LOCUS       sequence-181706-        6246 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses His- and HA-tagged ataxin-3 with a mutation in the 
            SUMO-interacting motif.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6246)
  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 6246)
  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: "EMBO J. 
            2017 Mar 8. pii: e201695151. doi: 10.15252/embj.201695151."
COMMENT     Sequence Label: 10xHis-ataxin-3 *SIM-HA
FEATURES             Location/Qualifiers
     source          1..6246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(45..633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(125..144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(804..1595,1596..1664))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1427..1446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1665..1769)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1844..1863)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2035..2414
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2415..2618
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2569..2589
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2620..2638
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2641..2659
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2669..2693
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2788..2817
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3931..3957
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3998..4015)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4004..4228
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4361..4380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4512..4559
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4567..5589
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4567..5589
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5719..5840
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5756..5775)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5810..5829
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5889..5905)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5889..5905)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5902..5924)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5913..5929
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5937..5943,5944..5961,5962..5967))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5982..6003
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6120..6137)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
       61 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
      121 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
      181 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
      241 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
      301 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
      361 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
      421 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
      481 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
      541 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
      601 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
      661 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
      721 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
      781 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
      841 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
      901 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
      961 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     1021 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     1081 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     1141 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     1201 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     1261 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     1321 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     1381 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     1441 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     1501 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     1561 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     1621 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     1681 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     1741 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     1801 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     1861 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     1921 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     1981 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
     2041 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     2101 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     2161 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     2221 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     2281 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     2341 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     2401 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     2461 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     2521 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     2581 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
     2641 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
     2701 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
     2761 ctctagcgtt taaacttaag aatggcgcac catcaccacc atcatcacca tcaccatggt
     2821 accggaggtt ccggtatgga gtccatcttc cacgagaaac aagaaggctc actttgtgct
     2881 caacattgcc tgaataactt attgcaagga gaatatttta gccctgtgga attatcctca
     2941 attgcacatc agctggatga ggaggagagg atgagaatgg cagaaggagg agttactagt
     3001 gaagattatc gcacgttttt acagcagcct tctggaaata tggatgacag tggttttttc
     3061 tctattcagg ttataagcaa tgccttgaaa gtttggggtt tagaactaat cctgttcaac
     3121 agtccagagt atcagaggct caggatcgat cctataaatg aaagatcatt tatatgcaat
     3181 tataaggaac actggtttac agttagaaaa ttaggaaaac agtggtttaa cttgaattct
     3241 ctcttgacgg gtccagaatt aatatcagat acatatcttg cacttttctt ggctcaatta
     3301 caacaggaag gttattctgc ctttgccgcc aagggtgatc tgccagattg cgaagctgac
     3361 caactcctgc agatgattag ggtccaacag atgcatcgac caaaacttat tggagaagaa
     3421 ttagcacaac taaaagagca aagagtccat aaaacagacc tggaacgagt gttagaagca
     3481 aatgatggct caggaatgtt agacgaagat gaggaggatt tgcagagggc tctggcacta
     3541 agtcgccaag aaattgacat ggaagatgag gaagcagatc tccgcagggc tattcagcta
     3601 agtatgcaag gtagttccag aaacatatct caagatatga cacagacatc aggtacaaat
     3661 cttacttcag aagagcttcg gaagagacga gaagcctact ttgaaaaaca gcagcaaaag
     3721 cagcaacagc agcagcagca gcagcagcag ggggacctat caggacagag ttcacatcca
     3781 tgtgaaaggc cagccaccag ttcaggagca cttgggagtg atctaggtga tgctatgagt
     3841 gaagaagaca tgcttcaggc agctgtgacc atgtctttag aaactgtcag aaatgatttg
     3901 aaaacagaag gaaaaaaagg cggcggatcc tacccatacg atgttccaga ttacgcttaa
     3961 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     4021 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4081 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4141 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     4201 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     4261 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4321 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4381 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4441 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4501 tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc
     4561 ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc
     4621 gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc
     4681 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
     4741 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac
     4801 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg
     4861 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg
     4921 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa
     4981 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
     5041 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat
     5101 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc
     5161 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag
     5221 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg
     5281 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
     5341 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt
     5401 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc
     5461 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat
     5521 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca
     5581 aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5641 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5701 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     5761 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     5821 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5881 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5941 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6001 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6061 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6121 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6181 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6241 aaaggc
//
