LOCUS       Exported                9487 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    HSP104-b/Leu(WT)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9487)
  AUTHORS   Schirmer EC, Homann OR, Kowal AS, Lindquist S
  TITLE     Dominant gain-of-function mutations in Hsp104p reveal crucial roles 
            for the middle region.
  JOURNAL   Mol Biol Cell 2004 May;15(5):2061-72. Epub 2004 Feb 20.
  PUBMED    14978213
REFERENCE   2  (bases 1 to 9487)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9487
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(663..1757)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1770..2174)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2474..2929)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2611..2632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2823..2842
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3059..3081
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3073..3090
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3074..3090
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3097..3116
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3097..3115
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5978..6642
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     primer_bind     complement(5991..6014)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    6309..6426
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     complement(6667..6683)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6668..6684)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(6713..6731)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6752..6768)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6752..6768)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6765..6787)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6776..6792
                     /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)."
     promoter        complement(6800..6830)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(6983..7000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7154..7742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7234..7253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7913..8773)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8536..8555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8774..8878)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    8915..9418
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     9460..9478
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
      241 accattatgg gaaatggttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca
      301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat
      361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
      421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
      481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
      541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg
      601 tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct
      661 ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg
      721 ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct
      781 tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac
      841 aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat
      901 ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc
      961 aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg
     1021 ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca
     1081 gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc
     1141 acagtttttc tccataatct tgaagaggcc aaaacattag ctttatccaa ggaccaaata
     1201 ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact
     1261 tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc
     1321 ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca
     1381 aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt
     1441 aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca
     1501 ctaccggtac cccatttagg accacccaca gcacctaaca aaacggcatc aaccttcttg
     1561 gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca
     1621 attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga
     1681 accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc
     1741 ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata
     1801 tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat
     1861 tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat
     1921 ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct
     1981 ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca
     2041 aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat
     2101 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga
     2161 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg
     2221 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt
     2281 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg
     2341 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta
     2401 gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2461 ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
     2521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg
     2941 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     3001 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     3061 cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa
     3121 ttggagctct tttgctcggg tgtcaagttc aattaaacaa attccattag ttttattaat
     3181 tatatatatt atattactga ttcttgttcg aaagttttta aaaatcacac tatattaaat
     3241 taatctaggt catcatcaat ttccatactg tcctcattat cgtcatcacc taacgtgtca
     3301 gcccctatag tagcttcgtg atttggtaga acttccagac attcttcagc ttcctcagga
     3361 acattttcat cacgagattt acccttcttc aagacgacat tgacagtttc cttatccttg
     3421 atttcattct ttaagatcct tagtgccagt ttgttcaaaa tttcgttttg aattaacctg
     3481 ttcagtggac gtgcacccat atcatcggaa taaccatatt tggccaagaa gtccttggcc
     3541 tcttgagtta aattcaactt gtaatgttta tcattttgct cgaatctctc ttcaatttcc
     3601 ttcaaacgaa tatccacgat cttatgaata gcttttctag atagcttgtt gaaaatgact
     3661 atactagaaa ttctgttcaa aaattctggt ctgaaatgtt gcctaacagc acccatgacc
     3721 aaattcttgg tagattcttg gatctttgat ccttgttgag aattgataaa ttcagcacct
     3781 agattggaag tcatgatgac aatacaattg gaacagtcga tcgtcttacc ttgaccacta
     3841 gtaattctac cgtcatccaa catttgtagc atgacagtca aaacatcagg atgtgccttt
     3901 tctacttcat cgaataacaa aacggagtat ggtttgtatt gcagttggtt agttaaaaag
     3961 ccaccttcat cgtacccgac ataacctgcc gtggtaccca acaacttaga gaccgcatac
