LOCUS       sequence-222144-        6129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human Parkin N273K 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-N273K
FEATURES             Location/Qualifiers
     source          1..6129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        111..414
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        415..618
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     569..589
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      657..666
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(663..665,666..668,669..1379)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(663..665,666..668,669..1379)
                     /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(708..729)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(969..988)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1316..1337
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2967..3088
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3004..3023)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3058..3077
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3095..3550)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3232..3253)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3444..3463
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3577..3681
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3683..4040
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3683..3874
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3706..3726)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3891..4026
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4075..4869
                     /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             4075..4869
                     /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(4129..4148)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4739..4758
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5057..5076)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5101..5148
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5477..6065
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5966..5985
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa
       61 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      121 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      181 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      241 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      301 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      361 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      421 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      481 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      541 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      601 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggtcgcca
      661 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      721 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
      781 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      841 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
      901 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
      961 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1021 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1081 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1141 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1201 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1261 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1321 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1381 ccggccggac tcagatctcg agctcaagct tcgaattcat gatagtgttt gtcaggttca
     1441 actccagcca tggtttccca gtggaggtcg attctgacac cagcatcttc cagctcaagg
     1501 aggtggttgc taagcgacag ggggttccgg ctgaccagtt gcgtgtgatt ttcgcaggga
     1561 aggagctgag gaatgactgg actgtgcaga attgtgacct ggatcagcag agcattgttc
     1621 acattgtgca gagaccgtgg agaaaaggtc aagaaatgaa tgcaactgga ggcgacgacc
     1681 ccagaaacgc ggcgggaggc tgtgagcggg agccccagag cttgactcgg gtggacctca
     1741 gcagctcagt cctcccagga gactctgtgg ggctggctgt cattctgcac actgacagca
     1801 ggaaggactc accaccagct ggaagtccag caggtagatc aatctacaac agcttttatg
     1861 tgtattgcaa aggcccctgt caaagagtgc agccgggaaa actcagggta cagtgcagca
     1921 cctgcaggca ggcaacgctc accttgaccc agggtccatc ttgctgggat gatgttttaa
     1981 ttccaaaccg gatgagtggt gaatgccaat ccccacactg ccctgggact agtgcagaat
     2041 ttttctttaa atgtggagca caccccacct ctgacaagga aacatcagta gctttgcacc
     2101 tgatcgcaac aaatagtcgg aacatcactt gcattacgtg cacagacgtc aggagccccg
     2161 tcctggtttt ccagtgcaac tcccgccacg tgatttgctt agactgtttc cacttatact
     2221 gtgtgacaag actcaaagat cggcagtttg ttcacgaccc tcaacttggc tactccctgc
     2281 cttgtgtggc tggctgtccc aactccttga ttaaagagct ccatcacttc aggattctgg
     2341 gagaagagca gtacaaccgg taccagcagt atggtgcaga ggagtgtgtc ctgcagatgg
     2401 ggggcgtgtt atgcccccgc cctggctgtg gagcggggct gctgccggag cctgaccaga
     2461 ggaaagtcac ctgcgaaggg ggcaatggcc tgggctgtgg gtttgccttc tgccgggaat
     2521 gtaaagaagc gtaccatgaa ggggagtgca gtgccgtatt tgaagcctca ggaacaacta
     2581 ctcaggccta cagagtcgat gaaagagccg ccgagcaggc tcgttgggaa gcagcctcca
     2641 aagaaaccat caagaaaacc accaagccct gtccccgctg ccatgtacca gtggaaaaaa
     2701 atggaggctg catgcacatg aagtgtccgc agccccagtg caggctcgag tggtgctgga
     2761 actgtggctg cgagtggaac cgcgtctgca tgggggacca ctggttcgac gtgtaggtcg
     2821 acggtaccgc gggcccggga tccaccggat ctagataact gatcataatc agccatacca
     2881 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     2941 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3001 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3061 gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta
     3121 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     3181 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     3241 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     3301 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     3361 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     3421 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     3481 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     3541 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3601 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3661 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     3721 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3781 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3841 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     3901 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3961 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4021 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4081 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4141 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4201 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     4261 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4321 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4381 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4441 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4501 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4561 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     4621 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4681 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4741 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4801 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4861 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4921 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4981 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5041 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     5101 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     5161 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     5221 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     5281 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     5341 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5401 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5461 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5521 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5581 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5641 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5701 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5761 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5821 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5881 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5941 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6001 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6061 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6121 acatgttct
//
