LOCUS       sequence-450956-        8890 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 8890)
  TITLE     Budding yeast expression plasmid for induced production of 
            fluorescently tagged cytoplasmic protein aggregates
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8890)
  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            "Unpublished"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*-mNeonGreen in pRS315
FEATURES             Location/Qualifiers
     source          1..8890
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(540..995)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(677..698)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     889..908
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1121..1143
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1135..1152
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1136..1152
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1159..1178
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1159..1177
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1341..1575
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             3158..3868
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELYIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      3413..3422
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4006..4024)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(4190..4208)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4229..4245)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4229..4245)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4242..4264)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4253..4269
                     /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(4277..4283,4284..4301,4302..4307))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4322..4343
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4460..4477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4631..5219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4711..4730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5390..6181,6182..6250))
                     /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     6013..6032
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6251..6355)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    6392..6895
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6936..6954
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6992..7014)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7114..7133
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(7627..8721)
                     /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
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(join(8722..8890,1..239))
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 ctctattcta tatgaaaagc cggttccggc gctctcacct ttcctttttc tcccaatttt
       61 tcagttgaaa aaggtatatg cgtcaggcga cctctgaaat taacaaaaaa tttccagtca
      121 tcgaatttga ttctgtgcga tagcgcccct gtgtgttctc gttatgttga ggaaaaaaat
      181 aatggttgct aagagattcg aactcttgca tcttacgata cctgagtatt cccacagtta
      241 actgcggtca agatatttct tgaatcaggc gccttagacc gctcggccaa acaaccaatt
      301 acttgttgag aaatagagta taattatcct ataaatataa cgtttttgaa cacacatgaa
      361 caaggaagta caggacaatt gattttgaag agaatgtgga ttttgatgta attgttggga
      421 ttccattttt aataaggcaa taatattagg tatgtagata tactagaagt tctcctcgac
      481 cggtcgatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga
      541 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
      601 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
      661 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
      721 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
      781 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
      841 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
      901 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
      961 acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac
     1021 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     1081 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     1141 acgacggcca gtgaattgta atacgactca ctatagggcg aattggagct ccaccgcggt
     1201 ggcggccgca tgttcatgta tgtatctgta tttaaaacac ttttgtatta tttttcctca
     1261 tatatgtgta taggtttata cggatgattt aattattact tcaccaccct ttatttcagg
     1321 ctgatatctt agccttgtta ctagttagaa aaagacattt ttgctgtcag tcactgtcaa
