     4681 taagagtgaa ttaaataagc acatagaaga tggcatggga agaaatttgg ctgatcggtg
     4741 caccagtgaa gtcaatgcct ccattctcca atctcagcaa gaaatcattg aaaatttgaa
     4801 gccactactt ccagctggta tacagaataa acttcataca ttaatccctt gcaaaaagtt
     4861 tgatctcagc tatgatctca attgccacaa gctgtgttcg gattttcaag aagacattgt
     4921 gtttcggttt tccctgggct ggtcttccct tgtacatcga ttcctgggtt ctacgaatgc
     4981 acagagggtg ctgcttgggc tgtcagagcc tatctttcag gtccctagat ctttagcttc
     5041 agctcctact gctccttcta acccagcagc accagataat gcagcccagg aagagctcat
     5101 gatcacactg atcacaggat tggcatccct cacgtctaga acctccatgg gcatcatagt
     5161 tgttgggggc gtgatttgga aaacagtggg ctggaaactc atctctgtca ccttaagtat
     5221 gtatggagct ttgtaccttt atgaaagact gacttggact actcgtgcga aagagagagc
     5281 gtttaagcag cagtttgtaa actatgcaac tgaaaaactg cagatgattg tgaagttcac
     5341 aagtgcaaac tgcagccacc aagtccagca agaaatggcc accacttttg ctcgactgtg
     5401 ccaacaagtt gatgttactc agaaacatct ggaagaagaa attgctagat tatccaaaga
     5461 gattgaccaa ctggagaaaa tacaaaacaa ttcaaagctc ttaagaaata aagctgttca
     5521 acttgaacgt gagctggaaa atttttcgaa gcagtttctc cacccgagca gtggagaatc
     5581 ccctagagga tcccgggctg actacaaaga ccatgacggt gattataaag atcatgacat
     5641 cgactacaag gatgacgatg acaagtagtg atcccgggtg gcatccctgt gacccctccc
     5701 cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa
     5761 aattaagttg catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg
     5821 gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt
     5881 gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg
     5941 ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca
     6001 ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg
     6061 tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac
     6121 aggcgtgaac cactgctccc ttccctgtcc ttctgatttt aaaataacta taccagcagg
     6181 aggacgtcca gacacagcat aggctacctg gccatgccca accggtggga catttgagtt
     6241 gcttgcttgg cactgtcctc tcatgcgttg ggtccactca gtagatgcct gttgaattgg
     6301 gtacgcggcc agcttggctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     6361 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     6421 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     6481 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     6541 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     6601 ctgagctatt ccagaagtag tgaggaggct tttttggagg aattgatcag cttgggatct
     6661 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     6721 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     6781 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     6841 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     6901 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     6961 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     7021 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     7081 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     7141 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     7201 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     7261 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     7321 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     7381 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     7441 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     7501 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     7561 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     7621 agcgcgggga tctcatgctg gagttcttcg cccaccccgg gctcgatccc ctcgcgagtt
     7681 ggttcagctg ctgcctgagg ctggacgacc tcgcggagtt ctaccggcag tgcaaatccg
     7741 tcggcatcca ggaaaccagc agcggctatc cgcgcatcca tgcccccgaa ctgcaggagt
     7801 ggggaggcac gatggccgct ttggtcgacc cggacgggac gctcctgcgc ctgatacaga
     7861 acgaattgct tgcaggcatc tcatgagtgt gtcttcccgt tttccgcctg aggtcactgc
     7921 gtggatggag cgctggcgcc tgctgcgcga cggcgagctg ctcaccaccc actcgccaag
     7981 ctggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc gatcataatc
     8041 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     8101 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     8161 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     8221 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat caattcccta
     8281 tagtgagtcg tattaaattc gtaatcatgt catagctgtt tcctgtgtga aattgttatc
     8341 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     8401 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     8461 acctgtcgtg ccagctgcat taatgaatcg gcc
//
