LOCUS       sequence-181696-        5781 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-tagged ataxin-3 with a mutation in the VCP-binding 
            motif.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5781)
  AUTHORS   Pfeiffer A, Luijsterburg MS, Acs K, Wiegant WW, Helfricht A, Herzog 
            LK, Minoia M, Bottcher C, Salomons FA, van Attikum H, Dantuma NP
  TITLE     Ataxin-3 consolidates the MDC1-dependent DNA double-strand break 
            response by counteracting the SUMO-targeted ubiquitin ligase RNF4.
  JOURNAL   EMBO J. 2017 Mar 8. pii: e201695151. doi: 10.15252/embj.201695151.
  PUBMED    28275011
REFERENCE   2  (bases 1 to 5781)
  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. 
            2017 Mar 8. pii: e201695151. doi: 10.15252/embj.201695151."
COMMENT     Sequence Label: GFP-ataxin-3 *VBM
FEATURES             Location/Qualifiers
     source          1..5781
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    130..251
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(167..186)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     221..240
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(258..713)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(395..416)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     607..626
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        740..844
                     /gene="bla"
                     /label=AmpR promoter
     promoter        846..1203
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        846..1037
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(869..889)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1054..1189
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1238..2032
                     /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             1238..2032
                     /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(1292..1311)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1902..1921
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2220..2239)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2264..2311
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2640..3228
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3129..3148
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3403..3706
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3707..3910
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3861..3881
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3949..3958
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3955..3957,3958..3960,3961..4671)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3955..3957,3958..3960,3961..4671)
                     /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(4000..4021)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4261..4280)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4608..4629
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ggatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta
       61 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
      121 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
      181 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
      241 aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
      301 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
      361 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
      421 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
      481 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
      541 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
      601 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
      661 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
      721 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
      781 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      841 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
      901 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
      961 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     1021 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     1081 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     1141 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     1201 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     1261 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     1321 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     1381 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     1441 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     1501 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     1561 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     1621 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     1681 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     1741 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     1801 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     1861 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     1921 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     1981 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     2041 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     2101 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     2161 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     2221 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     2281 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     2341 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     2401 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     2461 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     2521 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     2581 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2641 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2701 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2761 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     2821 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2881 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2941 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3001 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3061 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3121 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3181 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3241 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3301 ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc
     3361 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     3421 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     3481 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     3541 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     3601 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     3661 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     3721 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     3781 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     3841 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     3901 aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc caccatggtg
     3961 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     4021 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     4081 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     4141 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     4201 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     4261 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     4321 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     4381 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     4441 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     4501 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     4561 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     4621 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggactc
     4681 agatctcgag ctcaaatgga gtccatcttc cacgagaaac aagaaggctc actttgtgct
     4741 caacattgcc tgaataactt attgcaagga gaatatttta gccctgtgga attatcctca
     4801 attgcacatc agctggatga ggaggagagg atgagaatgg cagaaggagg agttactagt
     4861 gaagattatc gcacgttttt acagcagcct tctggaaata tggatgacag tggttttttc
     4921 tctattcagg ttataagcaa tgccttgaaa gtttggggtt tagaactaat cctgttcaac
     4981 agtccagagt atcagaggct caggatcgat cctataaatg aaagatcatt tatatgcaat
     5041 tataaggaac actggtttac agttagaaaa ttaggaaaac agtggtttaa cttgaattct
     5101 ctcttgacgg gtccagaatt aatatcagat acatatcttg cacttttctt ggctcaatta
     5161 caacaggaag gttattctat atttgtcgtt aagggtgatc tgccagattg cgaagctgac
     5221 caactcctgc agatgattag ggtccaacag atgcatcgac caaaacttat tggagaagaa
     5281 ttagcacaac taaaagagca aagagtccat aaaacagacc tggaacgagt gttagaagca
     5341 aatgatggct caggaatgtt agacgaagat gaggaggatt tgcagagggc tctggcacta
     5401 agtcgccaag aaattgacat ggaagatgag gaagcagatc tccgcagggc tattcagcta
     5461 agtatgcaag gtagttccag aaacatatct caagatatga cacagacatc aggtacaaat
     5521 cttacttcag aagagcttca caaccaccac gaagcctact ttgaaaaaca gcagcaaaag
     5581 cagcaacagc agcagcagca gcagcagcag ggggacctat caggacagag ttcacatcca
     5641 tgtgaaaggc cagccaccag ttcaggagca cttgggagtg atctaggtga tgctatgagt
     5701 gaagaagaca tgcttcaggc agctgtgacc atgtctttag aaactgtcag aaatgatttg
     5761 aaaacagaag gaaaaaaata a
//
