LOCUS       sequence-433568-        5308 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PGK EGFP-RAB3A-T86E.
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 Apr 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-T86E
FEATURES             Location/Qualifiers
     source          1..5308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        30..540
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     459..477
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      556..565
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(562..564,565..567,568..1278)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(562..564,565..567,568..1278)
                     /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(607..628)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(868..887)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1215..1236
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2110..2231
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2147..2166)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2201..2220
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2238..2693)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2375..2396)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2587..2606
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2720..2824
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2826..3183
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2826..3017
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2849..2869)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3034..3169
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3218..4012
                     /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             3218..4012
                     /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(3272..3291)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3882..3901
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4200..4219)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4244..4291
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4620..5208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5109..5128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccgccat gcattagtta ttaatccacg gggttggggt tgcgcctttt ccaaggcagc
       61 cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc
      121 gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat cttcgccgct
      181 acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga aggttccttg
      241 cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac gtctcactag taccctcgca
      301 gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt ggccaatagc
      361 ggctgctcag cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg
      421 tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct
      481 ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct ctctccccag
      541 gtggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
      601 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      661 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      721 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
      781 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      841 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      901 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
      961 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1021 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     1081 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1141 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     1201 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1261 atggacgagc tgtacaagtc cggactcaga tccaccatgg catccgccac agactcgcgc
     1321 tatgggcaga aggagtcctc ggatcagaac ttcgactaca tgttcaagat tctcatcatc
     1381 ggcaacagca gcgtgggcaa gacgtccttc ctcttccgct atgctgacga ctcgttcacg
     1441 cctgccttcg tcagcaccgt gggcatcgac ttcaaggtca agaccatcta tcgcaacgac
     1501 aagaggatca agctgcagat ctgggacaca gcagggcaag agcggtaccg ggaaatcacc
     1561 accgcatact accggggcgc catgggcttc atcctcatgt atgacatcac caacgaggaa
     1621 tccttcaatg cagtgcagga ctggtccacc cagatcaaga cctactcatg ggacaatgcc
     1681 caggtgctgc tggtaggaaa caagtgtgac atggaggatg agcgggtggt gtcatcagaa
     1741 cgtggccggc agctagctga ccaccttggg ttcgagttct ttgaggcaag cgccaaggac
     1801 aacattaacg tcaagcagac ctttgagcgc ctggtggatg tcatctgcga gaagatgtcc
     1861 gagtcgttgg acacggcgga ccctgcggtc acaggcgcca agcagggccc acagctcagt
     1921 gaccagcagg tgccaccgca ccaggactgc gcctgctgac tcgacggtac cgcgggcccg
     1981 ggatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta
     2041 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     2101 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2161 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     2221 aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     2281 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     2341 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     2401 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     2461 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     2521 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     2581 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     2641 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     2701 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2761 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2821 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     2881 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     2941 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3001 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3061 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3121 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3181 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     3241 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3301 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3361 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     3421 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3481 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3541 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3601 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3661 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3721 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     3781 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3841 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     3901 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     3961 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4021 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     4081 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4141 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     4201 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     4261 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     4321 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     4381 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     4441 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     4501 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     4561 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4621 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4681 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4741 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     4801 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4861 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4921 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4981 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5041 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5101 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5161 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5221 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5281 ttatcccctg attctgtgga taaccgta
//
