LOCUS       sequence-350115-        5554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP fused to human Tollip cDNA. Localisation and expression in 
            mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5554)
  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 5554)
  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
FEATURES             Location/Qualifiers
     source          1..5554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     164..185
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1240..1361
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1277..1296)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1331..1350
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1368..1823)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1505..1526)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1717..1736
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1850..1954
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1956..2313
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1956..2147
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1979..1999)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2164..2299
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2348..3142
                     /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             2348..3142
                     /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(2402..2421)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3012..3031
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3330..3349)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3374..3421
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3750..4338
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4239..4258
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4513..4816
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4817..5020
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4971..4991
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      5059..5068
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5065..5067,5068..5070,5071..5554,1..227)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5065..5067,5068..5070,5071..5554,1..227)
                     /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(5110..5131)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5371..5390)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
       61 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
      121 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
      181 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtac tcagatctcg
      241 agctcaagct taccatggcg accaccgtca gcactcagcg cgggccggtg tacatcggtg
      301 agctcccgca ggacttcctc cgcatcacgc ccacacagca gcagcggcag gtccagctgg
      361 acgcccaggc ggcccagcag ctgcagtacg gaggcgcagt gggcaccgtg ggccgactga
      421 acatcacggt ggtacaggca aagttggcca agaattacgg catgacccgc atggacccct
      481 actgccgact gcgcctgggc tacgcggtgt acgagacgcc cacggcacac aatggcgcca
      541 agaatccccg ctggaataag gtcatccact gcacggtgcc cccaggcgtg gactctttct
      601 atctcgagat cttcgatgag agagccttct ccatggacga ccgcattgcc tggacccaca
      661 tcaccatccc agagtccctg aggcagggca aggtggagga caagtggtac agcctgagcg
      721 ggaggcaggg ggacgacaag gagggcatga tcaacctcgt catgtcctac gcgctgcttc
      781 cagctgccat ggtgatgcca ccccagcccg tggtcctgat gccaacagtg taccagcagg
      841 gcgttggcta tgtgcccatc acagggatgc ccgctgtctg tagccccggc atggtgcccg
      901 tggccctgcc cccggccgcc gtgaacgccc agccccgctg tagcgaggag gacctgaaag
      961 ccatccagga catgttcccc aacatggacc aggaggtgat ccgctccgtg ctggaagccc
     1021 agcgagggaa caaggatgcc gccatcaact ccctgctgca gatgggggag gagccatagg
     1081 aattctgcag tcgacggtac cgcgggcccg ggatccaccg gatctagata actgatcata
     1141 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     1201 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     1261 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     1321 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt
     1381 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     1441 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     1501 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     1561 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     1621 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     1681 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     1741 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     1801 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1861 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1921 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     1981 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2041 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2101 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2161 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2221 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     2281 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     2341 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     2401 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     2461 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     2521 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     2581 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     2641 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     2701 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     2761 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     2821 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     2881 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     2941 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     3001 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     3061 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     3121 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     3181 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3241 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3301 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
     3361 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
     3421 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     3481 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     3541 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
     3601 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3661 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3721 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3781 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3841 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3901 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3961 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4021 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4081 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4141 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4201 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4261 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4321 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4381 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4441 accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     4501 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     4561 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     4621 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     4681 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     4741 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     4801 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     4861 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     4921 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     4981 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
     5041 ccgctagcgc taccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     5101 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     5161 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     5221 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     5281 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     5341 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     5401 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     5461 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     5521 atcatggccg acaagcagaa gaacggcatc aagg
//
