LOCUS       sequence-4-expor        6783 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 6783)
  AUTHORS   Liu JJ, Lindquist S
  TITLE     Oligopeptide-repeat expansions modulate 'protein-only' inheritance 
            in yeast.
  JOURNAL   Nature 1999 Aug 5;400(6744):573-6.
  PUBMED    10448860
REFERENCE   2  (bases 1 to 6783)
  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: "Nature"; 
            date: "1999-08-5"; volume: "400(6744)"; pages: "573-6"
COMMENT     Sequence Label: p316CUP1-3SGFP.SG
FEATURES             Location/Qualifiers
     source          1..6783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     promoter        196..416
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             417..1220
                     /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"
     rep_origin      complement(1351..1806)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1488..1509)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1700..1719
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1936..1958
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1950..1967
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1951..1967
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1974..1993
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1974..1992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2010..2726)
                     /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="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2050..2070)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     2665..2693
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(3545..3779)
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     primer_bind     complement(3963..3979)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3964..3980)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(4009..4027)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4048..4064)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4048..4064)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4061..4083)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4072..4088
                     /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(join(4096..4102,4103..4120,4121..4126))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4279..4296)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4450..5038)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4530..4549)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5209..6000,6001..6069))
                     /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     5832..5851
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6070..6174)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    6211..6714
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6756..6774
                     /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 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
      241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
      961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
     1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg
     1981 actcactata gggcgaattg gagctctcat ttgtatagtt catccatgcc atgtgtaatc
     2041 ccagcagctg ttacaaactc aagaaggacc atgtggtctc tcttttcgtt gggatctttc
     2101 gaaagggcag attgtgtgga caggtaatgg ttgtctggta aaaggacagg gccatcgcca
     2161 attggagtat tttgttgata atggtctgct agttgaacgc ttccatcttc aatgttgtgt
     2221 ctaattttga agttagcttt gattccattc ttttgtttgt ctgccatgat gtatacattg
     2281 tgtgagttat agttgtattc caatttgtgc ccaagaatgt ttccatcttc tttaaaatca
     2341 atacctttta actcgattct attaacaagg gtatcacctt caaacttgac ttcagcacgt
     2401 gtcttgtagt tcccgtcatc tttgaaaaat atagttcttt cctgtacata accttcgggc
     2461 atggcactct tgaaaaagtc atgccgtttc atatgatccg ggtatcttga aaagcactga
     2521 acaccataag tgaaagtagt gacaagtgtt ggccatggaa caggtagttt tccagtagtg
     2581 caaataaatt taagggtaag ttttccgtat gttgcatcac cttcaccctc tccactgaca
     2641 gaaaatttgt gcccattaac atcaccatct aattcaacaa gaattgggac aactccagtg
     2701 aagagttctt ctcctttgct agccatgagc tcatcgttaa caacttcgtc atccacttct
     2761 tcttcctgtt ccttgatcaa ggcatcagca ctggtaacat tggcattgtt ggtattatgt
     2821 gttgattcag agattttaag gtcttctacc tttggaagtt ccgatttttc ctccgtcttt
     2881 tcttcagtct ggactggttt ctcctccttt ttaactggtt cttcgacctt tgttggctct
     2941 ttagttggtt ctttggtttc agcagacttc tcttcctctt tcttatcaga ttcggcaggt
     3001 tttgtgccaa ccttcttggt agcattggcc aacttgatac cggaactgga gacaagcttc
     3061 aaagtcttct ttggtttggg agcggcctgc ttttgttgct tttgaaagtc gttcaaagac
     3121 ataccttgag actgtggttg gaaaccagct tgatatcctt gcaaattgtt attgtagttg
     3181 aagtttttgt aatttccacg gccaccttgt ggattgaatt gctgctgata accgccttga
     3241 ggattgtact gttgatagcc tccttgagga ttatactgtt gctggtaacc ggcgtcggga
     3301 ttgtactgtt gatagccacc ttgttggtac ccagaataac cttggtaatt ttggtagtac
     3361 ccacctgcag gttgggcttg agcattgtaa gcttgataac cttggtatct gttgttacct
     3421 tgttgttggt taccgttctg gctgtattgc tggtagtttt gctgattgtt gccttggttt
     3481 gaatccgaca tggatcctga ttgattgatt gattgtacag tttgtttttc ttaatatcta
     3541 tttcgatgac ttctatatga tattgcacta acaagaagat attataatgc aattgataca
     3601 agacaaggag ttatttgctt ctcttttata tgattctgac aatccatatt gcgttggtag
     3661 tcttttttgc tggaacggtt cagcggaaaa gacgcatcgc tctttttgct tctagaagaa
     3721 atgccagcaa aagaatctct tgacagtgac tgacagcaaa aatgtctttt tctaactagt
     3781 aacaaggcta agatatcagc ctgaaataaa gggtggtgaa gtaataatta aatcatccgt
     3841 ataaacctat acacatatat gaggaaaaat aatacaaaag tgttttaaat acagatacat
     3901 acatgaacat atgcacgtat agcgcccaaa tgtcggtaat gggaattcga tatcaagctt
     3961 atcgataccg tcgacctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg
     4021 gttaattccg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4081 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4141 atgagtgagg taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4201 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4261 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4321 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4381 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4441 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4501 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4561 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4621 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4681 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4741 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4801 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4861 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     4921 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4981 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5041 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5101 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5161 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5221 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5281 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5341 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5401 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5461 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5521 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5581 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5641 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5701 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5761 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5821 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5881 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5941 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6001 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6061 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6121 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6181 tccccgaaaa gtgccacctg ggtccttttc atcacgtgct ataaaaataa ttataattta
     6241 aattttttaa tataaatata taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa
     6301 aatagttttt gttttccgaa gatgtaaaag actctagggg gatcgccaac aaatactacc
     6361 ttttatcttg ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt
     6421 agaagaccac acacgaaaat cctgtgattt tacattttac ttatcgttaa tcgaatgtat
     6481 atctatttaa tctgcttttc ttgtctaata aatatatatg taaagtacgc tttttgttga
     6541 aattttttaa acctttgttt attttttttt cttcattccg taactcttct accttcttta
     6601 tttactttct aaaatccaaa tacaaaacat aaaaataaat aaacacagag taaattccca
     6661 aattattcca tcattaaaag atacgaggcg cgtgtaagtt acaggcaagc gatccgtcct
     6721 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     6781 gtc
//
