LOCUS       sequence-393626-        7474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of H153D mutation in PPM1H.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7474)
  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 7474)
  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: CMV EGFP-PPM1H-H153D
FEATURES             Location/Qualifiers
     source          1..7474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(549..1340,1341..1409))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1172..1191
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1410..1514)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1589..1608)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1780..2159
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2160..2363
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2314..2334
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(2405..2407,2408..2410,2411..2413,2414..3124)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MHVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     CDS             join(2405..2407,2408..2410,2411..2413,2414..3124)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MHVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(2453..2474)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2714..2733)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3061..3082
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4904..4930
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4971..4988)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4977..5201
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5334..5353)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5485..5532
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             5540..6562
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    6692..6813
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6729..6748)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6783..6802
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6862..6878)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6862..6878)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6875..6897)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6886..6902
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6910..6916,6917..6934,6935..6940))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6955..6976
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7093..7110)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7264..7474,1..378))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7344..7363)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
       61 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
      121 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
      181 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
      241 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
      301 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
      361 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
      421 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
      481 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
      541 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
      601 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
      661 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
      721 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
      781 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
      841 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
      901 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
      961 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     1021 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     1081 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     1141 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     1201 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     1261 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     1321 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     1381 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     1441 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     1501 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     1561 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     1621 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     1681 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     1741 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
     1801 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1861 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1921 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1981 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc
     2041 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     2101 ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     2161 tgatgcggtt ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc
     2221 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     2281 ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt
     2341 gggaggtcta tataagcaga gctcgtttag tgaaccgtca gaattggcca ccggatctgc
     2401 caccatgcat gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2461 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     2521 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     2581 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     2641 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2701 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     2761 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2821 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     2881 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     2941 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     3001 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     3061 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     3121 caagggatcc atgctcactc gagtgaaatc tgccgtggcc aatttcatgg gcggcatcat
     3181 ggctggcagc tcaggctccg agcacggcgg cggcagctgc ggaggctcgg acctgcccct
     3241 gcgtttcccc tacgggcggc cagagttcct ggggctgtct caggacgagg tggagtgcag
     3301 cgccgaccac atcgcccgcc ccatcctcat cctcaaggag actcggcggc tgccctgggc
     3361 cactggctac gcagaggtta tcaatgccgg gaagagcaca cacaatgaag accaagccag
     3421 ctgtgaggtg ctcactgtga agaagaaggc aggggccgtg acctcaaccc caaacaggaa
     3481 ctcatccaag agacggtcct cccttcccaa tggggaaggg ctgcagctga aggagaactc
     3541 ggaatccgag ggtgtttcct gccactattg gtcgctgttt gacggggacg cggggtccgg
     3601 ggccgcggtg gtggcgtcac gcctgctgca gcaccacatc acggagcagc tgcaggacat
     3661 cgtggacatc ctgaagaact ccgccgtcct gccccctacc tgcctggggg aggagcctga
     3721 gaacacgccc gccaacagcc ggactctgac ccgggcagcc tccctgcgcg gaggggtggg
     3781 ggccccgggc tcccccagca cgccccccac acgcttcttt accgagaaga agattcccca
     3841 tgagtgcctg gtcatcggag cgcttgaaag tgcattcaag gaaatggacc tacagataga
     3901 acgagagagg agttcatata atatatctgg tggctgcacg gccctcattg tgatttgcct
     3961 tttggggaag ctgtatgttg caaatgctgg ggatagcagg gccataatca tcagaaatgg
     4021 agaaattatc cccatgtctt cagaatttac ccccgagacg gagcgccagc gacttcagta
     4081 cctggcattc atgcagcctc acttgctggg aaatgagttc acacatttgg agtttccaag
     4141 gagagtacag agaaaggagc ttggaaagaa gatgctctac agggacttta atatgacagg
     4201 ctgggcatac aaaaccattg aggatgagga cttgaagttc ccccttatat atggagaagg
     4261 caagaaggcc cgggtaatgg caactattgg agtgaccagg ggacttgggg accatgacct
     4321 gaaggtgcat gactccaaca tctacattaa accattcctg tcttcagctc cagaggtaag
     4381 aatctacgat ctttcaaaat atgatcatgg atcagatgat gtgctgatct tggccactga
     4441 tggactctgg gacgttttat caaatgaaga agtagcagaa gcaatcactc agtttcttcc
     4501 taactgtgat ccagatgatc ctcacaggta cacactggca gctcaggacc tggtgatgcg
     4561 tgcccggggt gtgctgaagg acagaggatg gcggatatca gcagttcttg aagtactccg
     4621 gatgcagctc tcgggccacg tgatgaaatg ctaggcggct gtcaaacctc cactctaaca
     4681 cttctctctc cgggtcatcc atcccatgca ggctcacagg gtgtaacaag ctggtgttct
     4741 ctcctttatt ggcctcttct accttatctg gctcaactgg tactagcttg tagcaccatc
     4801 caaaggtcag tggatatcta atgaccgact gggctcagga gacgacattt ctgtatatgt
     4861 cattccttta atacatggaa acaagctgtc atgagcggcc gcttacccat acgatgtgcc
     4921 agattacgcc taagtcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     4981 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     5041 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     5101 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     5161 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     5221 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     5281 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     5341 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5401 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5461 ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag
     5521 tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     5581 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
     5641 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     5701 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     5761 ggaagtgctt gacattgggg agttcagcga gagcctgacc tattgcatct cccgccgtgc
     5821 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     5881 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     5941 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     6001 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     6061 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     6121 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     6181 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     6241 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     6301 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     6361 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     6421 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     6481 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     6541 tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta
     6601 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     6661 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     6721 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     6781 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     6841 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     6901 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     6961 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     7021 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     7081 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     7141 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     7201 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     7261 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     7321 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     7381 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     7441 aagcgtggcg ctttctcata gctcacgctg tagg
//
