LOCUS       sequence-393644-        5199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Q67L mutation in ARF6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5199)
  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 5199)
  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 ARF6(Q67L)-CFP
FEATURES             Location/Qualifiers
     source          1..5199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        112..622
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     541..559
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      1235..1244
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1241..1243,1244..1246,1247..1960)
                     /codon_start=1
                     /product="enhanced CFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1286..1307)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1547..1566)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1894..1915
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2083..2204
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2120..2139)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2174..2193
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2211..2666)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2348..2369)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2560..2579
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2693..2797
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2799..3156
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2799..2990
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2822..2842)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3007..3142
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3191..3985
                     /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             3191..3985
                     /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(3245..3264)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3855..3874
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4173..4192)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4217..4264
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4593..5181
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5082..5101
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
       61 cctgattctg tggataaccg tattaccgcc atgcattagt tattaatcca cggggttggg
      121 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg
      181 ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag
      241 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc
      301 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc
      361 acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc
      421 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga
      481 aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg
      541 gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg
      601 aatcaccgac ctctctcccc aggggatccg cggccgctag cgctaccgga ctcagatctc
      661 gagctcaagc ttcgaattca tggggaaggt gctatccaaa atcttcggga acaaggaaat
      721 gcggatcctc atgttgggcc tggacgcggc cggcaagaca acaatcctgt acaagttgaa
      781 gctgggccag tcggtgacca ccattcccac tgtgggtttc aacgtggaga cggtgactta
      841 caaaaatgtc aagttcaacg tatgggatgt gggcggcctt gacaagatcc ggccgctctg
      901 gcggcattac tacactggga cccaaggtct catcttcgta gtggactgcg ccgaccgcga
      961 ccgcatcgat gaggctcgcc aggagctgca ccgcattatc aatgaccggg agatgaggga
     1021 cgccataatc ctcatcttcg ccaacaagca ggacctgccc gatgccatga aaccccacga
     1081 gatccaggag aaactgggcc tgacccggat tcgggacagg aactggtatg tgcagccctc
     1141 ctgtgccacc tcaggggacg gactctatga ggggctcaca tggttaacct ctaactacaa
     1201 atctttggta ccgcgggccc gggatccacc ggtcgccacc atggtgagca agggcgagga
     1261 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1321 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1381 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgacctg
     1441 gggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1501 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1561 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1621 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacat
     1681 cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa
     1741 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1801 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa
     1861 actgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1921 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc
     1981 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     2041 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     2101 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2161 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag
     2221 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     2281 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     2341 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2401 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2461 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2521 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2581 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     2641 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     2701 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2761 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     2821 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2881 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2941 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3001 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3061 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     3121 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     3181 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     3241 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     3301 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     3361 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     3421 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     3481 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3541 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3601 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3661 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     3721 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3781 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     3841 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     3901 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3961 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4021 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4081 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4141 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     4201 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     4261 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     4321 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     4381 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     4441 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4501 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4561 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4621 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4681 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     4741 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4801 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4861 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4921 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4981 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5041 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5101 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5161 ggggggcgga gcctatggaa aaacgccagc aacgcggcc
//
