LOCUS       sequence-222657-        6124 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression of Htt fragment containing amino acids 2-90, 
            with A2C cysteine mutation and a 37 polyQ repeat.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6124)
  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 6124)
  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(A2C)-37Q
FEATURES             Location/Qualifiers
     source          1..6124
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      115..201
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     336..355
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        336..354
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    355..379
                     /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             394..416
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             427..444
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1286..1304)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1300..1347
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1407..1511
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1512..1580,1581..2372)
                     /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(1730..1749)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2414..2927)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2609..2630)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2821..2840
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3038..3626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3527..3546
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3780..3797
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3819..3838)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3979..4001
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3995..4186)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4743..5825)
                     /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     5787..5806
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5826..5903)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      join(6056..6124,1..18)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
       61 gggtggcggg caggacgccc gccataaact gccaggaatt aattccccag gcatcaaata
      121 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
      181 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
      241 ggagggtggc gggcaggacg cccgccataa actgccagga attggggatc ggaattaatt
      301 cccggtttaa accggggatc tcgatcccgc gaaattaata cgactcacta taggggaatt
      361 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
      421 catatgcatc atcatcatca ccatagcggt gcaattagcg gtgatagcct gattagcctg
      481 gcaagcaccg gtaaacgtgt tagcattaaa gatctgctgg atgagaaaga ttttgagatt
      541 tgggccatta acgagcagac catgaaactg gaaagcgcaa aagttagccg tgttttttgc
      601 accggtaaaa aactggtgta tatcctgaaa acccgtctgg gtcgtaccat taaagcaacc
      661 gcaaatcatc gttttctgac cattgatggt tggaaacgtc tggatgaact gagcctgaaa
      721 gaacatattg cactgcctcg taaactggaa agtagcagcc tgcaactgag tccggaaatt
      781 gaaaaactga gccagagcga tatctattgg gatagcattg ttagcattac cgaaaccggt
      841 gttgaagaag tttttgatct gaccgttccg ggtccgcaca attttgttgc aaatgatatt
      901 attgtgcaca actgcaccct ggaaaaactg atgaaagcat ttgaaagcct gaaaagcttt
      961 cagcagcagc aacaacagca gcaacagcaa cagcagcagc agcagcaaca acagcagcag
     1021 caacagcaac aacaacaaca acagcagcaa caacagcaac agcagcagca accgcctccg
     1081 cctccgccac ctcctcctcc accacagctg cctcagcctc cgcctcaggc acagcctctg
     1141 ctgccacagc ctcagccacc tccaccgcct ccacctccac ctccgggtcc ggcagttgca
     1201 gaagaaccgc tgcatcgtcc gtaactgcag gaaggggatc cggctgctaa caaagcccga
     1261 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1321 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ataactacgt
     1381 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1441 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1501 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1561 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1621 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1681 gttttcgccc cgaagaacgt ttcccaatga tgagcacttt taaagttctg ctatgtggcg
     1741 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1801 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1861 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1921 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1981 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2041 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2101 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2161 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2221 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2281 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2341 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2401 tttagattga tttaccccgg ttgataatca gaaaagcccc aaaaacagga agattgtata
     2461 agcaaatatt taaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2521 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2581 atagcccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2641 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2701 accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2761 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2821 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2881 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtaaa aggatctagg
     2941 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3001 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3061 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3121 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3181 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3241 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3301 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3361 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3421 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3481 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3541 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3601 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3661 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3721 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3781 gcgagtcagt gagcgaggaa gctatggtgc actctcagta caatctgctc tgatgccgca
     3841 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3901 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3961 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4021 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgcagcgat tcacagatgt
     4081 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4141 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4201 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4261 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4321 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag ccgaacgcca
     4381 gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga
     4441 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4501 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4561 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4621 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4681 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4741 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4801 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4861 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     4921 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     4981 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5041 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5101 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5161 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5221 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5281 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5341 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5401 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5461 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5521 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5581 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5641 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5701 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5761 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5821 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     5881 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     5941 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6001 atgccggcat gccgcccttt cgtcttcaag aattaattcc caattcccca ggcatcaaat
     6061 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     6121 cgct
//
