LOCUS       sequence-421779-        6033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expresssion of ligase-dead C431N 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(C431N)-A92mKO2
FEATURES             Location/Qualifiers
     source          1..6033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        32..335
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        336..539
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     490..510
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             875..1525
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="VSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVT
                     MAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSAS
                     VSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL
                     KLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             875..1525
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="VSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVT
                     MAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSAS
                     VSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL
                     KLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    2792..2913
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2829..2848)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2883..2902
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2920..3375)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3057..3078)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3269..3288
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3402..3506
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3508..3865
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3508..3699
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3531..3551)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3716..3851
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3900..4694
                     /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             3900..4694
                     /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(3954..3973)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4564..4583
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4882..4901)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4926..4973
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5302..5890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5791..5810
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
       61 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      121 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      181 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      241 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      301 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      361 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      421 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      481 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
      541 gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc accatgatag tgtttgtcag
      601 gttcaactcc agccatggtt tcccagtgga ggtcgattct gacaccagca tcttccagct
      661 caaggaggtg gttgctaagc gacagggggt tccggctgac cagttgcgtg tgattttcgc
      721 agggaaggag ctgaggaatg actggactgt gcagaattgt gacctggatc agcagagcat
      781 tgttcacatt gtgcagagac cgtggagaaa aggtcaagaa atgaatgcaa ctggaggcga
      841 cgaccccaga aacgcggcgg gtgctggttc tggcgtgagc gtgatcaagc ccgagatgaa
      901 gatgaggtac tacatggacg gctccgtcaa tgggcatgag ttcacaatcg agggtgaggg
      961 cacaggcaga ccttacgagg gacatcagga gatgacactg cgcgtcacaa tggccgaggg
     1021 cgggccaatg cctttcgcct tcgacctggt gtcccacgtg ttctgttacg gccacagagt
     1081 ttttaccaag tacccagaag agatcccaga ctatttcaag caggcctttc ctgagggcct
     1141 gtcctgggag aggtccctgg agttcgagga cggcggctcc gcctccgtga gcgcccacat
     1201 cagcctgagg ggcaacacct tctaccacaa gtccaagttc accggcgtga acttccccgc
     1261 cgacggcccc atcatgcaga accagagcgt ggactgggag ccctccaccg agaagatcac
     1321 cgccagcgac ggcgtgctga agggcgacgt gaccatgtac ctgaagctgg agggcggcgg
     1381 caaccacaag tgccagatga agaccaccta caaggccgcc aaggagatcc tggagatgcc
     1441 cggcgaccac tacatcggcc acaggctggt gaggaagacc gagggcaaca tcaccgagca
     1501 ggtggaggac gccgtggccc actcagcagg atcaggaggt ggaggctgtg agcgggagcc
     1561 ccagagcttg actcgggtgg acctcagcag ctcagtcctc ccaggagact ctgtggggct
     1621 ggctgtcatt ctgcacactg acagcaggaa ggactcacca ccagctggaa gtccagcagg
     1681 tagatcaatc tacaacagct tttatgtgta ttgcaaaggc ccctgtcaaa gagtgcagcc
     1741 gggaaaactc agggtacagt gcagcacctg caggcaggca acgctcacct tgacccaggg
     1801 tccatcttgc tgggatgatg ttttaattcc aaaccggatg agtggtgaat gccaatcccc
     1861 acactgccct gggactagtg cagaattttt ctttaaatgt ggagcacacc ccacctctga
     1921 caaggaaaca tcagtagctt tgcacctgat cgcaacaaat agtcggaaca tcacttgcat
     1981 tacgtgcaca gacgtcagga gccccgtcct ggttttccag tgcaactccc gccacgtgat
     2041 ttgcttagac tgtttccact tatactgtgt gacaagactc aatgatcggc agtttgtcca
     2101 cgaccctcag cttggctact ccctgccttg tgtggctggc tgtcccaact ccttgattaa
     2161 agagctccat cacttcagga ttctgggaga agagcagtac aaccggtacc agcagtatgg
     2221 tgcagaggag tgtgtcctgc agatgggggg cgtgttatgc ccccgccctg gctgtggagc
     2281 ggggctgctg ccggagcctg accagaggaa agtcacctgc gaagggggca atggcctggg
     2341 ctgtgggttt gccttctgcc gggaatgtaa agaagcgtac catgaagggg agtgcagtgc
     2401 cgtatttgaa gcctcaggaa caactactca ggcctacaga gtcgatgaaa gagccgccga
     2461 gcaggctcgt tgggaagcag cctccaaaga aaccatcaag aaaaccacca agccctgtcc
     2521 ccgctgccat gtaccagtgg aaaaaaatgg aggcaacatg cacatgaagt gtccgcagcc
     2581 ccagtgcagg ctcgagtggt gctggaactg tggctgcgag tggaaccgcg tctgcatggg
     2641 ggaccactgg ttcgacgtgt aaggatccac cggatctaga taactgatca taatcagcca
     2701 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     2761 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     2821 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2881 ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt
     2941 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3001 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     3061 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     3121 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     3181 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     3241 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     3301 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     3361 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3421 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3481 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     3541 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3601 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3661 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3721 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3781 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     3841 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     3901 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3961 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4021 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4081 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4141 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4201 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4261 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4321 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4381 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     4441 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4501 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4561 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4621 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4681 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4741 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4801 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4861 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     4921 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     4981 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     5041 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     5101 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     5161 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5221 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5281 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5341 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5401 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5461 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5521 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5581 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5641 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5701 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5761 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5821 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5881 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5941 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     6001 gcattagtta ttaatagtaa tcaattacgg ggt
//