     4021 ttctcgctta attcagaaca atcgaccctg atcatcatgt cctcatcatt aaacaaaaat
     4081 ccagcaactt ttttagccaa ttcagtttta ccggaaccgg acaaacctaa aaataagaag
     4141 gatgctggtt gccttggatt agctaaacct gatctagaca atctaacggc attggaaaca
     4201 gctttaatgg catccatttg gcccacgact tcagatgata agtcacgttc catatgaatc
     4261 aatttttcat tttcagattc tgacaacttc ttaacaggga taccagtcaa tcttgcagct
     4321 gtttcagaaa tggtgtctga gtcgaccaca ttttggatca tggagttggc accagcacgt
     4381 ctctcttcct cagcaacctg atcttcaagc ttttcgattt gctttttgat atctgggatg
     4441 gcgaagtacc ttaaatcagc ggcggtagca gtatcatatc tacgttcagc atcaagggcc
     4501 ttgttttcca gttcatccaa tttcttttta gcttgtgtca attcttcatg gccatgcttt
     4561 tcttcattgt aacgttgtct tagaggttcc aattcttctt gcaatgaagc ttccttctgc
     4621 ctagctaact ttaatctatc tttagtggtg gagtcggcat cttcatctct ctctagagct
     4681 tttatctcta cttgaatcaa ttgcaattga cgttccttgg aatccaattc ttctggctta
     4741 gaatctcttg cgacggcgac accagcacaa gaaatatcaa ctaaatccaa agcagaatct
     4801 ggcaatcttc tatatggcaa gtaacgcttg gctaattgag ccgcggtgac taaggcgcta
     4861 tccagaatcc ttacaccatg atgtatttca tactttggtt gcagacctct caatatggcc
     4921 actgtttgtc tcacacttgg ttcagcgact tcaattttct ggaatcttct ttcaaaggca
     4981 ccatcctttt ccacaataga tctatattcg ttattggtgg tggcaccgat gaccttcaat
     5041 tggcctctgg acaaagctgg cttcaagatg ttagcagcgt cgtccttacc attacccatt
     5101 aacatgtgaa tttcatcaat gaataacaca attagagtct ttgattcttc gatttccttc
     5161 aaaacacctt tgaatctttc ttcgaaatca cctttgtatt tagcacctgc ggttaatgcg
     5221 gccaaatcta gactgaacaa tttagcgcct tgtaagatag tgggaacgtc atcgtcaatg
     5281 attctttgag caacaccttc aataatagcg gtcttaccga tacctggctc accaattaaa
     5341 catgggttgg acttaattct tcttgctaaa actctaatag tgcttcttat ttcttcttca
     5401 cggccgatga cagggtcaag tttaccttga cgagcctgct cagtcatatc aatggcgtac
     5461 tttgataaat attccaaagg tgtgttcgta tcagcgccac gagagtcaat tctagtgtta
     5521 ccacgaagtt caagagcttg ttgcttgatg gcctcaatat ctacttgagc ttccttaaat
     5581 atttgctgaa tagacgaatc attgaataga gcaaacaata tatggtcttg cgctataaat
     5641 gagtccttct gttgtttttg aatcttagca gcgtcttgaa ggactttccc caaagcataa
     5701 cttggagtta tctccgcagg tgcaggttgc tgttgaggaa ttcttactag atttctatta
     5761 accactttct tgaaaagatc atagtcgtaa cggcccttct caattagatt ctgtaggtaa
     5821 gggactgatc catcttctgg cgtttcaatg aaggcagcta gaatatgtat aggttgtaat
     5881 tgtggatgtt gatgatccga agccaatttt tgagccaacg tcaaaatcgt tagagccctt
     5941 tctgtaaatt gcgtttggtc gttcatcccg gatccgggtt ttttctcctt gacgttaaag
     6001 tatagaggta tattaacaat tttttgttga tacttttatt acatttgaat aagaagtaat
     6061 acaaaccgaa aatgttgaaa gtattagtta aagtggttat gcagtttttg catttatata
     6121 tctgttaata gatcaaaaat catcgcttcg ctgattaatt accccagaaa taaggctaaa
     6181 aaactaatcg cattatcatc ctatggttgt taatttgatt cgttcatttg aaggtttgtg
     6241 gggccaggtt actgccaatt tttcctcttc ataaccataa aagctagtat tgtagaatct
     6301 ttattgttcg gagcagtgcg gcgcgaggca catctgcgtt tcaggaacgc gaccggtgaa
     6361 gacgaggacg cacggaggag agtcttcctt cggagggctg tcacccgctc ggcggcttct
     6421 aatccgtact tcaatatagc aatgagcagt taagcgtatt actgaaagtt ccaaagagaa
     6481 ggttttttta ggctaagata atggggctct ttacatttcc acaacatata agtaagatta
     6541 gatatggata tgtatatgga tatgtatatg gtggtaatgc catgtaatat gattattaaa
     6601 cttctttgcg tccatccaaa aaaaaagtaa gaatttttga aaattcgaat tcgatatcaa
     6661 gcttatcgat accgtcgacc tcgagggggg gcccggtacc cagcttttgt tccctttagt
     6721 gagggttaat tccgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     6781 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     6841 cctaatgagt gaggtaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6901 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     6961 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     7021 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     7081 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     7141 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     7201 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     7261 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     7321 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     7381 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     7441 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     7501 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     7561 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     7621 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     7681 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     7741 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7801 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     7861 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7921 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7981 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     8041 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     8101 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     8161 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     8221 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     8281 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     8341 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     8401 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     8461 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     8521 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     8581 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     8641 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     8701 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     8761 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     8821 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     8881 catttccccg aaaagtgcca cctgggtcct tttcatcacg tgctataaaa ataattataa
     8941 tttaaatttt ttaatataaa tatataaatt aaaaatagaa agtaaaaaaa gaaattaaag
     9001 aaaaaatagt ttttgttttc cgaagatgta aaagactcta gggggatcgc caacaaatac
     9061 taccttttat cttgctcttc ctgctctcag gtattaatgc cgaattgttt catcttgtct
     9121 gtgtagaaga ccacacacga aaatcctgtg attttacatt ttacttatcg ttaatcgaat
     9181 gtatatctat ttaatctgct tttcttgtct aataaatata tatgtaaagt acgctttttg
     9241 ttgaaatttt ttaaaccttt gtttattttt ttttcttcat tccgtaactc ttctaccttc
     9301 tttatttact ttctaaaatc caaatacaaa acataaaaat aaataaacac agagtaaatt
     9361 cccaaattat tccatcatta aaagatacga ggcgcgtgta agttacaggc aagcgatccg
     9421 tcctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     9481 tttcgtc
//
