LOCUS       sequence-368442-        5130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of ""dark MitoTrap"" - an FRB-domain targeted to the 
            mitochondria that is the same size as pMito-mCherry-FRB but is 
            non-fluorescent - in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5130)
  AUTHORS   Kuey C, Sittewelle M, Larocque G, Hernandez-Gonzalez M, Royle SJ
  TITLE     Recruitment of clathrin to intracellular membranes is sufficient for
            vesicle formation.
  JOURNAL   Elife. 2022 Jul 19;11. pii: 78929. doi: 10.7554/eLife.78929.
  PUBMED    35852853
REFERENCE   2  (bases 1 to 5130)
  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: "Elife."; 
            date: "2022-07-19"; volume: "11. pii: 78929. doi"; pages: " 
            10.7554/eLife.78929"
COMMENT     Sequence Label: pMito-dCherry-FRB
FEATURES             Location/Qualifiers
     source          1..5130
                     /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      755..764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             761..1465
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSNAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELY"
     primer_bind     complement(906..924)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1174..1193
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1469..1495
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1514..1792
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             1526..1795
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     polyA_signal    1918..2039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1955..1974)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2009..2028
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2046..2501)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2183..2204)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2395..2414
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2528..2632
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2634..2991
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2634..2825
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2657..2677)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2842..2977
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3026..3820
                     /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             3026..3820
                     /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(3080..3099)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3690..3709
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4008..4027)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4052..4099
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4428..5016
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4917..4936
                     /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 ccggactcag atctcgagct caagcttcga attcgccacc atgaagagct tcattacaag
      661 gaacaagaca gccattttgg caaccgttgc tgctacaggt actgccatcg gtgcctacta
      721 ttattacaac caattgcaac aggatccacc ggtcgccacc atggtgagca agggcgagga
      781 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
      841 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
      901 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
      961 ccctcagttc atgtacggct ccaacgccta cgtgaagcac cccgccgaca tccccgacta
     1021 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     1081 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     1141 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
     1201 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     1261 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
     1321 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
     1381 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
     1441 caccggcggc atggacgagc tgtacactta cccctacgac gtgcccgact acgcaggctc
     1501 cgcacgagtg gccatcctct ggcatgagat gtggcatgaa ggcctggaag aggcatctcg
     1561 tttgtacttt ggggaaagga acgtgaaagg catgtttgag gtgctggagc ccttgcatgc
     1621 tatgatggaa cggggccccc agactctgaa ggaaacatcc tttaatcagg cctatggtcg
     1681 agatttaatg gaggcccaag agtggtgcag gaagtacatg aaatcaggga atgtcaagga
     1741 cctcacccaa gcctgggacc tctattatca tgtgttccga cgaatctcaa agcaggccgg
     1801 ctgataatct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     1861 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     1921 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     1981 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     2041 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2101 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2161 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2221 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     2281 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     2341 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     2401 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     2461 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     2521 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2581 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     2641 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2701 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2761 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2821 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     2881 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     2941 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     3001 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3061 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3121 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3181 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     3241 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3301 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3361 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3421 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3481 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3541 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     3601 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3661 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3721 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     3781 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     3841 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     3901 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     3961 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     4021 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     4081 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     4141 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     4201 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     4261 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4321 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4381 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4441 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4501 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4561 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4621 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4681 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4741 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4801 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4861 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4921 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4981 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5041 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5101 tctgtggata accgtattac cgccatgcat
//
