LOCUS       sequence-222755-        6349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Htt fragment containing amino acids 2-90, 
            with P90C cysteine mutation and a 37 polyQ repeat.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6349)
  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 6349)
  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-Htt2-90-37Q-P90C
FEATURES             Location/Qualifiers
     source          1..6349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      33..119
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     254..273
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        254..272
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    273..297
                     /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             312..334
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             345..362
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1429..1447)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1443..1490
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1550..1654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1655..1723,1724..2515)
                     /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(1873..1892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2557..3070)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2752..2773)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2964..2983
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3181..3769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3670..3689
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3923..3940
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3962..3981)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4122..4144
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4138..4329)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4886..5968)
                     /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     5930..5949
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5969..6046)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      6199..6285
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cataaactgc caggaattaa ttccccaggc atcaaataaa acgaaaggct cagtcgaaag
       61 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
      121 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
      181 cgccataaac tgccaggaat tggggatcgg aattaattcc cggtttaaac cggggatctc
      241 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
      301 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcatcat catcatcacc
      361 atagcggtgc aattagcggt gatagcctga ttagcctggc aagcaccggt aaacgtgtta
      421 gcattaaaga tctgctggat gagaaagatt ttgagatttg ggccattaac gagcagacca
      481 tgaaactgga aagcgcaaaa gttagccgtg ttttttgcac cggtaaaaaa ctggtgtata
      541 tcctgaaaac ccgtctgggt cgtaccatta aagcaaccgc aaatcatcgt tttctgacca
      601 ttgatggttg gaaacgtctg gatgaactga gcctgaaaga acatattgca ctgcctcgta
      661 aactggaaag tagcagcctg caactgagtc cggaaattga aaaactgagc cagagcgata
      721 tctattggga tagcattgtt agcattaccg aaaccggtgt tgaagaagtt tttgatctga
      781 ccgttccggg tccgcacaat tttgttgcaa atgatattat tgtgcacaac gccaccctgg
      841 aaaaactgat gaaagcattt gaaagcctga aaagctttca gcagcagcaa caacagcagc
      901 aacagcaaca gcagcagcag cagcaacaac agcagcagca acagcaacaa caacaacaac
      961 agcagcaaca acagcaacag cagcagcaac cgcctccgcc tccgccacct cctcctccac
     1021 cacagctgcc tcagcctccg cctcaggcac agcctctgct gccacagcct cagccacctc
     1081 caccgcctcc acctccacct ccgggtccgg cagttgcaga agaaccgctg catcgttgct
     1141 aactgcagga agggagaacc gctgcatcgt tgctaactgc aggaagagaa ccgctgcatc
     1201 gttgctatct gcaggaaggg gccgctgcat cgttgctaac tgcaggaagg ggaccgctgc
     1261 atcgttgcta actgcaggaa ggggccgctg catcgttgct aactgcagga aggagaaccg
     1321 ctgcatcgtt gctaactgca ggaagggaac cgctgcatcg ttgctaactg caggaagggg
     1381 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1441 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     1501 gaactatatc cggataacta cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1561 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1621 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1681 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1741 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1801 caacagcggt aagatccttg agagttttcg ccccgaagaa cgtttcccaa tgatgagcac
     1861 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     1921 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1981 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2041 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2101 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2161 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     2221 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2281 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2341 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2401 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2461 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2521 agaccaagtt tactcatata tactttagat tgatttaccc cggttgataa tcagaaaagc
     2581 cccaaaaaca ggaagattgt ataagcaaat atttaaattg taaacgttaa tattttgtta
     2641 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     2701 aaaatccctt ataaatcaaa agaatagccc gagatagggt tgagtgttgt tccagtttgg
     2761 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     2821 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc
     2881 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     2941 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     3001 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     3061 cagggcgcgt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3121 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3181 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3241 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3301 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3361 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3421 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3481 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3541 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3601 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3661 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3721 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3781 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3841 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3901 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagctatgg tgcactctca
     3961 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4021 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4081 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4141 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4201 gtcgtgcagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4261 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4321 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4381 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4441 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4501 gggtcaatgc cagccgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc
     4561 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4621 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4681 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     4741 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     4801 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4861 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4921 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4981 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     5041 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5101 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5161 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5221 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5281 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5341 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5401 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5461 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5521 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5581 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5641 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5701 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5761 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5821 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5881 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5941 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     6001 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     6061 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     6121 accgccgccg caaggaatgg tgcatgccgg catgccgccc tttcgtcttc aagaattaat
     6181 tcccaattcc ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     6241 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     6301 ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgc
//
