LOCUS       sequence-450955-        7773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for cytoplasmic aggregates.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7773)
  TITLE     Budding yeast expression plasmid for induced production of 
            fluorescently tagged cytoplasmic protein aggregates
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7773)
  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: 
            "Unpublished"
COMMENT     Sequence Label: CUP1-∆ssCPY*-mGFP
FEATURES             Location/Qualifiers
     source          1..7773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        202..436
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             2019..2732
                     /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
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2049..2077)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2672..2692
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2873..2891)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(3057..3075)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3096..3112)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3096..3112)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3109..3131)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3120..3136
                     /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(3144..3150,3151..3168,3169..3174))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3189..3210
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3327..3344)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3498..4086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3578..3597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4257..5048,5049..5117))
                     /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     4880..4899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5118..5222)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5259..5762
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     5803..5821
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5859..5881)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5981..6000
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6024..6239
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             6240..7043
                     /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(7174..7629)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7311..7332)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7523..7542
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(7755..7773,1..4)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(7769..7773,1..13)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     join(7770..7773,1..13)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aacgacggcc agtgaattgt aatacgactc actatagggc gaattggagc tccaccgcgg
       61 tggcggccgc atgttcatgt atgtatctgt atttaaaaca cttttgtatt atttttcctc
      121 atatatgtgt ataggtttat acggatgatt taattattac ttcaccaccc tttatttcag
      181 gctgatatct tagccttgtt actagttaga aaaagacatt tttgctgtca gtcactgtca
      241 agagattctt ttgctggcat ttcttctaga agcaaaaaga gcgatgcgtc ttttccgctg
      301 aaccgttcca gcaaaaaaga ctaccaacgc aatatggatt gtcagaatca tataaaagag
      361 aagcaaataa ctccttgtct tgtgtcaatt gcattataat atcttcttgt tagtgcaata
      421 tcatatagaa gtcatcgaaa tagatattaa gaaaaacaaa ctgtaacgaa ttcatgatct
      481 cattgcaaag accgttgggt ctagataagg acgttttgct gcaagctgcg gaaaaatttg
      541 gtttggacct cgacctggat catctcttga aggagttgga ctccaatgta ttggacgctt
      601 gggcccaaat agagcatttg tacccaaacc aggttatgag ccttgaaact tccactaagc
      661 caaaattccc tgaagcaatc aaaacgaaga aagactggga ctttgtggtc aagaatgacg
      721 caattgaaaa ctatcagctt cgtgtcaaca agattaagga ccctaaaatc ctgggcattg
      781 acccaaatgt cacacagtac acgggttact tggatgtgga agacgaggac aagcatttct
      841 tcttttggac ttttgaaagt agaaacgatc ctgcaaagga tccggtcatc ctttggttga
      901 acgggggtcc aggttgttct tcactaaccg ggctgttctt tgaattagga ccctcatcca
      961 ttggacctga tttgaaaccc atcgggaacc cttactcttg gaacagcaat gccaccgtga
     1021 tcttccttga ccagcctgtc aacgttgggt tctcgtattc cgggtcctca ggtgtttcca
     1081 acactgtcgc cgctggtaag gatgtctata acttcttgga gttgttcttc gatcagttcc
     1141 ctgaatacgt caacaagggc caagatttcc acatcgctag ggaatcctac gccggccatt
     1201 acatccctgt ttttgcctct gaaattttgt ctcacaagga cagaaacttc aacttaacct
     1261 ccgtcttgat cggaaatggc ctcactgacc cattgactca gtataactat tacgaaccaa
     1321 tggcctgtgg tgaaggtggc gaaccatctg ttttgccctc ggaggaatgc tctgctatgg
     1381 aagactcttt ggaacgttgt ttgggcttga tcgagtcgtg ctatgactcg caatccgtct
     1441 ggtcctgtgt tccagctacc atttattgta ataacgccca attggctcct taccaacgta
     1501 ccggcagaaa cgtttacgat atcaggaagg attgtgaagg tggcaatttg tgctacccaa
     1561 cgttacaaga tatcgacgac tacttaaacc aggactacgt caaagaagct gtcggtgcgg
     1621 aggttgacca ctacgaatcc tgtaacttcg atatcaacag aaatttcctg tttgcgggtg
     1681 attggatgaa gccttaccac accgccgtaa cagatctttt gaatcaagac ctacccattc
     1741 tggtatatgc aggcgataaa gatttcatct gtaactggtt gggtaataag gcgtggacgg
     1801 atgtcttgcc atggaagtac gacgaagaat ttgcaagcca aaaagtacgt aactggactg
     1861 cttctatcac cgacgaggtc gctggtgaag tcaaatccta caagcacttc acctatttga
     1921 gagtcttcaa tggtggccac atggttccat ttgacgtccc tgaaaacgcc ttaagtatgg
     1981 ttaacgaatg gatccacggt ggtttctcct tactcgagat gagtaaagga gaagaacttt
     2041 tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt
     2101 ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt aaatttattt
     2161 gcactactgg aaaactacct gttccatggc caacacttgt cactactttc ggttatggtg
     2221 ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc aagagtgcca
     2281 tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg aactacaaga
     2341 cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag ttaaaaggta
     2401 ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac tataactcac
