LOCUS       sequence-379615-        8493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To produce mutants mimicking phosphorylated 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-T564D
FEATURES             Location/Qualifiers
     source          1..8493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(8..25)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(179..767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(259..278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(938..1729,1730..1798))
                     /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     1561..1580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1799..1903)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1930..2385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2017..2036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2227..2248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2511..2533
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2525..2542
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2526..2542
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2703..3082
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3083..3286
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3237..3257
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             5561..5626
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    5637..6259
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5836..5855)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(6283..6303)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6301..6598
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             6652..7446
                     /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(6706..6725)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7316..7335
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    8098..8232
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8135..8154)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8189..8208
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8237..8256)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8238..8256)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8270..8286)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8270..8286)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8283..8305)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8294..8310
                     /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(8318..8324,8325..8342,8343..8348))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8363..8384
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
       61 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
      121 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
      181 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      241 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
      301 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
      361 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
      421 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
      481 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
      541 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
      601 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
      661 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
      721 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
      781 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
      841 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
      901 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
      961 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     1021 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     1081 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     1141 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     1201 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     1261 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     1321 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     1381 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     1441 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     1501 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     1561 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     1621 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     1681 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     1741 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     1801 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     1861 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     1921 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1981 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2041 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2101 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2161 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2221 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2281 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2341 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttccatt cgccattcag
     2401 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     2461 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     2521 acgttgtaaa acgacggcca gtgccaagct gatctataca ttgaatcaat attggcaatt
     2581 agccatatta gtcattggtt atatagcata aatcaatatt ggctattggc cattgcatac
     2641 gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat gaccgccatg
     2701 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     2761 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2821 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2881 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2941 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc
     3001 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt
     3061 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata
     3121 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     3181 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca
     3241 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     3301 tcagaattaa gcttccacca tggcagaaac ggtatctcca ctgaagcact tcgtgctggc
