LOCUS       sequence-384016-        7211 bp    DNA     circular SYN 14-APR-2025
DEFINITION  mammalian expression of TurboID tagged Mfn1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7211)
  AUTHORS   Nguyen TT, Voeltz GK
  TITLE     An ER phospholipid hydrolase drives ER-associated mitochondrial 
            constriction for fission and fusion.
  JOURNAL   Elife. 2022 Nov 30;11:e84279. doi: 10.7554/eLife.84279.
  PUBMED    36448541
REFERENCE   2  (bases 1 to 7211)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2022-11-30"; volume: "11:e84279. doi"; pages: " 
            10.7554/eLife.84279"
COMMENT     Sequence Label: V5-TurboID-Mfn1
FEATURES             Location/Qualifiers
     source          1..7211
                     /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"
     CDS             609..650
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             657..1613
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     polyA_signal    3999..4120
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4036..4055)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4090..4109
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4127..4582)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4264..4285)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4476..4495
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4609..4713
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4715..5072
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4715..4906
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4738..4758)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4923..5058
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5107..5901
                     /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             5107..5901
                     /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(5161..5180)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5771..5790
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6089..6108)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6133..6180
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6509..7097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6998..7017
                     /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 ccggtatggg caagcccatc cccaaccccc tgctgggcct ggacagcacc gctagcaaag
      661 acaatactgt gcctctgaag ctgatcgctc tcctggctaa tggcgagttc catagtggcg
      721 aacagctggg agaaaccctg ggcatgtcca gggccgctat caacaagcac attcagactc
      781 tgcgcgactg gggcgtggac gtgttcaccg tgcccggaaa gggctactct ctgcccgagc
      841 ctatcccgct gctgaacgct aaacagattc tgggacagct ggacggcggg agcgtggcag
      901 tcctgcctgt ggtcgactcc accaatcagt acctgctgga tcgaatcggc gagctgaaga
      961 gtggggatgc ttgcattgca gaatatcagc aggcagggag aggaagcaga gggaggaaat
     1021 ggttctctcc ttttggagct aacctgtacc tgagtatgtt ttggcgcctg aagcggggac
     1081 cagcagcaat cggcctgggc ccggtcatcg gaattgtcat ggcagaagcg ctgcgaaagc
     1141 tgggagcaga caaggtgcga gtcaaatggc ccaatgacct gtatctgcag gatagaaagc
     1201 tggcaggcat cctggtggag ctggccggaa taacaggcga tgctgcacag atcgtcattg
     1261 gcgccgggat taacgtggct atgaggcgcg tggaggaaag cgtggtcaat cagggctgga
     1321 tcacactgca ggaagcaggg attaacctgg acaggaatac tctggccgct acgctgatcc
     1381 gagagctgcg ggcagccctg gaactgttcg agcaggaagg cctggctcca tatctgccac
     1441 ggtgggagaa gctggataac ttcatcaata gacccgtgaa gctgatcatt ggggacaaag
     1501 agattttcgg gattagccgg gggattgata aacagggagc cctgctgctg gaacaggacg
     1561 gagttatcaa accctggatg ggcggagaaa tcagtctgcg gtctgccgaa aagctgcagt
     1621 ctcgagctca agcttcgaat tcgatggcag aacctgtttc tccactgaag cactttgtgc
     1681 tggctaagaa ggcgattact gcaatctttg accagttact ggagtttgtt actgaaggat
     1741 cacattttgt tgaagcaaca tataagaatc cggaacttga tcgaatagcc actgaagatg
     1801 atctggtaga aatgcaagga tataaagaca agctttccat cattggtgag gtgctatctc
     1861 ggagacacat gaaggtggca ttttttggca ggacaagcag tgggaagagc tctgttatca
     1921 atgcaatgtt gtgggataaa gttctcccta gtgggattgg ccatataacc aattgcttcc
     1981 taagtgttga aggaactgat ggagataaag cctatcttat gacagaagga tcagatgaaa
     2041 aaaagagtgt gaagacagtt aatcaactgg cccatgccct tcacatggac aaagatttga
     2101 aagctggctg tcttgtacgt gtgttttggc caaaagcaaa atgtgccctc ttgagagatg
     2161 acctggtgtt agtagacagt ccaggcacag atgtcactac agagctggat agctggattg
     2221 ataagttttg cctagatgct gatgtctttg ttttggtcgc aaactctgaa tcaacactaa
     2281 tgaatacgga aaaacacttt tttcacaagg tgaatgagcg gctttccaag cctaatattt
     2341 tcattctcaa taatcgttgg gatgcctctg catcagagcc agaatatatg gaagacgtac
     2401 gcagacagca catggaaaga tgcctgcatt tcttggtgga ggagctcaaa gttgtaaatg
     2461 ctttagaagc acagaatcgt atcttctttg tttcagcaaa ggaagttctt agtgctagaa
     2521 agcaaaaagc acaggggatg ccagaaagtg gtgtggcact tgctgaagga tttcatgcaa
     2581 gattacagga atttcagaat tttgaacaaa tctttgagga gtgtatctcg cagtcagcag
     2641 tgaaaacaaa gttcgaacag cacactatca gagctaaaca gatactagct actgtgaaaa
