LOCUS       sequence-450962-        8895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for cytoplasmic aggregates under LEU2 selection
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8895)
  TITLE     Budding yeast expression plasmid for induced production of 
            fluorescently tagged cytoplasmic protein aggregates
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8895)
  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            "Unpublished"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 in pRS315
FEATURES             Location/Qualifiers
     source          1..8895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             37..1131
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDKASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLELSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(1624..1643)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1743..1765
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1803..1821)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    1862..2365
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        2402..2506
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2507..2575,2576..3367)
                     /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(2725..2744)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3538..4126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4027..4046
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4280..4297
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4414..4435
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4450..4455,4456..4473,4474..4480)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4488..4504
                     /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     4493..4515
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4512..4528
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4512..4528
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4549..4567
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     4733..4751
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(4892..5605)
                     /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(4932..4952)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     5547..5575
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(7188..7422)
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     primer_bind     complement(7585..7604)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7586..7604)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7611..7628)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7611..7627)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7620..7642)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7768..8223
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7855..7874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8065..8086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        join(8524..8895,1..36)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 atatatatat atatatttca aggatatacc attctaatgt ctgcccctaa gaagatcgtc
       61 gttttgccag gtgaccacgt tggtcaagaa atcacagccg aagccattaa ggttcttaaa
      121 gctatttctg atgttcgttc caatgtcaag ttcgatttcg aaaatcattt aattggtggt
      181 gctgctatcg atgctacagg tgttccactt ccagatgagg cgctggaagc ctccaagaag
      241 gctgatgccg ttttgttagg tgctgtgggt ggtcctaaat ggggtaccgg tagtgttaga
      301 cctgaacaag gtttactaaa aatccgtaaa gaacttcaat tgtacgccaa cttaagacca
      361 tgtaactttg catccgactc tcttttagac ttatctccaa tcaagccaca atttgctaaa
      421 ggtactgact tcgttgttgt cagagaatta gtgggaggta tttactttgg taagagaaag
      481 gaagacgatg gtgatggtgt cgcttgggat agtgaacaat acaccgttcc agaagtgcaa
      541 agaatcacaa gaatggccgc tttcatggcc ctacaacatg agccaccatt gcctatttgg
      601 tccttggata aagctaatgt tttggcctct tcaagattat ggagaaaaac tgtggaggaa
      661 accatcaaga acgaattccc tacattgaag gttcaacatc aattgattga ttctgccgcc
      721 atgatcctag ttaagaaccc aacccaccta aatggtatta taatcaccag caacatgttt
      781 ggtgatatca tctccgataa agcctccgtt atcccaggtt ccttgggttt gttgccatct
      841 gcgtccttgg cctctttgcc agacaagaac accgcatttg gtttgtacga accatgccac
      901 ggttctgctc cagatttgcc aaagaataag gtcaacccta tcgccactat cttgtctgct
      961 gcaatgatgt tggaattgtc attgaacttg cctgaagaag gtaaggccat tgaagatgca
     1021 gttaaaaagg ttttggatgc aggtatcaga actggtgatt taggtggttc caacagtacc
     1081 accgaagtcg gtgatgctgt cgccgaagaa gttaagaaaa tccttgctta aaaagattct
     1141 ctttttttat gatatttgta cataaacttt ataaatgaaa ttcataatag aaacgacacg
