LOCUS       sequence-309908-        6933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression and labeling of mutant mitofusin 1 (E209A) 
            deficient in GTP hydrolysis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6933)
  AUTHORS   Abrisch RG, Gumbin SC, Wisniewski BT, Lackner LL, Voeltz GK
  TITLE     Fission and fusion machineries converge at ER contact sites to 
            regulate mitochondrial morphology.
  JOURNAL   J Cell Biol. 2020 Apr 6;219(4). pii: 133843. doi: 
            10.1083/jcb.201911122.
  PUBMED    32328629
REFERENCE   2  (bases 1 to 6933)
  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 
            Biol."; date: "2020-04-6"; volume: "219(4). pii: 133843. doi"; 
            pages: " 10.1083/jcb.201911122"
COMMENT     Sequence Label: GFP-Mfn1-E209A
FEATURES             Location/Qualifiers
     source          1..6933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1329
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    3721..3842
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3758..3777)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3812..3831
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3849..4304)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3986..4007)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4198..4217
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4331..4435
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4437..4794
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4437..4628
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4460..4480)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4645..4780
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4829..5623
                     /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             4829..5623
                     /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(4883..4902)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5493..5512
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5811..5830)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5855..5902
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6231..6819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6720..6739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgcc gagctgttca ccggcatcgt gcccatcctg
      661 atcgagctga atggcgatgt gaatggccac aagttcagcg tgagcggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcctgtg
      781 ccctggccca ccctggtgac caccctgagc tacggcgtgc agtgcttctc acgctacccc
      841 gatcacatga agcagcacga cttcttcaag agcgccatgc ctgagggcta catccaggag
      901 cgcaccatct tcttcgagga tgacggcaac tacaagtcgc gcgccgaggt gaagttcgag
      961 ggcgataccc tggtgaatcg catcgagctg accggcaccg atttcaagga ggatggcaac
     1021 atcctgggca ataagatgga gtacaactac aacgcccaca atgtgtacat catgaccgac
     1081 aaggccaaga atggcatcaa ggtgaacttc aagatccgcc acaacatcga ggatggcagc
     1141 gtgcagctgg ccgaccacta ccagcagaat acccccatcg gcgatggccc tgtgctgctg
     1201 cccgataacc actacctgtc cacccagagc gccctgtcca aggaccccaa cgagaagcgc
     1261 gatcacatga tctacttcgg cttcgtgacc gccgccgcca tcacccacgg catggatgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attcgatggc agaacctgtt
     1381 tctccactga agcactttgt gctggctaag aaggcgatta ctgcaatctt tgaccagtta
     1441 ctggagtttg ttactgaagg atcacatttt gttgaagcaa catataagaa tccggaactt
     1501 gatcgaatag ccactgaaga tgatctggta gaaatgcaag gatataaaga caagctttcc
     1561 atcattggtg aggtgctatc tcggagacac atgaaggtgg cattttttgg caggacaagc
     1621 agtgggaaga gctctgttat caatgcaatg ttgtgggata aagttctccc tagtgggatt
     1681 ggccatataa ccaattgctt cctaagtgtt gaaggaactg atggagataa agcctatctt
     1741 atgacagaag gatcagatga aaaaaagagt gtgaagacag ttaatcaact ggcccatgcc
     1801 cttcacatgg acaaagattt gaaagctggc tgtcttgtac gtgtgttttg gccaaaagca
     1861 aaatgtgccc tcttgagaga tgacctggtg ttagtagaca gtccaggcac agatgtcact
     1921 acagagctgg atagctggat tgataagttt tgcctagatg ctgatgtctt tgttttggtc
     1981 gcaaactctg catcaacact aatgaatacg gaaaaacact tttttcacaa ggtgaatgag
     2041 cggctttcca agcctaatat tttcattctc aataatcgtt gggatgcctc tgcatcagag
     2101 ccagaatata tggaagacgt acgcagacag cacatggaaa gatgcctgca tttcttggtg
     2161 gaggagctca aagttgtaaa tgctttagaa gcacagaatc gtatcttctt tgtttcagca
     2221 aaggaagttc ttagtgctag aaagcaaaaa gcacagggga tgccagaaag tggtgtggca
     2281 cttgctgaag gatttcatgc aagattacag gaatttcaga attttgaaca aatctttgag
     2341 gagtgtatct cgcagtcagc agtgaaaaca aagttcgaac agcacactat cagagctaaa
     2401 cagatactag ctactgtgaa aaacataatg gattcagtaa acctggcagc tgaagataaa
     2461 aggcattatt cagtggaaga gagggaagac caaattgata gactggactt tattcgaaac
     2521 cagatgaacc ttttaacact ggatgttaag aaaaaaatca aggaggttac cgaggaggtg
     2581 gcaaacaaag tttcatgtgc aatgacagat gaaatttgtc gactgtctgt tttggttgat
     2641 gaattttgtt cagagtttca tcctaatcca gatgtattaa aaatatataa aagtgaatta
     2701 aataagcaca tagaggatgg tatgggaaga aatttggctg atcgatgcac cgatgaagta
     2761 aacgccttag tgcttcagac ccagcaagaa attattgaaa atttgaagcc attacttcca
     2821 gctggtatac aggataaact acatacactg atcccttgca agaaatttga tctcagttat
     2881 aatctaaatt accacaagtt atgttcagat tttcaagagg atattgtatt tcgtttttcc
     2941 ctgggctggt cttcccttgt acatcgattt ttgggcccta gaaatgctca aagggtgctc
     3001 ctaggattat cagagcctat ctttcagctc cctagatctt tagcttctac tcccactgct
     3061 cctaccactc cagcaacgcc agataatgca tcacaggaag aactcatgat tacattagta
     3121 acaggattgg cgtccgttac atctagaact tctatgggca tcattattgt tggaggagtg
     3181 atttggaaaa ctataggctg gaaactccta tctgtttcat taactatgta tggagctttg
     3241 tatctttatg aaagactgag ctggaccacc catgccaagg agcgagcctt taaacagcag
     3301 tttgtaaact atgcaactga aaaactgagg atgattgtta gctccacgag tgcaaactgc
     3361 agtcaccaag taaaacaaca aatagctacc acttttgctc gcctgtgcca acaagttgat
     3421 attactcaaa aacagctgga agaagaaatt gctagattac ccaaagaaat agatcagttg
     3481 gagaaaatac aaaacaattc aaagctctta agaaataaag ctgttcaact tgaaaatgag
     3541 ctggagaatt ttactaagca gtttctacct tcaagcaatg aagaatccta aggatccacc
     3601 ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     3661 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     3721 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3781 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3841 taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     3901 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     3961 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     4021 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     4081 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     4141 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     4201 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     4261 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     4321 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4381 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga
     4441 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     4501 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     4561 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4621 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4681 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4741 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga
     4801 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     4861 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4921 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4981 cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc
     5041 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     5101 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     5161 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     5221 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     5281 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     5341 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     5401 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     5461 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     5521 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     5581 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     5641 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     5701 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     5761 cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg
     5821 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
     5881 cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     5941 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     6001 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     6061 gccatagcct caggttactc atatatactt tagattgatt taaaacttca tttttaattt
     6121 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6181 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6241 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6301 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6361 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6421 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6481 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6541 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6601 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6661 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6721 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6781 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6841 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6901 gattctgtgg ataaccgtat taccgccatg cat
//
