LOCUS       sequence-370367-        5965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant protein expression and purification. Encodes ataxin-3 
            full length with 3 UIMs and 77Q in the Poly-Q track fused with 
            His-Tag and TEV cleavage sequence (N-terminal).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5965)
  AUTHORS   Figueiredo F, Lopes-Marques M, Almeida B, Matscheko N, Martins PM, 
            Silva A, Macedo-Ribeiro S
  TITLE     A Robust Assay to Monitor Ataxin-3 Amyloid Fibril Assembly.
  JOURNAL   Cells. 2022 Jun 19;11(12). pii: cells11121969. doi: 
            10.3390/cells11121969.
  PUBMED    35741099
REFERENCE   2  (bases 1 to 5965)
  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: "Cells."; 
            date: "2022-06-19"; volume: "11(12). pii: cells11121969. doi"; 
            pages: " 10.3390/cells11121969"
COMMENT     Sequence Label: ATX3-77Q
FEATURES             Location/Qualifiers
     source          1..5965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             71..93
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             113..130
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(1434..1458)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1511..1529)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1525..1572
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1938..1943,1944..1960,1961..1966))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1993..2011)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2079..2183
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2184..2252,2253..3044)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPTGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2402..2421)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3215..3803
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3704..3723
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3957..3974
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3989..4128
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4054..4073)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4214..4236
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4230..4421)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5883..5902
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc
       61 tagaaataat tttgtttaac tttaagaagg agatatacat atgtcgtact accatcacca
      121 tcaccatcac ctcgaaaacc tgtattttca gggcatggag tccatcttcc acgagaaaca
      181 agaaggctca ctttgtgctc aacattgcct gaataactta ttgcaaggag aatattttag
      241 ccctgtggaa ttatcctcaa ttgcacatca gctggatgag gaggagagga tgagaatggc
      301 agaaggagga gttactagtg aagattatcg cacgttttta cagcagcctt ctggaaatat
      361 ggatgacagt ggttttttct ctattcaggt tataagcaat gccttgaaag tttggggttt
      421 agaactaatc ctgttcaaca gtccagagta tcagaggctc aggatcgatc ctataaatga
      481 aagatcattt atatgcaatt ataaggaaca ctggtttaca gttagaaaat taggaaaaca
      541 gtggtttaac ttgaattctc tcttgacggg tccagaatta atatcagata catatcttgc
      601 acttttcttg gctcaattac aacaggaagg ttattctata tttgtcgtta agggtgatct
      661 gccagattgc gaagctgacc aactcctgca gatgattagg gtccaacaga tgcatcgacc
      721 aaaactgatt ggcgaagaac tggctcaact gaaagaacag cgtgtgcata agaccgacct
      781 ggaacgtgtc ctggaagcaa atgacggcag cggcatgctg gatgaagacg aagaagatct
      841 gcagcgtgcc ctggcactgt ctcgtcagga aattgatatg gaagacgaag aagcagatct
      901 gcgtcgcgct attcagctgt caatgcaggg cagctctcgt aacatctcgc aggacatgac
      961 ccagacgagc ggtaccaatc tgacgtctga agaactgcgc aaacgtcgcg aagcatattt
     1021 tgaaaaacag caacagaagc aacaacagca gcaacaacaa caacaacagc agcagcagca
     1081 gcaacaacaa cagcaacaac aacagcaaca gcagcaacag caacagcaac agcagcaaca
     1141 acagcaacag caacaacagc aacagcaaca acagcaacaa cagcaacaac agcaacaaca
     1201 gcaacaacag caacaacagc aacagcaaca acagcagcag caacagcagc aacaacaaca
     1261 gagagatctg tcaggccaga gttcccatcc gtgtgaacgt ccggccacct caagcggtgc
     1321 actgggtagt gatctgggtg acgccatgtc cgaagaagac atgctgcagg cagcagtgac
     1381 gatgtccctg gaaaccgtgc gtaacgacct gaaaaccgaa ggcaaaaaat aagacccagc
     1441 tttcttgtac aaagtggttg attcgaggct gctaacaaag cccgaaagga agctgagttg
     1501 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1561 aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg gtgtggtcgc
     1621 catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg ggcggcggcc
     1681 aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca acgcatatag
     1741 cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat cccgcaagag
     1801 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     1861 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     1921 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt
     1981 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2041 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2101 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2161 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2221 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2281 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2341 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2401 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     2461 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2521 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2581 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2641 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2701 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     2761 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2821 aagttgcagg accacttctg cgctcggccc ttccgactgg ctggtttatt gctgataaat
     2881 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2941 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3001 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3061 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3121 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3181 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3241 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3301 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3361 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3421 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3481 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3541 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3601 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3661 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3721 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3781 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3841 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3901 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3961 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4021 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4081 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4141 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4201 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4261 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4321 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4381 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4441 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4501 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4561 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4621 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4681 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4741 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4801 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4861 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     4921 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     4981 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     5041 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     5101 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     5161 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     5221 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     5281 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     5341 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     5401 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     5461 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     5521 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     5581 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     5641 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgatcgacg ctctccctta
     5701 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     5761 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     5821 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5881 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     5941 acgatgcgtc cggcgtagag gatcg
//