     1201 aaattacaaa atggaatatg ttcatagggt agacgaaact atatacgcaa tctacataca
     1261 tttatcaaga aggagaaaaa ggaggatgta aaggaataca ggtaagcaaa ttgatactaa
     1321 tggctcaacg tgataaggaa aaagaattgc actttaacat taatattgac aaggaggagg
     1381 gcaccacgca aaaagttagg tgtaacagaa aatcatgaaa ctatgattcc taatttatat
     1441 attggaggat tttctctaaa aaaaaaaaat acaacaaata aaaaacactc aatgacctga
     1501 ccatttgatg gagtttaagt caataccttc ttgaaccatt tcccataatg gtgaaagttc
     1561 cctcaagaat tttactctgt cagaaacggc cttaacgacg tagtcgatat ggtgcactct
     1621 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     1681 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     1741 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     1801 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     1861 ggatcgcttg cctgtaactt acacgcgcct cgtatctttt aatgatggaa taatttggga
     1921 atttactctg tgtttattta tttttatgtt ttgtatttgg attttagaaa gtaaataaag
     1981 aaggtagaag agttacggaa tgaagaaaaa aaaataaaca aaggtttaaa aaatttcaac
     2041 aaaaagcgta ctttacatat atatttatta gacaagaaaa gcagattaaa tagatataca
     2101 ttcgattaac gataagtaaa atgtaaaatc acaggatttt cgtgtgtggt cttctacaca
     2161 gacaagatga aacaattcgg cattaatacc tgagagcagg aagagcaaga taaaaggtag
     2221 tatttgttgg cgatccccct agagtctttt acatcttcgg aaaacaaaaa ctattttttc
     2281 tttaatttct ttttttactt tctattttta atttatatat ttatattaaa aaatttaaat
     2341 tataattatt tttatagcac gtgatgaaaa ggacccaggt ggcacttttc ggggaaatgt
     2401 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2461 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2521 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2581 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     2641 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     2701 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     2761 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     2821 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     2881 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     2941 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3001 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3061 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3121 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3181 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3241 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3301 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3361 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3421 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3481 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3541 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3601 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3661 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     3721 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3781 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3841 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3901 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3961 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4021 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4081 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4141 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4201 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4261 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     4321 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4381 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     4441 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     4501 acaatttcac acaggaaaca gctatgacca tgattacgcc aagctcggaa ttaaccctca
     4561 ctaaagggaa caaaagctgg gtaccgtaca cgcgtttgta cagaaaaaaa agaaaaattt
     4621 gaaatataaa taacgttctt aatactaaca taactataaa aaaataaata gggacctaga
     4681 cttcaggttg tctaactcct tccttttcgg ttagagcgga tgtgggggga gggcgtgaat
     4741 gtaagcgtga cataactaat tacatgatat cgacaaagga aaaggggcct gtttactcac
     4801 aggctttttt caagtaggta attaagtcgt ttctgtcttt ttccttcttc aacccggtac
     4861 cggatccaga tattctatgg caaagctttt atttgtatag ttcatccatg ccatgtgtaa
     4921 tcccagcagc tgttacaaac tcaagaagga ccatgtggtc tctcttttcg ttgggatctt
     4981 tcgaaagttt agattgtgtg gacaggtaat ggttgtctgg taaaaggaca gggccatcgc
     5041 caattggagt attttgttga taatggtctg ctagttgaac gcttccatct tcaatgttgt
     5101 gtctaatttt gaagttaact ttgattccat tcttttgttt gtctgccatg atgtatacat
     5161 tgtgtgagtt atagttgtat tccaatttgt gtccaagaat gtttccatct tctttaaaat
