LOCUS       sequence-420302-        5546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP fused with pOTC-3H, a leader peptide from 
            mitochondrial matrix enzyme ornithine transcarbamylase and a 
            thermostable three helix-bundle.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5546)
  AUTHORS   Wu S, Hernandez Villegas NC, Sirkis DW, Thomas-Wright I, 
            Wade-Martins R, Schekman R
  TITLE     Unconventional secretion of alpha-synuclein mediated by 
            palmitoylated DNAJC5 oligomers.
  JOURNAL   Elife. 2023 Jan 10;12:e85837. doi: 10.7554/eLife.85837.
  PUBMED    36626307
REFERENCE   2  (bases 1 to 5546)
  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: "Elife."; 
            date: "2023-01-10"; volume: "12:e85837. doi"; pages: " 
            10.7554/eLife.85837"
COMMENT     Sequence Label: pCMV-pOTC-3H-GFP
FEATURES             Location/Qualifiers
     source          1..5546
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1399..1416
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      1426..1435
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1432..1434,1435..1437,1438..2151)
                     /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(1432..1434,1435..1437,1438..2148)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1477..1498)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1738..1757)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2085..2106
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2274..2395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2311..2330)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2365..2384
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2402..2857)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2539..2560)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2751..2770
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2884..2988
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2990..3347
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2990..3181
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3013..3033)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3198..3333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3382..4176
                     /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             3382..4176
                     /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(3436..3455)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4046..4065
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4364..4383)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4408..4455
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4784..5372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5273..5292
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggactcag atctcgaggc caccatgctg tttaatctga ggatcctgtt aaacaatgca
      601 gcttttagaa atggtcacaa cttcatggtt cgaaattttc ggtgtggaca accactacaa
      661 aataaagtgc agatgaatga ggacgacatg aaaaagctct acaagcaaat ggtgcaggag
      721 ctggagaagg cccgagatag aatggaaaaa ttgtataaag aaatggtcga actcatacag
      781 aaagcgatag agcttatgcg gaagatcttt caggaggtga agcaagaagt tgaaaaggcc
      841 attgaggaga tgaaaaaact ttacgacgaa gcgaaaaaga agattgagca gatgatccag
      901 cagataaaac agggtgggga taagcagaag atggaggagc ttctcaaacg cgcaaaggaa
      961 gaaatgaaaa aggtaaaaga taaaatggaa aagttgctcg aaaaacttaa gcaaattatg
     1021 caggaagcta agcagaaaat ggagaaactt ttgaaacagc tcaaagagga gatgaagaag
     1081 atgaaagaga agatggaaaa actgctgaaa gagatgaagc aacgaatgga agaggtcaaa
     1141 aagaagatgg atggcgatga tgagctcttg gaaaagatta agaaaaacat cgatgacctc
     1201 aagaagatcg ccgaagatct catcaagaag gctgaagaga acatcaagga agcgaagaag
     1261 atcgctgaac agcttgtaaa gagggccaaa caacttatag agaaagcgaa gcaggtagct
     1321 gaggagttga taaagaagat cctgcaactg attgagaaag ccaaggaaat tgcggagaaa
     1381 gtcctgaagg ggttggaaca tcaccatcac caccatgcac cggtcgccac catggtgagc
     1441 aagggcgagg agctgttcac cggcgtggtg cccatcctgg tcgagctgga cggcgacgta
     1501 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1561 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1621 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1681 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1741 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1801 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1861 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1921 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1981 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2041 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2101 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc
     2161 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     2221 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     2281 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2341 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
     2401 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2461 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2521 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2581 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2641 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2701 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2761 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2821 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     2881 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2941 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     3001 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3061 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3181 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3241 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     3301 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     3361 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3421 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3481 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3541 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     3601 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3661 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3721 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3781 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3841 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3901 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3961 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4021 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4081 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4141 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4201 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4261 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4321 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     4381 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     4441 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     4501 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     4561 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     4621 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     4681 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4741 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4801 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4861 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4921 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4981 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5041 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5101 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5161 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5221 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5281 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5341 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5401 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5461 tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta
     5521 gttcatagcc catatatgga gttccg
//
