LOCUS       sequence-222145-        6129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human Parkin S65A 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-S65A
FEATURES             Location/Qualifiers
     source          1..6129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      609..618
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(615..617,618..620,621..1331)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(615..617,618..620,621..1331)
                     /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(660..681)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(921..940)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1268..1289
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2919..3040
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2956..2975)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3010..3029
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3047..3502)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3184..3205)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3396..3415
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3529..3633
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3635..3992
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3635..3826
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3658..3678)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3843..3978
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4027..4821
                     /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             4027..4821
                     /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(4081..4100)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4691..4710
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5009..5028)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5053..5100
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5429..6017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5918..5937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      601 taccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      661 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      721 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      781 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      841 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      901 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      961 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1021 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1081 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1141 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1201 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1261 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1321 agctgtacaa gtccggccgg actcagatct cgagctcaag cttcgaattc atgatagtgt
     1381 ttgtcaggtt caactccagc catggtttcc cagtggaggt cgattctgac accagcatct
     1441 tccagctcaa ggaggtggtt gctaagcgac agggggttcc ggctgaccag ttgcgtgtga
     1501 ttttcgcagg gaaggagctg aggaatgact ggactgtgca gaattgtgac ctggatcagc
     1561 aggccattgt tcacattgtg cagagaccgt ggagaaaagg tcaagaaatg aatgcaactg
     1621 gaggcgacga ccccagaaac gcggcgggag gctgtgagcg ggagccccag agcttgactc
     1681 gggtggacct cagcagctca gtcctcccag gagactctgt ggggctggct gtcattctgc
     1741 acactgacag caggaaggac tcaccaccag ctggaagtcc agcaggtaga tcaatctaca
     1801 acagctttta tgtgtattgc aaaggcccct gtcaaagagt gcagccggga aaactcaggg
     1861 tacagtgcag cacctgcagg caggcaacgc tcaccttgac ccagggtcca tcttgctggg
     1921 atgatgtttt aattccaaac cggatgagtg gtgaatgcca atccccacac tgccctggga
     1981 ctagtgcaga atttttcttt aaatgtggag cacaccccac ctctgacaag gaaacatcag
     2041 tagctttgca cctgatcgca acaaatagtc ggaacatcac ttgcattacg tgcacagacg
     2101 tcaggagccc cgtcctggtt ttccagtgca actcccgcca cgtgatttgc ttagactgtt
     2161 tccacttata ctgtgtgaca agactcaatg atcggcagtt tgttcacgac cctcaacttg
     2221 gctactccct gccttgtgtg gctggctgtc ccaactcctt gattaaagag ctccatcact
     2281 tcaggattct gggagaagag cagtacaacc ggtaccagca gtatggtgca gaggagtgtg
     2341 tcctgcagat ggggggcgtg ttatgccccc gccctggctg tggagcgggg ctgctgccgg
     2401 agcctgacca gaggaaagtc acctgcgaag ggggcaatgg cctgggctgt gggtttgcct
     2461 tctgccggga atgtaaagaa gcgtaccatg aaggggagtg cagtgccgta tttgaagcct
     2521 caggaacaac tactcaggcc tacagagtcg atgaaagagc cgccgagcag gctcgttggg
     2581 aagcagcctc caaagaaacc atcaagaaaa ccaccaagcc ctgtccccgc tgccatgtac
     2641 cagtggaaaa aaatggaggc tgcatgcaca tgaagtgtcc gcagccccag tgcaggctcg
     2701 agtggtgctg gaactgtggc tgcgagtgga accgcgtctg catgggggac cactggttcg
     2761 acgtgtaggt cgacggtacc gcgggcccgg gatccaccgg atctagataa ctgatcataa
     2821 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     2881 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     2941 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3001 attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat tgtaagcgtt
     3061 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3121 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3181 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3241 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     3301 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     3361 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3421 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3481 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3541 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3601 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     3661 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3721 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3781 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3841 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3901 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3961 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     4021 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4081 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4141 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4201 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4261 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4321 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4381 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4441 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4501 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     4561 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4621 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4681 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4741 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4801 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4861 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4921 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4981 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     5041 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
     5101 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
     5161 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
     5221 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
     5281 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5341 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5401 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5461 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5521 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5581 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5641 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5701 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5761 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5821 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5881 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5941 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6001 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6061 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6121 ccgccatgc
//
