LOCUS       sequence-222660-        6292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Htt fragment containing amino acids 2-90, 
            with P90C cysteine mutation and a 49 polyQ repeat.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6292)
  AUTHORS   Warner JB 4th, Ruff KM, Tan PS, Lemke EA, Pappu RV, Lashuel HA
  TITLE     Monomeric Huntingtin Exon 1 Has Similar Overall Structural Features 
            for Wild-Type and Pathological Polyglutamine Lengths.
  JOURNAL   J Am Chem Soc. 2017 Oct 18;139(41):14456-14469. doi: 
            10.1021/jacs.7b06659. Epub 2017 Oct 9.
  PUBMED    28937758
REFERENCE   2  (bases 1 to 6292)
  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"; doi: 
            "10.1021/jacs.7b06659"; journalName: "J Am Chem Soc"; date: 
            "2017-10-18- 18"; volume: "139"; issue: "41"; pages: "14456-14469"
COMMENT     Sequence Label: pTWIN1-His6-Ssp-Htt2-90-49Q-P90C
FEATURES             Location/Qualifiers
     source          1..6292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      36..122
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     257..276
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        257..275
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    276..300
                     /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)."
     RBS             315..337
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             348..365
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1375..1393)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1389..1436
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1496..1600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1601..1669,1670..2461)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1819..1838)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2503..3016)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2698..2719)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2910..2929
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3127..3715
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3616..3635
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3869..3886
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3908..3927)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4068..4090
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4084..4275)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4832..5914)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5876..5895
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5915..5992)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      6145..6231
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag gctcagtcga
       61 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
      121 atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac
      181 gcccgccata aactgccagg aattggggat cggaattaat tcccggttta aaccggggat
      241 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
      301 cctctagaaa taattttgtt taactttaag aaggagatat acatatgcat catcatcatc
      361 accatagcgg tgcaattagc ggtgatagcc tgattagcct ggcaagcacc ggtaaacgtg
      421 ttagcattaa agatctgctg gatgagaaag attttgagat ttgggccatt aacgagcaga
      481 ccatgaaact ggaaagcgca aaagttagcc gtgttttttg caccggtaaa aaactggtgt
      541 atatcctgaa aacccgtctg ggtcgtacca ttaaagcaac cgcaaatcat cgttttctga
      601 ccattgatgg ttggaaacgt ctggatgaac tgagcctgaa agaacatatt gcactgcctc
      661 gtaaactgga aagtagcagc ctgcaactga gtccggaaat tgaaaaactg agccagagcg
      721 atatctattg ggatagcatt gttagcatta ccgaaaccgg tgttgaagaa gtttttgatc
      781 tgaccgttcc gggtccgcac aattttgttg caaatgatat tattgtgcac aacgccaccc
      841 tggaaaaact gatgaaagca tttgaaagcc tgaaaagctt tcagcagcag caacaacagc
      901 agcaacagca acagcagcag cagcagcaac aacagcagca gcaacagcaa caacaacaac
      961 aacagcagca acaacagcaa cagcagcaac aacaacagca acagcagcag cagcaacaac
     1021 agcaacagcc tccgcctccg ccaccgcctc ctccacctcc gcagctgccg caacctcctc
     1081 ctcaggcaca gccgctgctg ccgcagcctc aacctcctcc accacctcca cctccaccgc
     1141 ctggtccggc agttgcagaa gaaccgctgc atcgttgcta actgcaggaa gggggaaccg
     1201 ctgcatcgtt gctaactgca ggaaggggaa ccgctgcatc gttgctaact gcaggaaggg
     1261 gccgctgcat cgttgctaac tgcaggaagg gagaaccgct gcatcgttgc taactgcagg
     1321 aaggggatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1381 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1441 aaggaggaac tatatccgga taactacgtc aggtggcact tttcggggaa atgtgcgcgg
     1501 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1561 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1621 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1681 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1741 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt tcccaatgat
     1801 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     1861 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1921 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     1981 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2041 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2101 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     2161 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2221 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2281 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2341 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2401 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2461 actgtcagac caagtttact catatatact ttagattgat ttaccccggt tgataatcag
     2521 aaaagcccca aaaacaggaa gattgtataa gcaaatattt aaattgtaaa cgttaatatt
     2581 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2641 atcggcaaaa tcccttataa atcaaaagaa tagcccgaga tagggttgag tgttgttcca
     2701 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2761 gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttggggtcg
     2821 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2881 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2941 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3001 ccgctacagg gcgcgtaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3061 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3121 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3181 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3241 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3301 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3361 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3421 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3481 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3541 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3601 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3661 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3721 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3781 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3841 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag ctatggtgca
     3901 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3961 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4021 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4081 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4141 agcgtggtcg tgcagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4201 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4261 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4321 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4381 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4441 cactcagggt caatgccagc cgaacgccag caagacgtag cccagcgcgt cggccgccat
     4501 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     4561 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4621 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     4681 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4741 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     4801 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4861 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4921 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     4981 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     5041 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     5101 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5161 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     5221 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5281 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5341 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5401 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5461 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5521 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5581 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5641 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     5701 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     5761 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     5821 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     5881 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     5941 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6001 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     6061 ttgagcaccg ccgccgcaag gaatggtgca tgccggcatg ccgccctttc gtcttcaaga
     6121 attaattccc aattccccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     6181 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     6241 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cc
//
