LOCUS       sequence-222140-        6129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human Parkin F146A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6129)
  AUTHORS   Tang MY, Vranas M, Krahn AI, Pundlik S, Trempe JF, Fon EA
  TITLE     Structure-guided mutagenesis reveals a hierarchical mechanism of 
            Parkin activation.
  JOURNAL   Nat Commun. 2017 Mar 9;8:14697. doi: 10.1038/ncomms14697.
  PUBMED    28276439
REFERENCE   2  (bases 1 to 6129)
  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: "Nat 
            Commun."; date: "2017-03-9"; volume: "8:14697. doi"; pages: " 
            10.1038/ncomms14697"
COMMENT     Sequence Label: eGFP-pk-F146A
FEATURES             Location/Qualifiers
     source          1..6129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        96..399
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        400..603
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     554..574
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      642..651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(648..650,651..653,654..1364)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(648..650,651..653,654..1364)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(693..714)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(954..973)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1301..1322
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2952..3073
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2989..3008)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3043..3062
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3080..3535)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3217..3238)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3429..3448
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3562..3666
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3668..4025
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3668..3859
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3691..3711)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3876..4011
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4060..4854
                     /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             4060..4854
                     /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(4114..4133)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4724..4743
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5042..5061)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5086..5133
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5462..6050
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5951..5970
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg
       61 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      121 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      181 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      241 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      301 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      361 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      421 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      481 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      541 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      601 gctggtttag tgaaccgtca gatccgctag cgctaccggt cgccaccatg gtgagcaagg
      661 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
      721 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
      781 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
      841 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
      901 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
      961 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1021 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1081 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1141 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1201 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1261 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1321 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccggc cggactcaga
     1381 tctcgagctc aagcttcgaa ttcatgatag tgtttgtcag gttcaactcc agccatggtt
     1441 tcccagtgga ggtcgattct gacaccagca tcttccagct caaggaggtg gttgctaagc
     1501 gacagggggt tccggctgac cagttgcgtg tgattttcgc agggaaggag ctgaggaatg
     1561 actggactgt gcagaattgt gacctggatc agcagagcat tgttcacatt gtgcagagac
     1621 cgtggagaaa aggtcaagaa atgaatgcaa ctggaggcga cgaccccaga aacgcggcgg
     1681 gaggctgtga gcgggagccc cagagcttga ctcgggtgga cctcagcagc tcagtcctcc
     1741 caggagactc tgtggggctg gctgtcattc tgcacactga cagcaggaag gactcaccac
     1801 cagctggaag tccagcaggt agatcaatct acaacagcgc ttatgtgtat tgcaaaggcc
     1861 cctgtcaaag agtgcagccg ggaaaactca gggtacagtg cagcacctgc aggcaggcaa
     1921 cgctcacctt gacccagggt ccatcttgct gggatgatgt tttaattcca aaccggatga
     1981 gtggtgaatg ccaatcccca cactgccctg ggactagtgc agaatttttc tttaaatgtg
     2041 gagcacaccc cacctctgac aaggaaacat cagtagcttt gcacctgatc gcaacaaata
     2101 gtcggaacat cacttgcatt acgtgcacag acgtcaggag ccccgtcctg gttttccagt
     2161 gcaactcccg ccacgtgatt tgcttagact gtttccactt atactgtgtg acaagactca
     2221 atgatcggca gtttgttcac gaccctcaac ttggctactc cctgccttgt gtggctggct
     2281 gtcccaactc cttgattaaa gagctccatc acttcaggat tctgggagaa gagcagtaca
     2341 accggtacca gcagtatggt gcagaggagt gtgtcctgca gatggggggc gtgttatgcc
     2401 cccgccctgg ctgtggagcg gggctgctgc cggagcctga ccagaggaaa gtcacctgcg
     2461 aagggggcaa tggcctgggc tgtgggtttg ccttctgccg ggaatgtaaa gaagcgtacc
     2521 atgaagggga gtgcagtgcc gtatttgaag cctcaggaac aactactcag gcctacagag
     2581 tcgatgaaag agccgccgag caggctcgtt gggaagcagc ctccaaagaa accatcaaga
     2641 aaaccaccaa gccctgtccc cgctgccatg taccagtgga aaaaaatgga ggctgcatgc
     2701 acatgaagtg tccgcagccc cagtgcaggc tcgagtggtg ctggaactgt ggctgcgagt
     2761 ggaaccgcgt ctgcatgggg gaccactggt tcgacgtgta ggtcgacggt accgcgggcc
     2821 cgggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt
     2881 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2941 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3001 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3061 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3121 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3181 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3241 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3301 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3361 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3421 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3481 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     3541 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3601 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3661 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3721 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3781 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3841 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3901 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3961 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4021 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     4081 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4141 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4201 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     4261 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4321 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4381 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4441 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4501 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4561 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     4621 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4681 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4741 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4801 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4861 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4921 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4981 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     5041 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     5101 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     5161 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     5221 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     5281 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     5341 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5401 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5461 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5521 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5581 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5641 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5701 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5761 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5821 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5881 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5941 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6001 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6061 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6121 cgttatccc
//
