LOCUS       sequence-379622-        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-T564A
FEATURES             Location/Qualifiers
     source          1..8493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(4..26)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    15..31
                     /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(39..45,46..63,64..69))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    84..105
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(222..239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(393..981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(473..492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1152..1943,1944..2012))
                     /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     1775..1794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2013..2117)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2144..2599
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2231..2250)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2441..2462
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2725..2747
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2739..2756
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2740..2756
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2917..3296
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3297..3500
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3451..3471
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             5775..5840
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    5851..6473
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(6050..6069)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(6497..6517)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6515..6812
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             6866..7660
                     /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(6920..6939)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7530..7549
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    8312..8446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8349..8368)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8403..8422
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8451..8470)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8452..8470)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(join(8484..8493,1..7))
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(join(8484..8493,1..7))
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
       61 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
      121 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      181 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
      241 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
      301 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
      361 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
      421 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
      481 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
      541 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
      601 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
      661 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
      721 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
      781 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
      841 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
      901 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
      961 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     1021 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     1081 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     1141 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     1201 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     1261 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     1321 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     1381 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     1441 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     1501 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     1561 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     1621 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     1681 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     1741 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     1801 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     1861 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     1921 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     1981 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     2041 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     2101 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc
     2161 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2221 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2281 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2341 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2401 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2461 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2521 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2581 atattaacgc ttacaatttc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     2641 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     2701 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca
     2761 agctgatcta tacattgaat caatattggc aattagccat attagtcatt ggttatatag
     2821 cataaatcaa tattggctat tggccattgc atacgttgta tctatatcat aatatgtaca
     2881 tttatattgg ctcatgtcca atatgaccgc catgttgaca ttgattattg actagttatt
     2941 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     3001 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     3061 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     3121 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtccgc
     3181 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     3241 tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     3301 tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     3361 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     3421 caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt gtacggtggg