     2701 acataatgga ttcagtaaac ctggcagctg aagataaaag gcattattca gtggaagaga
     2761 gggaagacca aattgataga ctggacttta ttcgaaacca gatgaacctt ttaacactgg
     2821 atgttaagaa aaaaatcaag gaggttaccg aggaggtggc aaacaaagtt tcatgtgcaa
     2881 tgacagatga aatttgtcga ctgtctgttt tggttgatga attttgttca gagtttcatc
     2941 ctaatccaga tgtattaaaa atatataaaa gtgaattaaa taagcacata gaggatggta
     3001 tgggaagaaa tttggctgat cgatgcaccg atgaagtaaa cgccttagtg cttcagaccc
     3061 agcaagaaat tattgaaaat ttgaagccat tacttccagc tggtatacag gataaactac
     3121 atacactgat cccttgcaag aaatttgatc tcagttataa tctaaattac cacaagttat
     3181 gttcagattt tcaagaggat attgtatttc gtttttccct gggctggtct tcccttgtac
     3241 atcgattttt gggccctaga aatgctcaaa gggtgctcct aggattatca gagcctatct
     3301 ttcagctccc tagatcttta gcttctactc ccactgctcc taccactcca gcaacgccag
     3361 ataatgcatc acaggaagaa ctcatgatta cattagtaac aggattggcg tccgttacat
     3421 ctagaacttc tatgggcatc attattgttg gaggagtgat ttggaaaact ataggctgga
     3481 aactcctatc tgtttcatta actatgtatg gagctttgta tctttatgaa agactgagct
     3541 ggaccaccca tgccaaggag cgagccttta aacagcagtt tgtaaactat gcaactgaaa
     3601 aactgaggat gattgttagc tccacgagtg caaactgcag tcaccaagta aaacaacaaa
     3661 tagctaccac ttttgctcgc ctgtgccaac aagttgatat tactcaaaaa cagctggaag
     3721 aagaaattgc tagattaccc aaagaaatag atcagttgga gaaaatacaa aacaattcaa
     3781 agctcttaag aaataaagct gttcaacttg aaaatgagct ggagaatttt actaagcagt
     3841 ttctaccttc aagcaatgaa gaatcctaag gatccaccgg atctagataa ctgatcataa
     3901 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     3961 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     4021 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4081 attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat tgtaagcgtt
     4141 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     4201 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     4261 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     4321 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     4381 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     4441 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     4501 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     4561 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4621 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4681 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     4741 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4801 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     4861 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     4921 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4981 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     5041 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     5101 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     5161 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     5221 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     5281 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     5341 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     5401 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     5461 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     5521 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     5581 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     5641 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     5701 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5761 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5821 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5881 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5941 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     6001 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     6061 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     6121 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
     6181 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
     6241 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
     6301 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
     6361 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6421 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6481 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6541 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6601 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6661 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6721 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6781 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6841 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6901 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6961 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7021 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7081 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7141 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7201 ccgccatgca t
//
