LOCUS       sequence-409179-        5965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant protein expression and purification. Encodes ataxin-3 
            full length with 3 UIMs and 13Q in the Poly-Q track-R388G 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-R388G
FEATURES             Location/Qualifiers
     source          1..5965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1179..1199)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1203..1220)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1240..1262
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(1293..1312)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1294..1312)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1396..1415)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             2877..3068
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3062..3084)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3170..3309
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3225..3244
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3324..3341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3495..4083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3575..3594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4254..5045,5046..5114))
                     /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     4877..4896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5115..5219)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5287..5305
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5332..5337,5338..5354,5355..5360)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(5726..5773)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5769..5787
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     protein_bind    5840..5864
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 tcactaccca gtgcaccgct tgaggtggcc ggacgttcac acggatggga actctggcct
       61 gacagatctc cctgttgttg ttgctgctgt tgctgctgct gttgttgctg ttgctgttgt
      121 tgctgttgtt gctgttgttg ctgttgttgc tgttgttgct gttgttgctg ttgctgttgt
      181 tgctgttgct gttgttgctg ctgttgctgt tgctgttgct gctgttgctg ttgttgttgc
      241 tgttgttgtt gctgctgctg ctgctgttgt tgttgttgtt gctgctgttg ttgcttctgt
      301 tgctgttttt caaaatatgc ttcgcgacgt ttgcgcagtt cttcagacgt cagattggta
      361 ccgctcgtct gggtcatgtc ctgcgagatg ttacgagagc tgccctgcat tgacagctga
      421 atagcgcgac gcagatctgc ttcttcgtct tccatatcaa tttcctgacg agacagtgcc
      481 agggcacgct gcagatcttc ttcgtcttca tccagcatgc cgctgccgtc atttgcttcc
      541 aggacacgtt ccaggtcggt cttatgcaca cgctgttctt tcagttgagc cagttcttcg
      601 ccaatcagtt ttggtcgatg catctgttgg accctaatca tctgcaggag ttggtcagct
      661 tcgcaatctg gcagatcacc cttaacgaca aatatagaat aaccttcctg ttgtaattga
      721 gccaagaaaa gtgcaagata tgtatctgat attaattctg gacccgtcaa gagagaattc
      781 aagttaaacc actgttttcc taattttcta actgtaaacc agtgttcctt ataattgcat
      841 ataaatgatc tttcatttat aggatcgatc ctgagcctct gatactctgg actgttgaac
      901 aggattagtt ctaaacccca aactttcaag gcattgctta taacctgaat agagaaaaaa
      961 ccactgtcat ccatatttcc agaaggctgc tgtaaaaacg tgcgataatc ttcactagta
     1021 actcctcctt ctgccattct catcctctcc tcctcatcca gctgatgtgc aattgaggat
     1081 aattccacag ggctaaaata ttctccttgc aataagttat tcaggcaatg ttgagcacaa
     1141 agtgagcctt cttgtttctc gtggaagatg gactccatgc cctgaaaata caggttttcg
     1201 aggtgatggt gatggtgatg gtagtacgac atatgtatat ctccttctta aagttaaaca
     1261 aaattatttc tagagggaaa ccgttgtggt ctccctatag tgagtcgtat taatttcgcg
     1321 ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
     1381 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
     1441 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     1501 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
     1561 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgatcgaccg
     1621 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     1681 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     1741 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
     1801 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc
     1861 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac
     1921 gtcttgctgg cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct
     1981 tccggcggca tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac
     2041 catcagggac agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcattgga
     2101 ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg
     2161 attgtaggcg ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc
     2221 cgggccacct cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa
     2281 gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga
     2341 acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg
     2401 ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     2461 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     2521 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     2581 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg
     2641 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
     2701 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac
     2761 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc
     2821 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga
     2881 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
     2941 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
     3001 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
     3061 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     3121 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     3181 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
     3241 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     3301 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3361 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3421 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3481 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3541 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3601 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3661 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3721 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3781 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3841 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3901 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     3961 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4021 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4081 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4141 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4201 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4261 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4321 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4381 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4441 gtcggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4501 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4561 gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4621 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4681 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4741 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4801 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4861 ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4921 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4981 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5041 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5101 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5161 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5221 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     5281 tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattctcatg tttgacagct
     5341 tatcatcgat aagctttaat gcggtagttt atcacagtta aattgctaac gcagtcaggc
     5401 accgtgtatg aaatctaaca atgcgctcat cgtcatcctc ggcaccgtca ccctggatgc
     5461 tgtaggcata ggcttggtta tgccggtact gccgggcctc ttgcgggata tcgtccattc
     5521 cgacagcatc gccagtcact atggcgtgct gctagcgcta tatgcgttga tgcaatttct
     5581 atgcgcaccc gttctcggag cactgtccga ccgctttggc cgccgcccag tcctgctcgc
     5641 ttcgctactt ggagccacta tcgactacgc gatcatggcg accacacccg tcctgtggat
     5701 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
     5761 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
     5821 tgttagcagc ctcgaatcaa ccactttgta caagaaagct gggtcttatt ttttgccttc
     5881 ggttttcagg tcgttacgca cggtttccag ggacatcgtc actgctgcct gcagcatgtc
     5941 ttcttcggac atggcgtcac ccaga
//