     1381 gagattcttt tgctggcatt tcttctagaa gcaaaaagag cgatgcgtct tttccgctga
     1441 accgttccag caaaaaagac taccaacgca atatggattg tcagaatcat ataaaagaga
     1501 agcaaataac tccttgtctt gtgtcaattg cattataata tcttcttgtt agtgcaatat
     1561 catatagaag tcatcgaaat agatattaag aaaaacaaac tgtaacgaat tcatgatctc
     1621 attgcaaaga ccgttgggtc tagataagga cgttttgctg caagctgcgg aaaaatttgg
     1681 tttggacctc gacctggatc atctcttgaa ggagttggac tccaatgtat tggacgcttg
     1741 ggcccaaata gagcatttgt acccaaacca ggttatgagc cttgaaactt ccactaagcc
     1801 aaaattccct gaagcaatca aaacgaagaa agactgggac tttgtggtca agaatgacgc
     1861 aattgaaaac tatcagcttc gtgtcaacaa gattaaggac cctaaaatcc tgggcattga
     1921 cccaaatgtc acacagtaca cgggttactt ggatgtggaa gacgaggaca agcatttctt
     1981 cttttggact tttgaaagta gaaacgatcc tgcaaaggat ccggtcatcc tttggttgaa
     2041 cgggggtcca ggttgttctt cactaaccgg gctgttcttt gaattaggac cctcatccat
     2101 tggacctgat ttgaaaccca tcgggaaccc ttactcttgg aacagcaatg ccaccgtgat
     2161 cttccttgac cagcctgtca acgttgggtt ctcgtattcc gggtcctcag gtgtttccaa
     2221 cactgtcgcc gctggtaagg atgtctataa cttcttggag ttgttcttcg atcagttccc
     2281 tgaatacgtc aacaagggcc aagatttcca catcgctagg gaatcctacg ccggccatta
     2341 catccctgtt tttgcctctg aaattttgtc tcacaaggac agaaacttca acttaacctc
     2401 cgtcttgatc ggaaatggcc tcactgaccc attgactcag tataactatt acgaaccaat
     2461 ggcctgtggt gaaggtggcg aaccatctgt tttgccctcg gaggaatgct ctgctatgga
     2521 agactctttg gaacgttgtt tgggcttgat cgagtcgtgc tatgactcgc aatccgtctg
     2581 gtcctgtgtt ccagctacca tttattgtaa taacgcccaa ttggctcctt accaacgtac
     2641 cggcagaaac gtttacgata tcaggaagga ttgtgaaggt ggcaatttgt gctacccaac
     2701 gttacaagat atcgacgact acttaaacca ggactacgtc aaagaagctg tcggtgcgga
     2761 ggttgaccac tacgaatcct gtaacttcga tatcaacaga aatttcctgt ttgcgggtga
     2821 ttggatgaag ccttaccaca ccgccgtaac agatcttttg aatcaagacc tacccattct
     2881 ggtatatgca ggcgataaag atttcatctg taactggttg ggtaataagg cgtggacgga
     2941 tgtcttgcca tggaagtacg acgaagaatt tgcaagccaa aaagtacgta actggactgc
     3001 ttctatcacc gacgaggtcg ctggtgaagt caaatcctac aagcacttca cctatttgag
     3061 agtcttcaat ggtggccaca tggttccatt tgacgtccct gaaaacgcct taagtatggt
     3121 taacgaatgg atccacggtg gtttctcctt actcgagatg gtgagcaagg gcgaggagga
     3181 taacatggcc tctctcccag cgacacatga gttatacatc tttggctcca tcaacggtgt
     3241 ggactttgac atggtgggtc agggcaccgg caatccaaat gatggttatg aggagttaaa
     3301 cctgaagtcc accaagggtg acctccagtt ctccccctgg attctggtcc ctcatatcgg
     3361 gtatggcttc catcagtacc tgccctaccc tgacgggatg tcgcctttcc aggccgccat
     3421 ggtagatggc tccggctacc aagtccatcg cacaatgcag tttgaagatg gtgcctccct
     3481 tactgttaac taccgctaca cctacgaggg aagccacatc aaaggagagg cccaggtgaa
     3541 ggggactggt ttccctgctg acggtcctgt gatgaccaac tcgctgaccg ctgcggactg
     3601 gtgcaggtcg aagaagactt accccaacga caaaaccatc atcagtacct ttaagtggag
     3661 ttacaccact ggaaatggca agcgctaccg gagcactgcg cggaccacct acacctttgc
     3721 caagccaatg gcggctaact atctgaagaa ccagccgatg tacgtgttcc gtaagacgga
     3781 gctcaagcac tccaagaccg agctcaactt caaggagtgg caaaaggcct ttaccgatgt
     3841 gatgggcatg gacgagctgt acaagtagaa gctttgccat agaatatctg gatccggtac
     3901 cgggttgaag aaggaaaaag acagaaacga cttaattacc tacttgaaaa aagcctgtga
     3961 gtaaacaggc cccttttcct ttgtcgatat catgtaatta gttatgtcac gcttacattc
     4021 acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta
     4081 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat
     4141 ttttcttttt tttctgtaca aacgcgtgta cggtacccag cttttgttcc ctttagtgag
     4201 ggttaattcc gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4261 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4321 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4381 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4441 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4501 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4561 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4621 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4681 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4741 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4801 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4861 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4921 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4981 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5041 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5101 