LOCUS       sequence-435166-        4602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PGK EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4602)
  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 4602)
  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
FEATURES             Location/Qualifiers
     source          1..4602
                     /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..1380)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(661..663,664..666,667..1377)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /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    1503..1624
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1540..1559)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1594..1613
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1631..2086)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1768..1789)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1980..1999
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2113..2217
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2219..2576
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2219..2410
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2242..2262)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2427..2562
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2611..3405
                     /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             2611..3405
                     /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(2665..2684)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3275..3294
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3593..3612)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3637..3684
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4013..4601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4502..4521
                     /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 gtacaagtaa
     1381 agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     1441 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     1501 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1561 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     1621 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     1681 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     1741 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     1801 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     1861 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     1921 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     1981 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     2041 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     2101 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2161 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     2221 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2281 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2341 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2401 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2461 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     2521 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     2581 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     2641 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2701 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2761 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     2821 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2881 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2941 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3001 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3061 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3121 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3181 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3241 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3301 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3361 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     3421 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     3481 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     3541 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     3601 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     3661 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     3721 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     3781 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     3841 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     3901 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3961 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4021 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4081 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4141 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4201 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4261 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4321 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4381 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4441 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4501 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4561 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aa
//
