LOCUS       sequence-421790-        6033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expresssion of Parkin, N-terminally tagged with 
            mKO2, M1 of Parkin intact.
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-mKO2-Parkin
FEATURES             Location/Qualifiers
     source          1..6033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        30..333
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        334..537
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     488..508
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      576..585
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             582..1232
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAH"
     CDS             582..1232
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAH"
     polyA_signal    2811..2932
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2848..2867)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2902..2921
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2939..3394)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3076..3097)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3288..3307
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3421..3525
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        3527..3718
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3550..3570)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3715..3849
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3898..4692
                     /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             3898..4692
                     /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(3952..3971)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4562..4581
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4880..4899)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4924..4971
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5300..5888
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5789..5808
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
       61 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      121 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      181 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      241 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      301 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      361 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      421 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      481 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
      541 ttagtgaacc gtcagatccg ctagcgctac cggtcgccac catggtgagc gtgatcaagc
      601 ccgagatgaa gatgaggtac tacatggacg gctccgtcaa tgggcatgag ttcacaatcg
      661 agggtgaggg cacaggcaga ccttacgagg gacatcagga gatgacactg cgcgtcacaa
      721 tggccgaggg cgggccaatg cctttcgcct tcgacctggt gtcccacgtg ttctgttacg
      781 gccacagagt ttttaccaag tacccagaag agatcccaga ctatttcaag caggcctttc
      841 ctgagggcct gtcctgggag aggtccctgg agttcgagga cggcggctcc gcctccgtga
      901 gcgcccacat cagcctgagg ggcaacacct tctaccacaa gtccaagttc accggcgtga
      961 acttccccgc cgacggcccc atcatgcaga accagagcgt ggactgggag ccctccaccg
     1021 agaagatcac cgccagcgac ggcgtgctga agggcgacgt gaccatgtac ctgaagctgg
     1081 agggcggcgg caaccacaag tgccagatga agaccaccta caaggccgcc aaggagatcc
     1141 tggagatgcc cggcgaccac tacatcggcc acaggctggt gaggaagacc gagggcaaca
     1201 tcaccgagca ggtggaggac gccgtggccc actactcaat gttgccttcc ctgggctccg
     1261 gactcagatc tcgagctcaa gctatgatag tgtttgtcag gttcaactcc agccatggtt
     1321 tcccagtgga ggtcgattct gacaccagca tcttccagct caaggaggtg gttgctaagc
     1381 gacagggggt tccggctgac cagttgcgtg tgattttcgc agggaaggag ctgaggaatg
     1441 actggactgt gcagaattgt gacctggatc agcagagcat tgttcacatt gtgcagagac
     1501 cgtggagaaa aggtcaagaa atgaatgcaa ctggaggcga cgaccccaga aacgcggcgg
     1561 gaggctgtga gcgggagccc cagagcttga ctcgggtgga cctcagcagc tcagtcctcc
     1621 caggagactc tgtggggctg gctgtcattc tgcacactga cagcaggaag gactcaccac
     1681 cagctggaag tccagcaggt agatcaatct acaacagctt ttatgtgtat tgcaaaggcc
     1741 cctgtcaaag agtgcagccg ggaaaactca gggtacagtg cagcacctgc aggcaggcaa
     1801 cgctcacctt gacccagggt ccatcttgct gggatgatgt tttaattcca aaccggatga
     1861 gtggtgaatg ccaatcccca cactgccctg ggactagtgc agaatttttc tttaaatgtg
     1921 gagcacaccc cacctctgac aaggaaacat cagtagcttt gcacctgatc gcaacaaata
     1981 gtcggaacat cacttgcatt acgtgcacag acgtcaggag ccccgtcctg gttttccagt
     2041 gcaactcccg ccacgtgatt tgcttagact gtttccactt atactgtgtg acaagactca
     2101 atgatcggca gtttgtccac gaccctcagc ttggctactc cctgccttgt gtggctggct
     2161 gtcccaactc cttgattaaa gagctccatc acttcaggat tctgggagaa gagcagtaca
     2221 accggtacca gcagtatggt gcagaggagt gtgtcctgca gatggggggc gtgttatgcc
     2281 cccgccctgg ctgtggagcg gggctgctgc cggagcctga ccagaggaaa gtcacctgcg
     2341 aagggggcaa tggcctgggc tgtgggtttg ccttctgccg ggaatgtaaa gaagcgtacc
     2401 atgaagggga gtgcagtgcc gtatttgaag cctcaggaac aactactcag gcctacagag
     2461 tcgatgaaag agccgccgag caggctcgtt gggaagcagc ctccaaagaa accatcaaga
     2521 aaaccaccaa gccctgtccc cgctgccatg taccagtgga aaaaaatgga ggctgcatgc
     2581 acatgaagtg tccgcagccc cagtgcaggc tcgagtggtg ctggaactgt ggctgcgagt
     2641 ggaaccgcgt ctgcatgggg gaccactggt tcgacgtgta aggatccacc ggatctagat
     2701 aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     2761 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     2821 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     2881 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtaa
     2941 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     3001 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3061 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3121 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     3181 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3241 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     3301 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     3361 cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg
     3421 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3481 caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga
     3541 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3601 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3661 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3721 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3781 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     3841 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     3901 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3961 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4021 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     4081 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4141 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4201 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4261 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4321 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4381 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     4441 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4501 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4561 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4621 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4681 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4741 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4801 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4861 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     4921 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     4981 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     5041 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     5101 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
     5161 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5221 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5281 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5341 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5401 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5461 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5521 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5581 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5641 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5701 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5761 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5821 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5881 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5941 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc
     6001 attagttatt aatagtaatc aattacgggg tca
//