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5161 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5221 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5281 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5341 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5401 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5461 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5521 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5581 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5641 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5701 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5761 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5821 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5881 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5941 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6001 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6061 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6121 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6181 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6241 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6301 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6361 ttccccgaaa agtgccacct gggtcctttt catcacgtgc tataaaaata attataattt
     6421 aaatttttta atataaatat ataaattaaa aatagaaagt aaaaaaagaa attaaagaaa
     6481 aaatagtttt tgttttccga agatgtaaaa gactctaggg ggatcgccaa caaatactac
     6541 cttttatctt gctcttcctg ctctcaggta ttaatgccga attgtttcat cttgtctgtg
     6601 tagaagacca cacacgaaaa tcctgtgatt ttacatttta cttatcgtta atcgaatgta
     6661 tatctattta atctgctttt cttgtctaat aaatatatat gtaaagtacg ctttttgttg
     6721 aaatttttta aacctttgtt tatttttttt tcttcattcc gtaactcttc taccttcttt
     6781 atttactttc taaaatccaa atacaaaaca taaaaataaa taaacacaga gtaaattccc
     6841 aaattattcc atcattaaaa gatacgaggc gcgtgtaagt tacaggcaag cgatccgtct
     6901 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     6961 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     7021 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     7081 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     7141 tgcaccatat cgactacgtc gttaaggccg tttctgacag agtaaaattc ttgagggaac
     7201 tttcaccatt atgggaaatg gttcaagaag gtattgactt aaactccatc aaatggtcag
     7261 gtcattgagt gttttttatt tgttgtattt tttttttttt agagaaaatc ctccaatata
     7321 taaattagga atcatagttt catgattttc tgttacacct aactttttgt gtggtgccct
     7381 cctccttgtc aatattaatg ttaaagtgca attctttttc cttatcacgt tgagccatta
     7441 gtatcaattt gcttacctgt attcctttac atcctccttt ttctccttct tgataaatgt
     7501 atgtagattg cgtatatagt ttcgtctacc ctatgaacat attccatttt gtaatttcgt
     7561 gtcgtttcta ttatgaattt catttataaa gtttatgtac aaatatcata aaaaaagaga
     7621 atctttttaa gcaaggattt tcttaacttc ttcggcgaca gcatcaccga cttcggtggt
     7681 actgttggaa ccacctaaat caccagttct gatacctgca tccaaaacct ttttaactgc
     7741 atcttcaatg gccttacctt cttcaggcaa gttcaatgac aatttcaaca tcattgcagc
     7801 agacaagata gtggcgatag ggttgacctt attctttggc aaatctggag cagaaccgtg
     7861 gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc aaagaggcca aggacgcaga
     7921 tggcaacaaa cccaaggaac ctgggataac ggaggcttca tcggagatga tatcaccaaa
     7981 catgttgctg gtgattataa taccatttag gtgggttggg ttcttaacta ggatcatggc
     8041 ggcagaatca atcaattgat gttgaacctt caatgtaggg aattcgttct tgatggtttc
     8101 ctccacagtt tttctccata atcttgaaga ggccaaaaca ttagctttat ccaaggacca
     8161 aataggcaat ggtggctcat gttgtagggc catgaaagcg gccattcttg tgattctttg
     8221 cacttctgga acggtgtatt gttcactatc ccaagcgaca ccatcaccat cgtcttcctt
     8281 tctcttacca aagtaaatac ctcccactaa ttctctgaca acaacgaagt cagtaccttt
     8341 agcaaattgt ggcttgattg gagataagtc taaaagagag tcggatgcaa agttacatgg
     8401 tcttaagttg gcgtacaatt gaagttcttt acggattttt agtaaacctt gttcaggtct
     8461 aacactaccg gtaccccatt taggaccacc cacagcacct aacaaaacgg catcagcctt
     8521 cttggaggct tccagcgcct catctggaag tggaacaccc gtagcatcga tagcagcacc
     8581 accaattaaa tgattttcga aatcgaactt gacattggaa cgaacatcag aaatagcttt
     8641 aagaacctta atggcttcgg ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac
     8701 gatcttctta ggggcagaca ttagaatggt atatccttga aatatatata tatatattgc
     8761 tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat tgctaaccac ctattggaaa
     8821 aaacaatagg tccttaaata atattgtcaa cttcaagtat tgtgatgcaa gcatttagtc
     8881 atgaacgctt
//
