LOCUS       sequence-370313-        5785 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant protein expression and purification. Encodes ataxin-3 
            Josephin domain fused with His-Tag (N-terminal).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5785)
  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 5785)
  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-JD
FEATURES             Location/Qualifiers
     source          1..5785
                     /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"
     protein_bind    140..164
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1254..1278)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1331..1349)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1345..1392
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1758..1763,1764..1780,1781..1786))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1813..1831)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1899..2003
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2004..2072,2073..2864)
                     /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(2222..2241)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3035..3623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3524..3543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3777..3794
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3809..3948
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3874..3893)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4034..4056
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4050..4241)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5703..5722
                     /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 ctcgaatcaa caagtttgta caaaaaagca ggctggatgg agtccatctt
      181 ccacgagaaa caagaaggct cactttgtgc tcaacattgc ctgaataact tattgcaagg
      241 agaatatttt agccctgtgg aattatcctc aattgcacat cagctggatg aggaggagag
      301 gatgagaatg gcagaaggag gagttactag tgaagattat cgcacgtttt tacagcagcc
      361 ttctggaaat atggatgaca gtggtttttt ctctattcag gttataagca atgccttgaa
      421 agtttggggt ttagaactaa tcctgttcaa cagtccagag tatcagaggc tcaggatcga
      481 tcctataaat gaaagatcat ttatatgcaa ttataaggaa cactggttta cagttagaaa
      541 attaggaaaa cagtggttta acttgaattc tctcttgacg ggtccagaat taatatcaga
      601 tacatatctt gcacttttct tggctcaatt acaacaggaa ggttattcta tatttgtcgt
      661 taagggtgat ctgccagatt gcgaagctga ccaactccta cagatgatta ggtgacaaca
      721 gatgcatcga ccaaaactta ttggagaaga attagcacaa ctaaaagagc aaagagtcca
      781 taaaacagac ctggaacgag tgttagaagc aaatgatggc tcaggaatgt tagacgaaga
      841 tgaggaggat ttgcagaggg ctctggcact aagtcgccaa gaaattgaca tggaagatga
      901 ggaagcagat ctccgcaggg ctattcagct aagtatgcaa ggtagttcca gaaacatatc
      961 tcaagatatg acacagacat caggtacaaa tcttacttca gaagagcttc ggaagagacg
     1021 agaagcctac tttgaaaaac agcagcaaaa gcagcaacag cagcagcagc agcagcagca
     1081 gggggaccta tcaggacaga gttcacatcc atgtgaaagg ccagccacca gttcaggagc
     1141 acttgggagt gatctaggtg atgctatgag tgaagaagac atgcttcagg cagctgtgac
     1201 catgtcttta gaaactgtca gaaatgattt gaaaacagaa ggaaaaaaat aagacccagc
     1261 tttcttgtac aaagtggttg attcgaggct gctaacaaag cccgaaagga agctgagttg
     1321 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1381 aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg gtgtggtcgc
     1441 catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg ggcggcggcc
     1501 aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca acgcatatag
     1561 cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat cccgcaagag
     1621 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     1681 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     1741 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt
     1801 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     1861 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1921 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     1981 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2041 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2101 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2161 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2221 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     2281 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2341 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2401 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2461 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2521 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     2581 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2641 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2701 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2761 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2821 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     2881 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     2941 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3001 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3061 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3121 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3181 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3241 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3301 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3361 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3421 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3481 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3541 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3601 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3661 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3721 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3781 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3841 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3901 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3961 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4021 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4081 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4141 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4201 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4261 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4321 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4381 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4441 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4501 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4561 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4621 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4681 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     4741 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     4801 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     4861 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     4921 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     4981 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     5041 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     5101 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     5161 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     5221 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     5281 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     5341 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     5401 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     5461 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgatcgacg ctctccctta
     5521 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     5581 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     5641 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5701 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     5761 acgatgcgtc cggcgtagag gatcg
//
