LOCUS       sequence-222643-        6082 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression of Htt fragment containing amino acids 2-90, 
            with A2C cysteine mutation and a 23 polyQ repeat.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6082)
  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 6082)
  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)-23Q
FEATURES             Location/Qualifiers
     source          1..6082
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..173
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        154..172
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    173..197
                     /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             212..234
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             245..262
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1062..1080)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1076..1123
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1183..1287
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1288..1356,1357..2148)
                     /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(1506..1525)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2190..2703)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2385..2406)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2597..2616
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      2814..3402
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3303..3322
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3556..3573
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3595..3614)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3755..3777
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3771..3962)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4519..5601)
                     /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     5563..5582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5602..5679)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      5832..5918
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(6015..6082,1..19)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg
       61 agggtggcgg gcaggacgcc cgccataaac tgccaggaat tggggatcgg aattaattcc
      121 cggtttaaac cggggatctc gatcccgcga aattaatacg actcactata ggggaattgt
      181 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca
      241 tatgcatcat catcatcacc atagcggtgc aattagcggt gatagcctga ttagcctggc
      301 aagcaccggt aaacgtgtta gcattaaaga tctgctggat gagaaagatt ttgagatttg
      361 ggccattaac gagcagacca tgaaactgga aagcgcaaaa gttagccgtg ttttttgcac
      421 cggtaaaaaa ctggtgtata tcctgaaaac ccgtctgggt cgtaccatta aagcaaccgc
      481 aaatcatcgt tttctgacca ttgatggttg gaaacgtctg gatgaactga gcctgaaaga
      541 acatattgca ctgcctcgta aactggaaag tagcagcctg caactgagtc cggaaattga
      601 aaaactgagc cagagcgata tctattggga tagcattgtt agcattaccg aaaccggtgt
      661 tgaagaagtt tttgatctga ccgttccggg tccgcacaat tttgttgcaa atgatattat
      721 tgtgcacaac tgcaccctgg aaaaactgat gaaagcattt gaaagcctga aaagctttca
      781 gcaacaacag cagcaacagc aacagcagca gcagcagcaa caacagcagc agcaacagca
      841 acaacaaccg cctccgcctc cgccacctcc tcctccacca cagctgcctc agcctccgcc
      901 tcaggcacag cctctgctgc cacagcctca gccacctcca ccgcctccac ctccacctcc
      961 gggtccggca gttgcagaag aaccgctgca tcgtccgtaa ctgcaggaag gggatccggc
     1021 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1081 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1141 atccggataa ctacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1201 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1261 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1321 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1381 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1441 ggtaagatcc ttgagagttt tcgccccgaa gaacgtttcc caatgatgag cacttttaaa
     1501 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     1561 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1621 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1681 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1741 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1801 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     1861 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     1921 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     1981 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2041 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2101 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2161 gtttactcat atatacttta gattgattta ccccggttga taatcagaaa agccccaaaa
     2221 acaggaagat tgtataagca aatatttaaa ttgtaaacgt taatattttg ttaaaattcg
     2281 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2341 cttataaatc aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga
     2401 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2461 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     2521 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2581 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2641 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2701 cgtaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2761 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2821 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2881 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2941 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3001 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3061 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3121 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3181 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3241 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3301 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3361 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3421 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3481 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3541 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcta tggtgcactc tcagtacaat
     3601 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3661 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3721 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3781 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc
     3841 agcgattcac agatrtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3901 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3961 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     4021 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     4081 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     4141 tgccagccga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     4201 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4261 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4321 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4381 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4441 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4501 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4561 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4621 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4681 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4741 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4801 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4861 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4921 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4981 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     5041 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5101 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5161 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5221 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5281 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5341 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5401 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5461 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5521 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5581 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5641 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5701 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5761 ccgcaaggaa tggtgcatgc cggcatgccg ccctttcgtc ttcaagaatt aattcccaat
     5821 tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     5881 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     5941 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggaatt
     6001 aattccccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     6061 tctgttgttt gtcggtgaac gc
//
