LOCUS       sequence-17-expo        7178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7178)
  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 7178)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol 
            Cell"; date: "2004-05"; volume: "15(5)"; pages: "2061-72. Epub 2004 
            Feb 20"
COMMENT     Sequence Label: GFP-CDC12(WT)
FEATURES             Location/Qualifiers
     source          1..7178
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    70..573
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        610..714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(715..783,784..1575)
                     /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     complement(933..952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1746..2334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2235..2254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2488..2505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2658..2663,2664..2681,2682..2688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /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)."
     primer_bind     2701..2723
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2720..2736
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2804..2820
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2805..2821
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3126..3839
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3156..3184)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3779..3799
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(5186..5205)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5187..5205)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5212..5229)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5212..5228)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5221..5243)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5373..5828
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5460..5479)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5670..5691
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5959..6762)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6763..6983)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7009..7028)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7128..7150
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
      121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
      181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
      241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
      301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
      361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
      421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
      481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
      541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
      601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcggaatt
     2761 aaccctcact aaagggaaca aaagctgggt accgggcccc ccctcgaggt cgacggtatc
     2821 gataagcttg atatcgaatt cctgcagccc gggggatccg atgcagtatc acgattagca
     2881 attcagctat gagttatgtt gctctttgtt ttgtttatgg aaattgtcct atggtaagtc
     2941 tctttttttt gcaatcgtga ttacagaaaa aaaacagggc gctggaaaag tgaagaatcc
     3001 gaaatttttt tcgaaatcac cattgtttgt tttgagtaga tcaaagtctt gaaaggtgca
     3061 gcaagatata ggatcttgac ctgaagagta ttgataacga actacatcac atattgtatc
     3121 aaataatgag caaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag
     3181 atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat
     3241 acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt ccatggccaa
     3301 cacttgtcac tacttttacg tatggtgttc aatgcttttc aagataccca gatcatatga
     3361 aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa agaactatat
     3421 ttttcaaaga tgacgggaac tacaagacac gtgctgaagt caagtttgaa ggtgataccc
     3481 ttgttaatag aatcgagtta aaaggtattg attttaaaga agatggaaac attcttggac
     3541 acaaattgga atacaactat aactcacaca atgtatacat catggcagac aaacaaaaga
     3601 atggaatcaa agttaacttc aaaattagac acaacattga agatggaagc gttcaactag
     3661 cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc
     3721 attacctgtc cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg
     3781 tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaag
     3841 gatccgctgc cactgcgact gctgcccctg ttcctcctcc tgtagggata tccaatcttc
     3901 ccaaccagcg ttataaaatt gttaatgaag aaggtggtac attcaccgtg atgttatgtg
     3961 gtgagagcgg attgggtaaa actacattta ttaatacctt atttcagact gtcttgaaaa
     4021 gggcagatgg tcagcaacac cgccaagaac ccatcagaaa gactgtagaa atcgacatca
     4081 caagagcttt gctggaagag aagcatttcg aattacgcgt taatgtcatc gacaccccag
     4141 gttttggaga taacgtgaat aacaacaaag cctggcaacc attggtggat tttattgatg
     4201 atcaacacga ttcttatatg cgtcaagagc agcaaccgta ccgtactaag aaatttgact
     4261 tgagagttca tgccgttctg tacttcatca ggcccactgg acatggttta aagccaattg
     4321 acattgagac tatgaagaga ttgtcaacca gagccaatct gattccagtt attgcgaaag
     4381 ctgatacatt gactgcccaa gaattacaac agttcaagtc cagaatcagg caagtaatag
     4441 aagctcaaga aattcgtatt ttcacgcctc cattagacgc tgattctaag gaagatgcca
     4501 aaagtggcag taatccagat tctgccgctg ttgaacatgc aagacagttg atagaagcaa
     4561 tgccatttgc catagtaggg tctgaaaaaa aatttgacaa tgggcaaggc actcaggtag
     4621 ttgcaaggaa gtatccttgg ggacttgtgg aaatcgaaaa cgattctcat tgtgatttcc
     4681 gtaagctcag agccctgtta ctgaggactt atctattaga tttgatttct accacccaag
     4741 aaatgcatta cgaaacatac agaagattaa gacttgaggg tcacgagaac accggggagg
     4801 ggaatgaaga tttcactctt ccagcaattg caccagcaag gaaattgtca cacaatccta
     4861 gatacaaaga agaagaaaat gcattgaaga agtatttcac tgaccaagtc aaagctgagg
     4921 agcaaagatt tagacaatgg gagcaaaaca ttgttaacga aagaatcaga ctgaacggtg
     4981 acttggaaga aattcaagga aaggtaaaaa aactagaaga gcaggtcaaa agcttgcaag
     5041 taaaaaaatc ccatttaaaa tgatgattaa ttaatgtctt cctctttgtc tcgtcaattt
     5101 caacgcctat ctctttgtct cttcttttgt cgcagcaata cgcacatatc tagagcggcc
     5161 gccaccgcgg tggagctcca attcgcccta tagtgagtcg tattacaatt cactggccgt
     5221 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     5281 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     5341 acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg cattaagcgc
     5401 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     5461 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5521 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5581 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5641 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5701 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5761 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     5821 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5881 gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt
     5941 tacttataat acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc
     6001 tgcttttctg taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct
     6061 tccaacaata ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg
     6121 acccaatgcg tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta
     6181 accttcatct cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc
     6241 gctcttcgca atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca
     6301 tgacaattct gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg
     6361 cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca
     6421 ttctgctatt ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc
     6481 aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt
     6541 aactgtgccc tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat
     6601 tttgggacct aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga
     6661 agcacacaag tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact
     6721 aggatgagta gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct
     6781 gcaggttttt gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac
     6841 actacatatg cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg
     6901 ttcggagatt accgaatcaa aaaaatttca aagaaaccga aatcaaaaaa aagaataaaa
     6961 aaaaaatgat gaattgaatt gaaaagctgt ggtatggtgc actctcagta caatctgctc
     7021 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     7081 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     7141 gtgtcagagg ttttcaccgt catcaccgaa acgcgcga
//