     5221 caataccttt taactcgatt ctattaacaa gggtatcacc ttcaaacttg acttcagcac
     5281 gtgtcttgta gttcccgtca tctttgaaaa atatagttct ttcctgtaca taaccttcgg
     5341 gcatggcact cttgaaaaag tcatgctgtt tcatatgatc tgggtatctc gcaaagcatt
     5401 gaacaccata accgaaagta gtgacaagtg ttggccatgg aacaggtagt tttccagtag
     5461 tgcaaataaa tttaagggta agttttccgt atgttgcatc accttcaccc tctccactga
     5521 cagaaaattt gtgcccatta acatcaccat ctaattcaac aagaattggg acaactccag
     5581 tgaaaagttc ttctccttta ctcatctcga gtaaggagaa accaccgtgg atccattcgt
     5641 taaccatact taaggcgttt tcagggacgt caaatggaac catgtggcca ccattgaaga
     5701 ctctcaaata ggtgaagtgc ttgtaggatt tgacttcacc agcgacctcg tcggtgatag
     5761 aagcagtcca gttacgtact ttttggcttg caaattcttc gtcgtacttc catggcaaga
     5821 catccgtcca cgccttatta cccaaccagt tacagatgaa atctttatcg cctgcatata
     5881 ccagaatggg taggtcttga ttcaaaagat ctgttacggc ggtgtggtaa ggcttcatcc
     5941 aatcacccgc aaacaggaaa tttctgttga tatcgaagtt acaggattcg tagtggtcaa
     6001 cctccgcacc gacagcttct ttgacgtagt cctggtttaa gtagtcgtcg atatcttgta
     6061 acgttgggta gcacaaattg ccaccttcac aatccttcct gatatcgtaa acgtttctgc
     6121 cggtacgttg gtaaggagcc aattgggcgt tattacaata aatggtagct ggaacacagg
     6181 accagacgga ttgcgagtca tagcacgact cgatcaagcc caaacaacgt tccaaagagt
     6241 cttccatagc agagcattcc tccgagggca aaacagatgg ttcgccacct tcaccacagg
     6301 ccattggttc gtaatagtta tactgagtca atgggtcagt gaggccattt ccgatcaaga
     6361 cggaggttaa gttgaagttt ctgtccttgt gagacaaaat ttcagaggca aaaacaggga
     6421 tgtaatggcc ggcgtaggat tccctagcga tgtggaaatc ttggcccttg ttgacgtatt
     6481 cagggaactg atcgaagaac aactccaaga agttatagac atccttacca gcggcgacag
     6541 tgttggaaac acctgaggac ccggaatacg agaacccaac gttgacaggc tggtcaagga
     6601 agatcacggt ggcattgctg ttccaagagt aagggttccc gatgggtttc aaatcaggtc
     6661 caatggatga gggtcctaat tcaaagaaca gcccggttag tgaagaacaa cctggacccc
     6721 cgttcaacca aaggatgacc ggatcctttg caggatcgtt tctactttca aaagtccaaa
     6781 agaagaaatg cttgtcctcg tcttccacat ccaagtaacc cgtgtactgt gtgacatttg
     6841 ggtcaatgcc caggatttta gggtccttaa tcttgttgac acgaagctga tagttttcaa
     6901 ttgcgtcatt cttgaccaca aagtcccagt ctttcttcgt tttgattgct tcagggaatt
     6961 ttggcttagt ggaagtttca aggctcataa cctggtttgg gtacaaatgc tctatttggg
     7021 cccaagcgtc caatacattg gagtccaact ccttcaagag atgatccagg tcgaggtcca
     7081 aaccaaattt ttccgcagct tgcagcaaaa cgtccttatc tagacccaac ggtctttgca
     7141 atgagatcat gaattcgtta cagtttgttt ttcttaatat ctatttcgat gacttctata
     7201 tgatattgca ctaacaagaa gatattataa tgcaattgac acaagacaag gagttatttg
     7261 cttctctttt atatgattct gacaatccat attgcgttgg tagtcttttt tgctggaacg
     7321 gttcagcgga aaagacgcat cgctcttttt gcttctagaa gaaatgccag caaaagaatc
     7381 tcttgacagt gactgacagc aaaaatgtct ttttctaact agtaacaagg ctaagatatc
     7441 agcctgaaat aaagggtggt gaagtaataa ttaaatcatc cgtataaacc tatacacata
     7501 tatgaggaaa aataatacaa aagtgtttta aatacagata catacatgaa catgcggccg
     7561 ccaccgcggt ggagctccaa ttcgccctat agtgagtcgt attacaattc actggccgtc
     7621 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     7681 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     7741 cagttgcgca gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg
     7801 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     7861 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     7921 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     7981 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     8041 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     8101 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     8161 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     8221 tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatcgac
     8281 cggtcgagga gaacttctag tatatctaca tacctaatat tattgcctta ttaaaaatgg
     8341 aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc tgtactccct
     8401 tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat ttctcaacaa
     8461 gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat cttgaccgca
     8521 gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca
     8581 ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc
     8641 gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact
     8701 gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag
     8761 agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat attatttaag
     8821 gacctattgt tttttccaat aggtggttag caatcgtctt actgtctaac ttttcttacc
     8881 ttttacattt cagca
//
