LOCUS       sequence-218585-        6480 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of human Ataxin-3 with C-terminal strep-HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6480)
  AUTHORS   Hulsmann J, Kravic B, Weith M, Gstaiger M, Aebersold RH, Collins BC,
            Meyer H
  TITLE     AP-SWATH reveals direct involvement of VCP/p97 in integrated stress 
            response signaling through facilitating CReP/PPP1R15B degradation.
  JOURNAL   Mol Cell Proteomics. 2018 Mar 29. pii: RA117.000471. doi: 
            10.1074/mcp.RA117.000471.
  PUBMED    29599191
REFERENCE   2  (bases 1 to 6480)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell 
            Proteomics. 2018 Mar 29. pii: RA117.000471. doi: 
            10.1074/mcp.RA117.000471."
COMMENT     Sequence Label:  pcDNA5FRT/TO-Ataxin3-Strep-HA
FEATURES             Location/Qualifiers
     source          1..6480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        156..535
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        536..739
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     690..710
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    741..759
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    762..780
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     790..814
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    957..981
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2098..2122)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2169..2192
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2229..2252
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2283..2309
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             2283..2309
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2353..2370)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2359..2583
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2716..2735)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2867..2914
                     /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             2922..3944
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2922..3944
                     /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    4074..4195
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4111..4130)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4165..4184
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4244..4260)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4244..4260)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4257..4279)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4268..4284
                     /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(4292..4298,4299..4316,4317..4322))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4337..4358
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4475..4492)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4646..5234)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4726..4745)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5405..6196,6197..6265))
                     /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     6028..6047
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6266..6370)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6445..6464)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
       61 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
      121 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
      181 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      241 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      301 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      361 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      421 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      481 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      541 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      601 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      661 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      721 ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca gtgatagaga
      781 tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
      841 tgacctccat agaagacacc gggaccgatc cagcctccgg actctagcgt ttaaacttaa
      901 gcttggtacc gagctcggat ccactagtcc agtgtggtgg aattctgcag atatcaacaa
      961 gtttgtacaa aaaagcaggc tccaccatgg agtccatctt ccacgagaaa caagaaggct
     1021 cactttgtgc tcaacattgc ctgaataact tattgcaagg agaatatttt agccccgtgg
     1081 aattatcctc aattgcacat cagctggatg aggaggagag gatgagaatg gcagaaggag
     1141 gagttactag tgaagattat cgcacgtttt tacagcagcc ttctggaaat atggatgaca
     1201 gtggtttttt ctctattcag gttataagca atgccttgaa agtttggggt ttagaactaa
     1261 tcctgttcaa cagtccagag tatcagaggc tcaggatcga tcctataaat gaaagatcat
     1321 ttatatgcaa ttataaggaa cactggttta cagttagaaa attaggaaaa cagtggttta
     1381 acttgaattc tctcttgacg ggtccagaat taatatcaga tacatatctt gcacttttct
     1441 tggctcaatt acaacaggaa ggttattcta tatttgtcgt taagggtgat ctgccagatt
     1501 gcgaagctga ccaactcctg cagatgatta gggtccaaca gatgcatcga ccaaaactta
     1561 ttggagaaga attagcacaa ctaaaagagc aaagagtcca taaaacagac ctggaacgag
     1621 tgttagaagc aaatgatggc tcaggaatgt tagacgaaga tgaggaggat ttgcagaggg
     1681 ctctggcact aagtcgccaa gaaattgaca tggaagatga ggaagcagat ctccgcaggg
     1741 ctattcagct aagtatgcaa ggtagttcca gaaacatatc tcaagatatg acacagacat
     1801 caggtacaaa tcttacttca gaagagcttc ggaagagacg agaagcctac tttgaaaaac
     1861 agcagcaaaa gcagcaacag cagcagcagc agcagcagca gcagcagcag cagcagcagc
     1921 agcagcaggg ggacctatca ggacagagtt cacatccatg tgaaaggcca gccaccagtt
     1981 caggagcact tgggagtgat ctaggtgatg ctatgagtga agaagacatg cttcaggcag
     2041 ctgtgaccat gtctttagaa actgtcagaa atgatttgaa aacagaagga aaaaaagacc
     2101 cagctttctt gtacaaagtg gttgatatca aggccgccga catcaccagc ctgtacaaga
     2161 aagtggggtg gagccatccc cagttcgaga agggcggagg atctggcggc ggaagcggcg
     2221 gaggaagctg gtcccaccct cagtttgaga aaggcaccga gctgggcagc accatggcca
     2281 gctaccccta cgacgtgccc gactacgcct gatgactcga gtctagaggg cccgtttaaa
     2341 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2401 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2461 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2521 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2581 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2641 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2701 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2761 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2821 acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc
     2881 gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg
     2941 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     3001 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     3061 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     3121 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     3181 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     3241 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     3301 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     3361 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     3421 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     3481 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     3541 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     3601 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     3661 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     3721 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     3781 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     3841 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     3901 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt
     3961 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4021 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4081 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4141 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4201 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4261 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4321 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4381 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4441 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4501 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4561 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4621 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4681 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4741 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4801 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4861 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4921 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4981 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5041 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5101 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5161 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5221 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5281 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5341 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5401 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5461 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5521 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5581 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5641 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5701 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5761 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5821 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5881 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5941 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6001 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6061 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6121 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6181 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6241 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6301 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6361 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     6421 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
//