     3481 aggtctatat aagcagagct cgtttagtga accgtcagaa ttaagcttcc accatggcag
     3541 aaacggtatc tccactgaag cacttcgtgc tggcaaagaa ggccatcact gcgatcttcg
     3601 gccagttact ggagtttgtt actgagggct cacattttgt tgaagcaaca tacaggaacc
     3661 cggaacttga tcggatagca accgaggatg atctggtgga gatacagggc tacagaaaca
     3721 agctcgctgt cattggggag gtgctgtctc ggaggcatat gaaagtggca ttttttggca
     3781 ggacaagtag tggcaagagc tctgtcatca acgcaatgct gtgggataaa gtcctcccca
     3841 gcgggattgg tcacacaacc aactgcttcc tgagtgtgga gggaactgat ggagataaag
     3901 cctaccttat gaccgaaggg tcagatgaga agaaaagcgt gaagactgtt aatcagctgg
     3961 cccatgccct ccatatggat aaagacttga aagctggttg tcttgtgcac gtattttggc
     4021 ccaaagcaaa atgtgccctc ttgagagatg acctggtttt agtagacagc ccaggtacag
     4081 atgtcaccac agagctggac atctggattg ataagttctg ccttgatgct gatgtttttg
     4141 ttttggtggc aaactcggaa tcaacgctga tgaacacgga gaaacatttt ttccataagg
     4201 tgaatgagcg gctctccaag ccaaacattt ttatcctgaa taatcgttgg gatgcttctg
     4261 cttcagagcc agaatatatg gaggatgtgc gcagacagca catggaaaga tgtcttcact
     4321 tcttggtaga agagctcaag gttgtgagtc ctttagaagc tcggaatcgg atcttttttg
     4381 tttcagccaa ggaagttctc aactccagaa tgaataaagc ccaggggatg ccagaaggtg
     4441 gaggggcact tgcagaagga tttcaggcaa gattacagga gtttcaaaat tttgaacaaa
     4501 cttttgagga gtgtatctcg cagtcagcag tgaaaacaaa gtttgaacag cacactatca
     4561 gagctaaaca gatactagac actgtgaaaa acatactgga ctcagtaaac gtggcagcag
     4621 cagagaagag ggtttattca atggaagaga gggaagacca aatcgacaga ctggacttta
     4681 ttcgaaacca gatgaacctt ttaacaatgg atgttaagaa gaagatcaag gaggtcacgg
     4741 aggaggtggc aaacaaggtt tcttgtgcaa tgacagatga gatttgtcgc ctgtctgttt
     4801 tggttgatga gttttgttct gagtttcatc ctacccccag tgtgctgaaa gtgtataaga
     4861 gtgaattaaa taagcacata gaagatggca tgggaagaaa tttggctgat cggtgcacca
     4921 gtgaagtcaa tgcctccatt ctccaatctc agcaagaaat cattgaaaat ttgaagccac
     4981 tacttccagc tggtatacag aataaacttc atacattaat cccttgcaaa aagtttgatc
     5041 tcagctatga tctcaattgc cacaagctgt gttcggattt tcaagaagac attgtgtttc
     5101 ggttttccct gggctggtct tcccttgtac atcgattcct gggttctacg aatgcacaga
     5161 gggtgctgct tgggctgtca gagcctatct ttcaggtccc tagatcttta gcttcaactc
     5221 ctgctgctcc ttctaaccca gcagcaccag ataatgcagc ccaggaagag ctcatgatca
     5281 cactgatcac aggattggca tccctcacgt ctagaacctc catgggcatc atagttgttg
     5341 ggggcgtgat ttggaaaaca gtgggctgga aactcatctc tgtcacctta agtatgtatg
     5401 gagctttgta cctttatgaa agactgactt ggactactcg tgcgaaagag agagcgttta
     5461 agcagcagtt tgtaaactat gcaactgaaa aactgcagat gattgtgaag ttcacaagtg
     5521 caaactgcag ccaccaagtc cagcaagaaa tggccaccac ttttgctcga ctgtgccaac
     5581 aagttgatgt tactcagaaa catctggaag aagaaattgc tagattatcc aaagagattg
     5641 accaactgga gaaaatacaa aacaattcaa agctcttaag aaataaagct gttcaacttg
     5701 aacgtgagct ggaaaatttt tcgaagcagt ttctccaccc gagcagtgga gaatccccta
     5761 gaggatcccg ggctgactac aaagaccatg acggtgatta taaagatcat gacatcgact
     5821 acaaggatga cgatgacaag tagtgatccc gggtggcatc cctgtgaccc ctccccagtg
     5881 cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta
     5941 agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg
     6001 gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa
     6061 ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa
     6121 gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc
     6181 agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc
     6241 aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg
     6301 tgaaccactg ctcccttccc tgtccttctg attttaaaat aactatacca gcaggaggac
     6361 gtccagacac agcataggct acctggccat gcccaaccgg tgggacattt gagttgcttg
     6421 cttggcactg tcctctcatg cgttgggtcc actcagtaga tgcctgttga attgggtacg
     6481 cggccagctt ggctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     6541 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     6601 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     6661 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     6721 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     6781 ctattccaga agtagtgagg aggctttttt ggaggaattg atcagcttgg gatctgatca
     6841 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     6901 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     6961 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     7021 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     7081 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     7141 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     7201 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     7261 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     7321 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     7381 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     7441 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     7501 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     7561 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     7621 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     7681 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     7741 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     7801 ggggatctca tgctggagtt cttcgcccac cccgggctcg atcccctcgc gagttggttc
     7861 agctgctgcc tgaggctgga cgacctcgcg gagttctacc ggcagtgcaa atccgtcggc
     7921 atccaggaaa ccagcagcgg ctatccgcgc atccatgccc ccgaactgca ggagtgggga
     7981 ggcacgatgg ccgctttggt cgacccggac gggacgctcc tgcgcctgat acagaacgaa
     8041 ttgcttgcag gcatctcatg agtgtgtctt cccgttttcc gcctgaggtc actgcgtgga
     8101 tggagcgctg gcgcctgctg cgcgacggcg agctgctcac cacccactcg ccaagctgga
     8161 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgccgatca taatcagcca
     8221 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     8281 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     8341 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     8401 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaatt ccctatagtg
     8461 agtcgtatta aattcgtaat catgtcatag ctg
//
