LOCUS       sequence-222644-        6125 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression of Htt fragment containing amino acids 2-90, 
            with P90C cysteine mutation and a 15 polyQ repeat.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6125)
  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 6125)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Htt2-90-15Q-P90C
FEATURES             Location/Qualifiers
     source          1..6125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      44..130
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     265..284
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        265..283
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    284..308
                     /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             323..345
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             356..373
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1216..1234)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1230..1277
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1337..1441
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1442..1510,1511..2302)
                     /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(1660..1679)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2344..2857)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2539..2560)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2751..2770
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      2968..3556
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3457..3476
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3710..3727
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3749..3768)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3909..3931
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3925..4116)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4673..5755)
                     /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     5717..5736
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5756..5833)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      5986..6072
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aggacgcccg ccataaactg ccaggaatta attccccagg catcaaataa aacgaaaggc
       61 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
      121 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
      181 ggcaggacgc ccgccataaa ctgccaggaa ttggggatcg gaattaattc ccggtttaaa
      241 ccggggatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      301 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac atatgcatca
      361 tcatcatcac catagcggtg caattagcgg tgatagcctg attagcctgg caagcaccgg
      421 taaacgtgtt agcattaaag atctgctgga tgagaaagat tttgagattt gggccattaa
      481 cgagcagacc atgaaactgg aaagcgcaaa agttagccgt gttttttgca ccggtaaaaa
      541 actggtgtat atcctgaaaa cccgtctggg tcgtaccatt aaagcaaccg caaatcatcg
      601 ttttctgacc attgatggtt ggaaacgtct ggatgaactg agcctgaaag aacatattgc
      661 actgcctcgt aaactggaaa gtagcagcct gcaactgagt ccggaaattg aaaaactgag
      721 ccagagcgat atctattggg atagcattgt tagcattacc gaaaccggtg ttgaagaagt
      781 ttttgatctg accgttccgg gtccgcacaa ttttgttgca aatgatatta ttgtgcacaa
      841 cgccaccctg gaaaaactga tgaaagcatt tgaaagcctg aaaagctttc agcagcagca
      901 acaacagcag caacagcaac aacaacaaca gcagcctccg cctccgcctc ctccaccacc
      961 tcctccacag ctgcctcagc ctccaccgca ggcacagcct ctgctgccac agcctcagcc
     1021 acctccaccg ccaccgcctc cacctccggg tccagcagtt gcagaagaac cgctgcatcg
     1081 ttgctaactg caggaagggg gaaccgctgc atcgttgcta actgcaggaa ggggaaccgc
     1141 tgcatcgttg ctaactgcag gaaggggatc cggctgctaa caaagcccga aaggaagctg
     1201 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1261 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ataactacgt caggtggcac
     1321 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1381 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     1441 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     1501 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     1561 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     1621 cgaagaacgt ttcccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     1681 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     1741 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     1801 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     1861 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     1921 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     1981 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2041 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2101 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2161 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2221 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2281 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     2341 tttaccccgg ttgataatca gaaaagcccc aaaaacagga agattgtata agcaaatatt
     2401 taaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2461 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagcccgag
     2521 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2581 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2641 aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2701 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2761 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2821 acacccgccg cgcttaatgc gccgctacag ggcgcgtaaa aggatctagg tgaagatcct
     2881 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2941 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3001 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3061 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3121 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3181 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3241 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3301 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3361 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3421 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3481 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3541 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3601 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3661 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3721 gagcgaggaa gctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3781 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3841 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3901 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3961 gcagctgcgg taaagctcat cagcgtggtc gtgcagcgat tcacagatgt ctgcctgttc
     4021 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4081 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4141 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4201 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4261 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag ccgaacgcca gcaagacgta
     4321 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4381 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4441 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4501 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4561 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4621 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4681 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4741 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4801 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4861 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4921 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     4981 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5041 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5101 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5161 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5221 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5281 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5341 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5401 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5461 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5521 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5581 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5641 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5701 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5761 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5821 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5881 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgccggcat
     5941 gccgcccttt cgtcttcaag aattaattcc caattcccca ggcatcaaat aaaacgaaag
     6001 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     6061 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     6121 cgggc
//
