LOCUS       sequence-435171-        5308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PGK EGFP-RAB3A-WT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5308)
  AUTHORS   Dou D, Aiken J, Holzbaur ELF
  TITLE     RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of 
            synaptic vesicle precursors.
  JOURNAL   J Cell Biol. 2024 Jun 3;223(6):e202307092. doi: 
            10.1083/jcb.202307092. Epub 2024 Mar 21.
  PUBMED    38512027
REFERENCE   2  (bases 1 to 5308)
  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: "J Cell 
            Biol."; date: "2024-06-3"; volume: "223(6):e202307092. doi"; pages: 
            " 10.1083/jcb.202307092. Epub 2024 Mar 21"
COMMENT     Sequence Label: PGK EGFP-RAB3A-WT
FEATURES             Location/Qualifiers
     source          1..5308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        129..639
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     558..576
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      655..664
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(661..663,664..666,667..1377)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(661..663,664..666,667..1377)
                     /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(706..727)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(967..986)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1314..1335
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2209..2330
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2246..2265)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2300..2319
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2337..2792)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2474..2495)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2686..2705
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2819..2923
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2925..3282
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2925..3116
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2948..2968)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3133..3268
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3317..4111
                     /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             3317..4111
                     /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(3371..3390)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3981..4000
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4299..4318)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4343..4390
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4719..5307
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5208..5227
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
       61 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat
      121 taatccacgg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc
      181 ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc
      241 ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg
      301 acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg
      361 tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca
      421 atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc
      481 gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc
      541 ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc
      601 ggctccctcg ttgaccgaat caccgacctc tctccccagg tggatccacc ggtcgccacc
      661 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      721 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      781 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      841 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      901 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      961 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1021 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1081 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1141 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1201 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1261 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1321 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc
     1381 ggactcagat ccaccatggc atccgccaca gactcgcgct atgggcagaa ggagtcctcg
     1441 gatcagaact tcgactacat gttcaagatt ctcatcatcg gcaacagcag cgtgggcaag
     1501 acgtccttcc tcttccgcta tgctgacgac tcgttcacgc ctgccttcgt cagcaccgtg
     1561 ggcatcgact tcaaggtcaa gaccatctat cgcaacgaca agaggatcaa gctgcagatc
     1621 tgggacacag cagggcaaga gcggtaccgg accatcacca ccgcatacta ccggggcgcc
     1681 atgggcttca tcctcatgta tgacatcacc aacgaggaat ccttcaatgc agtgcaggac
     1741 tggtccaccc agatcaagac ctactcatgg gacaatgccc aggtgctgct ggtaggaaac
     1801 aagtgtgaca tggaggatga gcgggtggtg tcatcagaac gtggccggca gctagctgac
     1861 caccttgggt tcgagttctt tgaggcaagc gccaaggaca acattaacgt caagcagacc
     1921 tttgagcgcc tggtggatgt catctgcgag aagatgtccg agtcgttgga cacggcggac
     1981 cctgcggtca caggcgccaa gcagggccca cagctcagtg accagcaggt gccaccgcac
     2041 caggactgcg cctgctgact cgacggtacc gcgggcccgg gatccaccgg atctagataa
     2101 ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     2161 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     2221 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     2281 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat
     2341 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     2401 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2461 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2521 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2581 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2641 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2701 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2761 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     2821 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2881 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     2941 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3001 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3061 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3121 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3181 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     3241 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     3301 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3361 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3421 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3481 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3541 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3601 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3661 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3721 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3781 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3841 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3901 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3961 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4021 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4081 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4141 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4201 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4261 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     4321 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     4381 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     4441 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     4501 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     4561 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4621 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4681 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4741 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4801 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4861 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4921 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4981 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5041 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5101 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5161 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5221 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5281 tcgtcagggg ggcggagcct atggaaaa
//
