LOCUS       sequence-338245-        5177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Tom20-CR, an anchor protein of iCMM/NiCMM, in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5177)
  AUTHORS   Miyamoto T, Uosaki H, Mizunoe Y, Han SI, Goto S, Yamanaka D, Masuda 
            M, Yoneyama Y, Nakamura H, Hattori N, Takeuchi Y, Ohno H, Sekiya M, 
            Matsuzaka T, Hakuno F, Takahashi SI, Yahagi N, Ito K, Shimano H
  TITLE     Rapid manipulation of mitochondrial morphology in a living cell with
            iCMM.
  JOURNAL   Cell Rep Methods. 2021 Jul 22;1(4):100052. doi: 
            10.1016/j.crmeth.2021.100052. eCollection 2021 Aug 23.
  PUBMED    35475143
REFERENCE   2  (bases 1 to 5177)
  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: "Cell Rep 
            Methods."; date: "2021-07-22"; volume: "1(4):100052. doi"; pages: " 
            10.1016/j.crmeth.2021.100052. eCollection 2021 Aug 23"
COMMENT     Sequence Label: Tom20-CR
FEATURES             Location/Qualifiers
     source          1..5177
                     /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      612..621
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(750..752,753..755,756..1466)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(795..816)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1056..1075)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1403..1424
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1479..1757
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     polyA_signal    1965..2086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2002..2021)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2056..2075
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2093..2548)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2230..2251)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2442..2461
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2575..2679
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2681..3038
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2681..2872
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2704..2724)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2889..3024
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3073..3867
                     /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             3073..3867
                     /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(3127..3146)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3737..3756
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4055..4074)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4099..4146
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4475..5063
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4964..4983
                     /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 ccggtgaatt cgccaccatg gtgggtcgga acagcgccat cgccgcgggc gtgtgcggtg
      661 ccctcttcat agggtactgc atctactttg accgcaaaag acgaagtgac cccaacttcg
      721 gatccagtgc tggtggtagt gctggtggta tggtgagcaa gggcgaggag ctgttcaccg
      781 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      841 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      901 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctgg ggcgtgcagt
      961 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1021 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1081 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1141 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacatc agccacaacg
     1201 tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca
     1261 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1321 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1381 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1441 ctctcggcat ggacgagctg tacaagggag caagtggaat cctctggcat gagatgtggc
     1501 atgaaggcct ggaagaggca tctcgtttgt actttgggga aaggaacgtg aaaggcatgt
     1561 ttgaggtgct ggagcccttg catgctatga tggaacgggg cccccagact ctgaaggaaa
     1621 catcctttaa tcaggcctat ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt
     1681 acatgaaatc agggaatgtc aaggacctcc tccaagcctg ggacctctat tatcatgtgt
     1741 tccgacgaat ctcaaagagt gctggtggta gtgctggtgg tagtgctggt ggtactggtg
     1801 gtcctcgagc ttaagcttcg aactctgcag tcgacagatc caccggatct agataactga
     1861 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     1921 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     1981 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2041 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta
     2101 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     2161 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     2221 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     2281 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     2341 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     2401 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     2461 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     2521 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2581 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2641 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     2701 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     2761 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     2821 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     2881 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2941 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     3001 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     3061 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3121 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3181 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3241 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3301 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3361 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3421 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3481 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3541 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3601 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3661 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3721 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3781 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3841 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     3901 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3961 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4021 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     4081 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     4141 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     4201 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     4261 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     4321 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4381 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4441 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4501 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4561 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4621 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtatca ccgcctacat
     4681 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4741 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4801 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4861 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4921 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4981 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5041 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5101 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5161 gtattaccgc catgcat
//
