LOCUS       sequence-393638-        5065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of fluorescently-tagged marker for 
            mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5065)
  AUTHORS   Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur ELF
  TITLE     Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and 
            ARF6 GTPase disrupts the axonal transport of autophagosomes.
  JOURNAL   Cell Rep. 2023 May 1;42(5):112448. doi: 
            10.1016/j.celrep.2023.112448.
  PUBMED    37133994
REFERENCE   2  (bases 1 to 5065)
  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.";
            date: "2023-05-1"; volume: "42(5):112448. doi"; pages: " 
            10.1016/j.celrep.2023.112448"
COMMENT     Sequence Label: PGK 4xMito-mEmerald
FEATURES             Location/Qualifiers
     source          1..5065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        86..596
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     515..533
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     615..634
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        615..633
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             647..721
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             743..817
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             839..913
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             935..1009
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      1076..1085
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1082..1084,1085..1087,1088..1801)
                     /codon_start=1
                     /product="brightly fluorescent monomeric variant of GFP"
                     /label=mEmerald
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1127..1148)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1388..1407)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1735..1756
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1923..2044
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1960..1979)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2014..2033
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2051..2506)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2188..2209)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2400..2419
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2533..2637
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2639..2996
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2639..2830
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2662..2682)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2847..2982
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3031..3825
                     /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             3031..3825
                     /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(3085..3104)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3695..3714
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4013..4032)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4057..4104
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4433..5021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4922..4941
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
       61 cgccatgcat tagttattaa tccacggggt tggggttgcg ccttttccaa ggcagccctg
      121 ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc
      181 ctgggtctcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc
      241 ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt
      301 tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg
      361 gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct
      421 gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg
      481 gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg
      541 agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccagggga
      601 tccgcggccg ctagtaatac gactcactat agggagaccc aagcttatgt ccgtcctgac
      661 gccgctgctg ctgcggggct tgacaggctc ggcccggcgg ctcccagtgc cgcgcgccaa
      721 gatccattcg ttgggggatc ccatgtccgt cctgacgccg ctgctgctgc ggggcttgac
      781 aggctcggcc cggcggctcc cagtgccgcg cgccaagatc cattcgttgg ggaagcttat
      841 gtccgtcctg acgccgctgc tgctgcgggg cttgacaggc tcggcccggc ggctcccagt
      901 gccgcgcgcc aagatccatt cgttggggga tcccatgtcc gtcctgacgc cgctgctgct
      961 gcggggcttg acaggctcgg cccggcggct cccagtgccg cgcgccaaga tccattcgtt
     1021 ggggaagctt cgaattctgc agtcgacggt accgcgggcc cgggatccac cggtcgccac
     1081 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1141 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1201 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1261 cctcgtgacc accttgacct acggcgtgca gtgcttcgcc cgctaccccg accacatgaa
     1321 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1381 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1441 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1501 caagctggag tacaactaca acagccacaa ggtctatatc accgccgaca agcagaagaa
     1561 cggcatcaag gtgaacttca agacccgcca caacatcgag gacggcagcg tgcagctcgc
     1621 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1681 ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
     1741 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     1801 agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     1861 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     1921 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1981 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     2041 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     2101 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     2161 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     2221 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     2281 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     2341 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     2401 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     2461 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     2521 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2581 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     2641 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2701 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2761 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2821 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2881 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     2941 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     3001 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     3061 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3121 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     3181 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     3241 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3301 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     3361 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3421 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3481 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3541 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3601 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3661 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3721 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3781 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     3841 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     3901 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     3961 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     4021 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     4081 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     4141 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     4201 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     4261 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     4321 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4381 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4441 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4501 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4561 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4621 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4681 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4741 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4801 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4861 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4921 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4981 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5041 ttttacggtt cctggccttt tgctg
//
