LOCUS       sequence-370314-        5773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant protein expression and purification. Encodes ataxin-3 
            Josephin domain and UIM1 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 5773)
  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 5773)
  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-D1
FEATURES             Location/Qualifiers
     source          1..5773
                     /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(1242..1266)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1319..1337)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1333..1380
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1746..1751,1752..1768,1769..1774))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1801..1819)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1887..1991
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1992..2060,2061..2852)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2210..2229)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3023..3611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3512..3531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3765..3782
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3797..3936
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3862..3881)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4022..4044
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4038..4229)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5691..5710
                     /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 aaaacttatt ggagaagaat tagcacaact aaaagagcaa agagtccata aaacagacct
      781 ggaacgagtg ttagaagcaa atgatggctc aggaatgtta gacgaagatg aggaggattt
      841 gcagagggct ctggcactaa gtcgccaaga aattgacatg gaagatgagg aagcagatct
      901 ccgcagggct attcagctaa gtatgcaagg tagctcgaga tagatatctc aagatatgac
      961 acagacatca ggtacaaatc ttacttcaga agagcttcgg aagagacgag aagcctactt
     1021 tgaaaaacag cagcaaaagc agcaacagca gcagcagcag cagcagcagg gggacctatc
     1081 aggacagagt tcacatccat gtgaaaggcc agccaccagt tcaggagcac ttgggagtga
     1141 tctaggtgat gctatgagtg aagaagacat gcttcaggca gctgtgacca tgtctttaga
     1201 aactgtcaga aatgatttga aaacagaagg aaaaaaataa gacccagctt tcttgtacaa
     1261 agtggttgat tcgaggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1321 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1381 ctgaaaggag gaactatatc cggatatcca caggacgggt gtggtcgcca tgatcgcgta
     1441 gtcgatagtg gctccaagta gcgaagcgag caggactggg cggcggccaa agcggtcgga
     1501 cagtgctccg agaacgggtg cgcatagaaa ttgcatcaac gcatatagcg ctagcagcac
     1561 gccatagtga ctggcgatgc tgtcggaatg gacgatatcc cgcaagaggc ccggcagtac
     1621 cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag
     1681 cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact
     1741 accgcattaa agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg
     1801 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     1861 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1921 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1981 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2041 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2101 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2161 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2221 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2281 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2341 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2401 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2461 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2521 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2581 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2641 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2701 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2761 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2821 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2881 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2941 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3001 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3061 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3661 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3721 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3781 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3841 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3901 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3961 gacgcgccct gacgggcttg tctgctcccg acatccgctt acagacaagc tgtgaccgtc
     4021 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4081 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4141 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4201 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4261 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4321 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4381 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4441 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4501 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4561 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4621 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4681 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
     4741 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
     4801 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
     4861 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
     4921 caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg
     4981 cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga
     5041 gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca
     5101 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc
     5161 ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa
     5221 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     5281 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     5341 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     5401 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     5461 ctgggttgaa ggctctcaag ggcatcggtc gatcgacgct ctcccttatg cgactcctgc
     5521 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5581 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     5641 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     5701 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     5761 gcgtagagga tcg
//