     3361 aaagaaggcc atcactgcga tcttcggcca gttactggag tttgttactg agggctcaca
     3421 ttttgttgaa gcaacataca ggaacccgga acttgatcgg atagcaaccg aggatgatct
     3481 ggtggagata cagggctaca gaaacaagct cgctgtcatt ggggaggtgc tgtctcggag
     3541 gcatatgaaa gtggcatttt ttggcaggac aagtagtggc aagagctctg tcatcaacgc
     3601 aatgctgtgg gataaagtcc tccccagcgg gattggtcac acaaccaact gcttcctgag
     3661 tgtggaggga actgatggag ataaagccta ccttatgacc gaagggtcag atgagaagaa
     3721 aagcgtgaag actgttaatc agctggccca tgccctccat atggataaag acttgaaagc
     3781 tggttgtctt gtgcacgtat tttggcccaa agcaaaatgt gccctcttga gagatgacct
     3841 ggttttagta gacagcccag gtacagatgt caccacagag ctggacatct ggattgataa
     3901 gttctgcctt gatgctgatg tttttgtttt ggtggcaaac tcggaatcaa cgctgatgaa
     3961 cacggagaaa cattttttcc ataaggtgaa tgagcggctc tccaagccaa acatttttat
     4021 cctgaataat cgttgggatg cttctgcttc agagccagaa tatatggagg atgtgcgcag
     4081 acagcacatg gaaagatgtc ttcacttctt ggtagaagag ctcaaggttg tgagtccttt
     4141 agaagctcgg aatcggatct tttttgtttc agccaaggaa gttctcaact ccagaatgaa
     4201 taaagcccag gggatgccag aaggtggagg ggcacttgca gaaggatttc aggcaagatt
     4261 acaggagttt caaaattttg aacaaacttt tgaggagtgt atctcgcagt cagcagtgaa
     4321 aacaaagttt gaacagcaca ctatcagagc taaacagata ctagacactg tgaaaaacat
     4381 actggactca gtaaacgtgg cagcagcaga gaagagggtt tattcaatgg aagagaggga
     4441 agaccaaatc gacagactgg actttattcg aaaccagatg aaccttttaa caatggatgt
     4501 taagaagaag atcaaggagg tcacggagga ggtggcaaac aaggtttctt gtgcaatgac
     4561 agatgagatt tgtcgcctgt ctgttttggt tgatgagttt tgttctgagt ttcatcctac
     4621 ccccagtgtg ctgaaagtgt ataagagtga attaaataag cacatagaag atggcatggg
     4681 aagaaatttg gctgatcggt gcaccagtga agtcaatgcc tccattctcc aatctcagca
     4741 agaaatcatt gaaaatttga agccactact tccagctggt atacagaata aacttcatac
     4801 attaatccct tgcaaaaagt ttgatctcag ctatgatctc aattgccaca agctgtgttc
     4861 ggattttcaa gaagacattg tgtttcggtt ttccctgggc tggtcttccc ttgtacatcg
     4921 attcctgggt tctacgaatg cacagagggt gctgcttggg ctgtcagagc ctatctttca
     4981 ggtccctaga tctttagctt caactcctga tgctccttct aacccagcag caccagataa
     5041 tgcagcccag gaagagctca tgatcacact gatcacagga ttggcatccc tcacgtctag
     5101 aacctccatg ggcatcatag ttgttggggg cgtgatttgg aaaacagtgg gctggaaact
     5161 catctctgtc accttaagta tgtatggagc tttgtacctt tatgaaagac tgacttggac
     5221 tactcgtgcg aaagagagag cgtttaagca gcagtttgta aactatgcaa ctgaaaaact
     5281 gcagatgatt gtgaagttca caagtgcaaa ctgcagccac caagtccagc aagaaatggc
     5341 caccactttt gctcgactgt gccaacaagt tgatgttact cagaaacatc tggaagaaga
     5401 aattgctaga ttatccaaag agattgacca actggagaaa atacaaaaca attcaaagct
     5461 cttaagaaat aaagctgttc aacttgaacg tgagctggaa aatttttcga agcagtttct
     5521 ccacccgagc ggtggagaat cccctagagg atcccgggct gactacaaag accatgacgg
     5581 tgattataaa gatcatgaca tcgactacaa ggatgacgat gacaagtagt gatcccgggt
     5641 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc
     5701 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct
     5761 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct
     5821 gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca
     5881 ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg
     5941 gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt
     6001 tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc
     6061 ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt
     6121 taaaataact ataccagcag gaggacgtcc agacacagca taggctacct ggccatgccc
     6181 aaccggtggg acatttgagt tgcttgcttg gcactgtcct ctcatgcgtt gggtccactc
     6241 agtagatgcc tgttgaattg ggtacgcggc cagcttggct gtggaatgtg tgtcagttag
     6301 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     6361 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     6421 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     6481 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     6541 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     6601 gaattgatca gcttgggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     6661 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     6721 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     6781 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     6841 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     6901 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     6961 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     7021 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     7081 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     7141 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     7201 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     7261 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     7321 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     7381 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     7441 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     7501 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     7561 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccaccccg
     7621 ggctcgatcc cctcgcgagt tggttcagct gctgcctgag gctggacgac ctcgcggagt
     7681 tctaccggca gtgcaaatcc gtcggcatcc aggaaaccag cagcggctat ccgcgcatcc
     7741 atgcccccga actgcaggag tggggaggca cgatggccgc tttggtcgac ccggacggga
     7801 cgctcctgcg cctgatacag aacgaattgc ttgcaggcat ctcatgagtg tgtcttcccg
     7861 ttttccgcct gaggtcactg cgtggatgga gcgctggcgc ctgctgcgcg acggcgagct
     7921 gctcaccacc cactcgccaa gctggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     7981 taggctccgc cgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     8041 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     8101 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     8161 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     8221 catgtctgga tcaattccct atagtgagtc gtattaaatt cgtaatcatg tcatagctgt
     8281 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     8341 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     8401 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     8461 cggggagagg cggtttgcgt attgggcgct ctt
//
