LOCUS       sequence-416458-        5177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mitochondrial OM marker and ""anchor"" in FKBP-FRB CID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5177)
  AUTHORS   Nakamura H, Rho E, Lee CT, Itoh K, Deng D, Watanabe S, Razavi S, 
            Matsubayashi HT, Zhu C, Jung E, Rangamani P, Watanabe S, Inoue T
  TITLE     ActuAtor, a Listeria-inspired molecular tool for physical 
            manipulation of intracellular organizations through de novo actin 
            polymerization.
  JOURNAL   Cell Rep. 2023 Sep 2:113089. doi: 10.1016/j.celrep.2023.113089.
  PUBMED    37734382
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. 
            2023 Sep 2:113089. doi: 10.1016/j.celrep.2023.113089."
COMMENT     Sequence Label: Tom20-ECFP-FRB
FEATURES             Location/Qualifiers
     source          1..5177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(383..971)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(463..482)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    1300..1347
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1372..1391
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(1579..2373)
                     /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             complement(1579..2373)
                     /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(1690..1709)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2300..2319
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2408..2765)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2422..2557
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2574..2765
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2722..2742
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(2767..2871)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2898..3353
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2985..3004)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3195..3216
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    3360..3481
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3371..3390)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3425..3444
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3689..3967)
                     /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"
     CDS             complement(join(3980..4690,4691..4693,4694..4696))
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4022..4043)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4371..4390
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4630..4651
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4825..4834
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4878..5081)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4907..4927)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        join(5082..5177,1..208)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg
       61 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
      121 tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg
      181 cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga
      241 ccccgtaatt gattactatt aataactaat gcatggcggt aatacggtta tccacagaat
      301 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
      361 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      421 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
      481 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
      541 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
      601 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
      661 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
      721 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgata
      781 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
      841 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
      901 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
      961 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     1021 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     1081 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaacct gaggctatgg
     1141 cagggcctgc cgccccgacg ttggctgcga gccctgggcc ttcacccgaa cttggggggt
     1201 ggggtgggga aaaggaagaa acgcgggcgt attggcccca atggggtctc ggtggggtat
     1261 cgacagagtg ccagccctgg gaccgaaccc cgcgtttatg aacaaacgac ccaacaccgt
     1321 gcgttttatt ctgtcttttt attgccgtca tagcgcgggt tccttccggt attgtctcct
     1381 tccgtgtttc agttagcctc cccctagggt gggcgaagaa ctccagcatg agatccccgc
     1441 gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga
     1501 aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt
     1561 cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     1621 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag
     1681 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag
     1741 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca
     1801 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct tgagcctggc
     1861 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
     1921 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
     1981 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
     2041 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag
     2101 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
     2161 ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc
     2221 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
     2281 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
     2341 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg
     2401 atcgatcttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     2461 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     2521 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     2581 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     2641 actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     2701 gggagcctgg ggactttcca caccctaact gacacacatt ccacagctgg ttctttccgc
     2761 ctcaggactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     2821 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     2881 ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2941 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3001 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3061 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3121 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3181 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3241 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3301 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttacgcctt
     3361 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     3421 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     3481 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     3541 ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcagt tatctagatc
     3601 cggtggatct gtcgactgca gagttcgaag cttgagctcg aggaccacca gtaccaccag
     3661 cactaccacc agcactacca ccagcactct ttgagattcg tcggaacaca tgataataga
     3721 ggtcccaggc ttggaggagg tccttgacat tccctgattt catgtacttc ctgcaccact
     3781 cttgggcctc cattaaatct cgaccatagg cctgattaaa ggatgtttcc ttcagagtct
     3841 gggggccccg ttccatcata gcatgcaagg gctccagcac ctcaaacatg cctttcacgt
     3901 tcctttcccc aaagtacaaa cgagatgcct cttccaggcc ttcatgccac atctcatgcc
     3961 agaggattcc acttgctccc ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg
     4021 tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg
     4081 actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt
     4141 tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga
     4201 agttggcctt gatgccgttc ttctgcttgt cggcggtgat atagacgttg tggctgatgt
     4261 agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca
     4321 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt
     4381 tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact
     4441 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccccagg
     4501 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact
     4561 tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt
     4621 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
     4681 cgcccttgct caccatacca ccagcactac caccagcact ggatccgaag ttggggtcac
     4741 ttcgtctttt gcggtcaaag tagatgcagt accctatgaa gagggcaccg cacacgcccg
     4801 cggcgatggc gctgttccga cccaccatgg tggcgaattc accggtagcg ctagcggatc
     4861 tgacggttca ctaaaccagc tctgcttata tagacctccc accgtacacg cctaccgccc
     4921 atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc
     4981 caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac
     5041 cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact
     5101 aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc
     5161 caggcgggcc atttacc
//