     2461 acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac ttcaaaatta
     2521 gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa aatactccaa
     2581 ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa tctaaacttt
     2641 cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta acagctgctg
     2701 ggattacaca tggcatggat gaactataca aataaaagct ttgccataga atatctggat
     2761 ccggtaccgg gttgaagaag gaaaaagaca gaaacgactt aattacctac ttgaaaaaag
     2821 cctgtgagta aacaggcccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct
     2881 tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg
     2941 aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat
     3001 ttcaaatttt tctttttttt ctgtacaaac gcgtgtacgg tacccagctt ttgttccctt
     3061 tagtgagggt taattccgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3121 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3181 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3241 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3301 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3361 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3421 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3481 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3541 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3601 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3661 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3721 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3781 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3841 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3901 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3961 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4021 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4081 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4141 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4201 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4261 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4321 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4381 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4441 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4501 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4561 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4621 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4681 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4741 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4801 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4861 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4921 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4981 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5041 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5101 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5161 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5221 cgcacatttc cccgaaaagt gccacctggg tccttttcat cacgtgctat aaaaataatt
     5281 ataatttaaa ttttttaata taaatatata aattaaaaat agaaagtaaa aaaagaaatt
     5341 aaagaaaaaa tagtttttgt tttccgaaga tgtaaaagac tctaggggga tcgccaacaa
     5401 atactacctt ttatcttgct cttcctgctc tcaggtatta atgccgaatt gtttcatctt
     5461 gtctgtgtag aagaccacac acgaaaatcc tgtgatttta cattttactt atcgttaatc
     5521 gaatgtatat ctatttaatc tgcttttctt gtctaataaa tatatatgta aagtacgctt
     5581 tttgttgaaa ttttttaaac ctttgtttat ttttttttct tcattccgta actcttctac
     5641 cttctttatt tactttctaa aatccaaata caaaacataa aaataaataa acacagagta
     5701 aattcccaaa ttattccatc attaaaagat acgaggcgcg tgtaagttac aggcaagcga
     5761 tccgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     5821 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     5881 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     5941 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     6001 ctgagagtgc accataccac cttttcaatt catcattttt tttttattct tttttttgat
     6061 ttcggtttcc ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga
     6121 aggagcacag acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt
     6181 gcccagtatt cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca
     6241 tgtcgaaagc tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc
     6301 tatttaatat catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca
     6361 ccaaggaatt actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac
     6421 atgtggatat cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat
     6481 ccgccaagta caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag
     6541 tcaaattgca gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg
     6601 cacacggtgt ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa
     6661 caaaggaacc tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta
     6721 ctggagaata tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg
     6781 gctttattgc tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga
     6841 cacccggtgt gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg
     6901 atgatgtggt ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg
     6961 gaagggatgc taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga
     7021 gaagatgcgg ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact
     7081 cacaaattag agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg
     7141 cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa
     7201 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     7261 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     7321 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     7381 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     7441 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     7501 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     7561 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     7621 agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     7681 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     7741 aacgccaggg ttttcccagt cacgacgttg taa
//
