LOCUS       sequence-222760-        6170 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression of Htt fragment containing amino acids 2-90, 
            with P90C cysteine mutation and a 43 polyQ repeat.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6170)
  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 6170)
  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-Htt2-90-43Q-P90C
FEATURES             Location/Qualifiers
     source          1..6170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      39..125
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     260..279
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        260..278
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    279..303
                     /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             318..340
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             351..368
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1256..1274)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1270..1317
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1377..1481
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1482..1550,1551..2342)
                     /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(1700..1719)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2384..2897)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2579..2600)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2791..2810
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3008..3596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3497..3516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3750..3767
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3789..3808)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3949..3971
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3965..4156)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4713..5795)
                     /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     5757..5776
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5796..5873)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      6026..6112
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcccgccata aactgccagg aattaattcc ccaggcatca aataaaacga aaggctcagt
       61 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
      121 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
      181 gacgcccgcc ataaactgcc aggaattggg gatcggaatt aattcccggt ttaaaccggg
      241 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
      301 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg catcatcatc
      361 atcaccatag cggtgcaatt agcggtgata gcctgattag cctggcaagc accggtaaac
      421 gtgttagcat taaagatctg ctggatgaga aagattttga gatttgggcc attaacgagc
      481 agaccatgaa actggaaagc gcaaaagtta gccgtgtttt ttgcaccggt aaaaaactgg
      541 tgtatatcct gaaaacccgt ctgggtcgta ccattaaagc aaccgcaaat catcgttttc
      601 tgaccattga tggttggaaa cgtctggatg aactgagcct gaaagaacat attgcactgc
      661 ctcgtaaact ggaaagtagc agcctgcaac tgagtccgga aattgaaaaa ctgagccaga
      721 gcgatatcta ttgggatagc attgttagca ttaccgaaac cggtgttgaa gaagtttttg
      781 atctgaccgt tccgggtccg cacaattttg ttgcaaatga tattattgtg cacaacgcca
      841 ccctggaaaa actgatgaaa gcatttgaaa gcctgaaaag ctttcagcag cagcaacaac
      901 agcagcaaca gcaacagcag cagcagcagc aacaacagca gcagcaacag caacaacaac
      961 aacaacagca gcaacaacag caacagcagc aacaacaaca gcaacagcaa caaccgcctc
     1021 cgcctccgcc acctcctcct ccaccacagc tgcctcagcc tccgcctcag gcacagcctc
     1081 tgctgccaca gcctcagcca cctccaccgc ctccaccccc acctccgggt ccggcagttg
     1141 cagaagaacc gctgcatcgt tgctaactgc agggaaccgc tgcatcgttg ctaactgcag
     1201 gaaggggatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1261 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1321 aaaggaggaa ctatatccgg ataactacgt caggtggcac ttttcgggga aatgtgcgcg
     1381 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1441 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1501 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1561 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1621 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt ttcccaatga
     1681 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     1741 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1801 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1861 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1921 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1981 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     2041 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2101 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2161 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2221 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2281 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2341 aactgtcaga ccaagtttac tcatatatac tttagattga tttaccccgg ttgataatca
     2401 gaaaagcccc aaaaacagga agattgtata agcaaatatt taaattgtaa acgttaatat
     2461 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2521 aatcggcaaa atcccttata aatcaaaaga atagcccgag atagggttga gtgttgttcc
     2581 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2641 cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc
     2701 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2761 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2821 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2881 gccgctacag ggcgcgtaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2941 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3001 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3061 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3121 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3181 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3241 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3301 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3361 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3421 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3481 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3541 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3601 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3661 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3721 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gctatggtgc
     3781 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     3841 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     3901 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3961 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     4021 cagcgtggtc gtgcagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     4081 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     4141 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     4201 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     4261 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     4321 tcactcaggg tcaatgccag ccgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     4381 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4441 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4501 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4561 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4621 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4681 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4741 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4801 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     4861 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     4921 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     4981 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5041 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5101 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5161 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5221 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5281 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5341 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5401 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5461 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5521 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5581 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5641 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     5701 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     5761 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     5821 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     5881 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5941 gttgagcacc gccgccgcaa ggaatggtgc atgccggcat gccgcccttt cgtcttcaag
     6001 aattaattcc caattcccca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     6061 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     6121 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac
//
