LOCUS       sequence-393642-        6149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of wild-type PPM1H.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6149)
  AUTHORS   Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur ELF
  TITLE     Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and 
            ARF6 GTPase disrupts the axonal transport of autophagosomes.
  JOURNAL   Cell Rep. 2023 May 1;42(5):112448. doi: 
            10.1016/j.celrep.2023.112448.
  PUBMED    37133994
REFERENCE   2  (bases 1 to 6149)
  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: "2023-05-1"; volume: "42(5):112448. doi"; pages: " 
            10.1016/j.celrep.2023.112448"
COMMENT     Sequence Label: PGK EGFP-PPM1H-WT
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        103..613
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     532..550
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      629..638
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(635..637,638..640,641..1351)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(635..637,638..640,641..1351)
                     /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(680..701)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(941..960)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1288..1309
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3024..3145
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3061..3080)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3115..3134
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3152..3607)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3289..3310)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3501..3520
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3634..3738
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3740..4097
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3740..3931
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3763..3783)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3948..4083
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4132..4926
                     /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             4132..4926
                     /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(4186..4205)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4796..4815
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5114..5133)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5158..5205
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5534..6122
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6023..6042
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
       61 gtggataacc gtattaccgc catgcattag ttattaatcc acggggttgg ggttgcgcct
      121 tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa
      181 acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg
      241 gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg
      301 ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac
      361 tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc
      421 tgtggccaat agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt
      481 gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc
      541 attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga
      601 cctctctccc caggtggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt
      661 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
      721 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
      781 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
      841 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
      901 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
      961 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1021 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1081 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1141 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1201 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     1261 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     1321 gatcactctc ggcatggacg agctgtacaa gggatccatg ctcactcgag tgaaatctgc
     1381 cgtggccaat ttcatgggcg gcatcatggc tggcagctca ggctccgagc acggcggcgg
     1441 cagctgcgga ggctcggacc tgcccctgcg tttcccctac gggcggccag agttcctggg
     1501 gctgtctcag gacgaggtgg agtgcagcgc cgaccacatc gcccgcccca tcctcatcct
     1561 caaggagact cggcggctgc cctgggccac tggctacgca gaggttatca atgccgggaa
     1621 gagcacacac aatgaagacc aagccagctg tgaggtgctc actgtgaaga agaaggcagg
     1681 ggccgtgacc tcaaccccaa acaggaactc atccaagaga cggtcctccc ttcccaatgg
     1741 ggaagggctg cagctgaagg agaactcgga atccgagggt gtttcctgcc actattggtc
     1801 gctgtttgac gggcacgcgg ggtccggggc cgcggtggtg gcgtcacgcc tgctgcagca
     1861 ccacatcacg gagcagctgc aggacatcgt ggacatcctg aagaactccg ccgtcctgcc
     1921 ccctacctgc ctgggggagg agcctgagaa cacgcccgcc aacagccgga ctctgacccg
     1981 ggcagcctcc ctgcgcggag gggtgggggc cccgggctcc cccagcacgc cccccacacg
     2041 cttctttacc gagaagaaga ttccccatga gtgcctggtc atcggagcgc ttgaaagtgc
     2101 attcaaggaa atggacctac agatagaacg agagaggagt tcatataata tatctggtgg
     2161 ctgcacggcc ctcattgtga tttgcctttt ggggaagctg tatgttgcaa atgctgggga
     2221 tagcagggcc ataatcatca gaaatggaga aattatcccc atgtcttcag aatttacccc
     2281 cgagacggag cgccagcgac ttcagtacct ggcattcatg cagcctcact tgctgggaaa
     2341 tgagttcaca catttggagt ttccaaggag agtacagaga aaggagcttg gaaagaagat
     2401 gctctacagg gactttaata tgacaggctg ggcatacaaa accattgagg atgaggactt
     2461 gaagttcccc cttatatatg gagaaggcaa gaaggcccgg gtaatggcaa ctattggagt
     2521 gaccagggga cttggggacc atgacctgaa ggtgcatgac tccaacatct acattaaacc
     2581 attcctgtct tcagctccag aggtaagaat ctacgatctt tcaaaatatg atcatggatc
     2641 agatgatgtg ctgatcttgg ccactgatgg actctgggac gttttatcaa atgaagaagt
     2701 agcagaagca atcactcagt ttcttcctaa ctgtgatcca gatgatcctc acaggtacac
     2761 actggcagct caggacctgg tgatgcgtgc ccggggtgtg ctgaaggaca gaggatggcg
     2821 gatatctaat gaccgactgg gctcaggaga cgacatttct gtatatgtca ttcctttaat
     2881 acatggaaac aagctgtcat gagcggccgc gactctagat cataatcagc cataccacat
     2941 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     3001 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     3061 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3121 tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa
     3181 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3241 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     3301 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3361 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3421 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3481 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3541 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3601 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3661 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3721 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     3781 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3841 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3901 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3961 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4021 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4081 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     4141 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4201 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4261 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     4321 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4381 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4441 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4501 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4561 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4621 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     4681 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4741 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4801 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4861 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4921 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4981 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     5041 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     5101 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     5161 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     5221 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     5281 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     5341 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     5401 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5461 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5521 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5581 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5641 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5701 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5761 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5821 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5881 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5941 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6001 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6061 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6121 aaaacgccag caacgcggcc tttttacgg
//
