LOCUS       sequence-350116-        5551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP fused to human Tollip cDNA carrying mutations in the CUE domain.
            Localisation and expression in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5551)
  AUTHORS   Ryan TA, Phillips EO, Collier CL, Jb Robinson A, Routledge D, Wood 
            RE, Assar EA, Tumbarello DA
  TITLE     Tollip coordinates Parkin-dependent trafficking of 
            mitochondrial-derived vesicles.
  JOURNAL   EMBO J. 2020 Jun 2;39(11):e102539. doi: 10.15252/embj.2019102539. 
            Epub 2020 Apr 20.
  PUBMED    32311122
REFERENCE   2  (bases 1 to 5551)
  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: "EMBO J."; 
            date: "2020-06-2"; volume: "39(11):e102539. doi"; pages: " 
            10.15252/embj.2019102539. Epub 2020 Apr 20"
COMMENT     Sequence Label: GFP-Tollip M240A,F241A
FEATURES             Location/Qualifiers
     source          1..5551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      623..632
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(629..631,632..634,635..1345)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(629..631,632..634,635..1345)
                     /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(674..695)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(935..954)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1282..1303
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2355..2476
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2392..2411)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2446..2465
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2483..2938)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2620..2641)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2832..2851
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2965..3069
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3071..3428
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3071..3262
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3094..3114)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3279..3414
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3463..4257
                     /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             3463..4257
                     /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(3517..3536)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4127..4146
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4445..4464)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4489..4536
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4865..5453
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5354..5373
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
      661 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      721 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      781 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
      841 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      901 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
      961 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     1021 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     1081 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     1141 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     1201 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     1261 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1321 tctcggcatg gacgagctgt acaagtactc agatctcgag ctcaagctta tggcgaccac
     1381 cgtcagcact cagcgcgggc cggtgtacat cggtgagctc ccgcaggact tcctccgcat
     1441 cacgcccaca cagcagcagc ggcaggtcca gctggacgcc caggcggccc agcagctgca
     1501 gtacggaggc gcagtgggca ccgtgggccg actgaacatc acggtggtac aggcaaagtt
     1561 ggccaagaat tacggcatga cccgcatgga cccctactgc cgactgcgcc tgggctacgc
     1621 ggtgtacgag acgcccacgg cacacaatgg cgccaagaat ccccgctgga ataaggtcat
     1681 ccactgcacg gtgcccccag gcgtggactc tttctatctc gagatcttcg atgagagagc
     1741 cttctccatg gacgaccgca ttgcctggac ccacatcacc atcccagagt ccctgaggca
     1801 gggcaaggtg gaggacaagt ggtacagcct gagcgggagg cagggggacg acaaggaggg
     1861 catgatcaac ctcgtcatgt cctacgcgct gcttccagct gccatggtga tgccacccca
     1921 gcccgtggtc ctgatgccaa cagtgtacca gcagggcgtt ggctatgtgc ccatcacagg
     1981 gatgcccgct gtctgtagcc ccggcatggt gcccgtggcc ctgcccccgg ccgccgtgaa
     2041 cgcccagccc cgctgtagcg aggaggacct gaaagccatc caggacgcgg cccccaacat
     2101 ggaccaggag gtgatccgct ccgtgctgga agcccagcga gggaacaagg atgccgccat
     2161 caactccctg ctgcagatgg gggaggagcc ataggaattc tgcagtcgac ggtaccgcgg
     2221 gcccgggatc caccggatct agataactga tcataatcag ccataccaca tttgtagagg
     2281 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     2341 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     2401 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     2461 tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta
     2521 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2581 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2641 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2701 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     2761 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2821 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2881 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca
     2941 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3001 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3061 aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa
     3121 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3181 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3241 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3301 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3361 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3421 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3481 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3541 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3601 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     3661 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3721 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3781 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3841 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3901 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3961 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     4021 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4081 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4141 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4201 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4261 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4321 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4381 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc
     4441 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     4501 gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg
     4561 gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt
     4621 ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg
     4681 gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac
     4741 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4801 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4861 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4921 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4981 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5041 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5101 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5161 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5221 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5281 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5341 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5401 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5461 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5521 ctgcgttatc ccctgattct gtggataacc g
//
