LOCUS       sequence-218129-        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        113..416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        417..620
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     571..591
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      659..668
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(665..667,668..670,671..1381)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(665..667,668..670,671..1381)
                     /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(710..731)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(971..990)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1318..1339
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2969..3090
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3006..3025)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3060..3079
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3097..3552)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3234..3255)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3446..3465
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3579..3683
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3685..4042
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3685..3876
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3708..3728)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3893..4028
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4077..4871
                     /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             4077..4871
                     /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(4131..4150)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4741..4760
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5059..5078)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5103..5150
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5479..6067
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5968..5987
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt
       61 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      121 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      181 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      241 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      301 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      361 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      421 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      481 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      541 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      601 aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc
      661 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      721 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      781 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      841 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      901 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      961 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1021 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1081 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1141 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1201 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1261 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1321 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1381 gtccggccgg actcagatct cgagctcaag cttcgaattc atgatagtgt ttgtcaggtt
     1441 caactccagc catggtttcc cagtggaggt cgattctgac accagcatct tccagctcaa
     1501 ggaggtggtt gctaagcgac agggggttcc ggctgaccag ttgcgtgtga ttttcgcagg
     1561 gaaggagctg aggaatgact ggactgtgca gaattgtgac ctggatcagc agagcattgt
     1621 tcacattgtg cagagaccgt ggagaaaagg tcaagaaatg aatgcaactg gaggcgacga
     1681 ccccagaaac gcggcgggag gctgtgagcg ggagccccag agcttgactc gggtggacct
     1741 cagcagctca gtcctcccag gagactctgt ggggctggct gtcattctgc acactgacag
     1801 caggaaggac tcaccaccag ctggaagtcc agcaggtaga tcaatctaca acagcgctta
     1861 tgtgtattgc aaaggcccct gtcaaagagt gcagccggga aaactcaggg tacagtgcag
     1921 cacctgcagg caggcaacgc tcaccttgac ccagggtcca tcttgctggg atgatgtttt
     1981 aattccaaac cggatgagtg gtgaatgcca atccccacac tgccctggga ctagtgcaga
     2041 atttttcttt aaatgtggag cacaccccac ctctgacaag gaaacatcag tagctttgca
     2101 cctgatcgca acaaatagtc ggaacatcac ttgcattacg tgcacagacg tcaggagccc
     2161 cgtcctggtt ttccagtgca actcccgcca cgtgatttgc ttagactgtt tccacttata
     2221 ctgtgtgaca agactcaatg atcggcagtt tgttcacgac cctcaacttg gctactccct
     2281 gccttgtgtg gctggctgtc ccaactcctt gattaaagag ctccatcact tcaggattct
     2341 gggagaagag cagtacaacc ggtaccagca gtatggtgca gaggagtgtg tcctgcagat
     2401 ggggggcgtg ttatgccccc gccctggctg tggagcgggg ctgctgccgg agcctgacca
     2461 gaggaaagtc acctgcgaag ggggcaatgg cctgggctgt gggtttgcct tctgccggga
     2521 atgtaaagaa gcgtaccatg aaggggagtg cagtgccgta tttgaagcct caggaacaac
     2581 tactcaggcc tacagagtcg atgaaagagc cgccgagcag gctcgttggg aagcagcctc
     2641 caaagaaacc atcaagaaaa ccaccaagcc ctgtccccgc tgccatgtac cagtggaaaa
     2701 aaatggaggc tgcatgcaca tgaagtgtcc gcagccccag tgcaggctcg agtggtgctg
     2761 gaactgtggc tgcgagtgga accgcgtctg catgggggac cactggttcg acgtgtaggt
     2821 cgacggtacc gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac
     2881 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2941 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     3001 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3061 tggtttgtcc aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt
     3121 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     3181 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     3241 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     3301 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     3361 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     3421 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     3481 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     3541 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3601 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3661 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     3721 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3781 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3841 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3901 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3961 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4021 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     4081 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     4141 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4201 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     4261 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4321 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4381 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4441 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4501 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4561 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     4621 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4681 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4741 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4801 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4861 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4921 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4981 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5041 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     5101 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     5161 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     5221 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     5281 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     5341 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5401 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5461 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5521 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5581 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5641 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5701 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5761 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5821 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5881 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5941 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6001 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6061 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6121 tcacatgtt
//
