LOCUS       sequence-421789-        6033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expresssion of Parkin, internally tagged with 
            mKO2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6033)
  AUTHORS   Waters CS, Angenent SB, Altschuler SJ, Wu LF
  TITLE     A PINK1 input threshold arises from positive feedback in the 
            PINK1/Parkin mitophagy decision circuit.
  JOURNAL   Cell Rep. 2023 Oct 31;42(10):113260. doi: 
            10.1016/j.celrep.2023.113260. Epub 2023 Oct 17.
  PUBMED    37851575
REFERENCE   2  (bases 1 to 6033)
  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: "2023-10-31"; volume: "42(10):113260. doi"; pages: " 
            10.1016/j.celrep.2023.113260. Epub 2023 Oct 17"
COMMENT     Sequence Label: pCMV-Parkin-A92mKO2
FEATURES             Location/Qualifiers
     source          1..6033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        33..336
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        337..540
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     491..511
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             876..1526
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="VSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVT
                     MAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSAS
                     VSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL
                     KLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             876..1526
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="VSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVT
                     MAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSAS
                     VSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL
                     KLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    2793..2914
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2830..2849)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2884..2903
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2921..3376)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3058..3079)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3270..3289
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3403..3507
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3509..3866
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3509..3700
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3532..3552)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3717..3852
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3901..4695
                     /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             3901..4695
                     /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(3955..3974)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4565..4584
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4883..4902)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4927..4974
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5303..5891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5792..5811
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
       61 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      121 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      181 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      241 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      301 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      361 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      421 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      481 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      541 ggtttagtga accgtcagat ccgctagcgc taccggtcgc caccatgata gtgtttgtca
      601 ggttcaactc cagccatggt ttcccagtgg aggtcgattc tgacaccagc atcttccagc
      661 tcaaggaggt ggttgctaag cgacaggggg ttccggctga ccagttgcgt gtgattttcg
      721 cagggaagga gctgaggaat gactggactg tgcagaattg tgacctggat cagcagagca
      781 ttgttcacat tgtgcagaga ccgtggagaa aaggtcaaga aatgaatgca actggaggcg
      841 acgaccccag aaacgcggcg ggtgctggtt ctggcgtgag cgtgatcaag cccgagatga
      901 agatgaggta ctacatggac ggctccgtca atgggcatga gttcacaatc gagggtgagg
      961 gcacaggcag accttacgag ggacatcagg agatgacact gcgcgtcaca atggccgagg
     1021 gcgggccaat gcctttcgcc ttcgacctgg tgtcccacgt gttctgttac ggccacagag
     1081 tttttaccaa gtacccagaa gagatcccag actatttcaa gcaggccttt cctgagggcc
     1141 tgtcctggga gaggtccctg gagttcgagg acggcggctc cgcctccgtg agcgcccaca
     1201 tcagcctgag gggcaacacc ttctaccaca agtccaagtt caccggcgtg aacttccccg
     1261 ccgacggccc catcatgcag aaccagagcg tggactggga gccctccacc gagaagatca
     1321 ccgccagcga cggcgtgctg aagggcgacg tgaccatgta cctgaagctg gagggcggcg
     1381 gcaaccacaa gtgccagatg aagaccacct acaaggccgc caaggagatc ctggagatgc
     1441 ccggcgacca ctacatcggc cacaggctgg tgaggaagac cgagggcaac atcaccgagc
     1501 aggtggagga cgccgtggcc cactcagcag gatcaggagg tggaggctgt gagcgggagc
     1561 cccagagctt gactcgggtg gacctcagca gctcagtcct cccaggagac tctgtggggc
     1621 tggctgtcat tctgcacact gacagcagga aggactcacc accagctgga agtccagcag
     1681 gtagatcaat ctacaacagc ttttatgtgt attgcaaagg cccctgtcaa agagtgcagc
     1741 cgggaaaact cagggtacag tgcagcacct gcaggcaggc aacgctcacc ttgacccagg
     1801 gtccatcttg ctgggatgat gttttaattc caaaccggat gagtggtgaa tgccaatccc
     1861 cacactgccc tgggactagt gcagaatttt tctttaaatg tggagcacac cccacctctg
     1921 acaaggaaac atcagtagct ttgcacctga tcgcaacaaa tagtcggaac atcacttgca
     1981 ttacgtgcac agacgtcagg agccccgtcc tggttttcca gtgcaactcc cgccacgtga
     2041 tttgcttaga ctgtttccac ttatactgtg tgacaagact caatgatcgg cagtttgtcc
     2101 acgaccctca gcttggctac tccctgcctt gtgtggctgg ctgtcccaac tccttgatta
     2161 aagagctcca tcacttcagg attctgggag aagagcagta caaccggtac cagcagtatg
     2221 gtgcagagga gtgtgtcctg cagatggggg gcgtgttatg cccccgccct ggctgtggag
     2281 cggggctgct gccggagcct gaccagagga aagtcacctg cgaagggggc aatggcctgg
     2341 gctgtgggtt tgccttctgc cgggaatgta aagaagcgta ccatgaaggg gagtgcagtg
     2401 ccgtatttga agcctcagga acaactactc aggcctacag agtcgatgaa agagccgccg
     2461 agcaggctcg ttgggaagca gcctccaaag aaaccatcaa gaaaaccacc aagccctgtc
     2521 cccgctgcca tgtaccagtg gaaaaaaatg gaggctgcat gcacatgaag tgtccgcagc
     2581 cccagtgcag gctcgagtgg tgctggaact gtggctgcga gtggaaccgc gtctgcatgg
     2641 gggaccactg gttcgacgtg taaggatcca ccggatctag ataactgatc ataatcagcc
     2701 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     2761 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2821 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2881 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt
     2941 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     3001 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     3061 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     3121 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     3181 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3241 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3301 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3361 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3421 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3481 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     3541 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3601 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3661 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3721 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3781 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3841 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     3901 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3961 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4021 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4081 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4141 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4201 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4261 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4321 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4381 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     4441 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4501 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4561 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4621 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4681 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4741 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4801 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4861 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     4921 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4981 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     5041 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     5101 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     5161 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5221 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5281 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5341 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5401 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5461 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5521 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5581 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5641 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5701 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5761 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5821 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5881 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5941 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     6001 tgcattagtt attaatagta atcaattacg ggg
//
