LOCUS       sequence-431078-        8130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LYS-Leu2 plasmid (218114) with the functional cassettes of pY2 
            (218115) inserted at the sfGFP dropout locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8130)
  AUTHORS   Martinusen SG, Slaton EW, Nelson SE, Pulgar MA, Besu JT, Simas CF, 
            Denard CA
  TITLE     Modular and integrative activity reporters enhance biochemical 
            studies in the yeast ER.
  JOURNAL   Protein Eng Des Sel. 2024 Jan 29;37:gzae008. doi: 
            10.1093/protein/gzae008.
  PUBMED    38696722
REFERENCE   2  (bases 1 to 8130)
  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: "Protein Eng
            Des Sel."; date: "2024-01-29"; volume: "37:gzae008. doi"; pages: " 
            10.1093/protein/gzae008"
COMMENT     Sequence Label: pY2-LYS-IV
FEATURES             Location/Qualifiers
     source          1..8130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    111..228
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     rep_origin      complement(1902..2490)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1982..2001)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2704..2723)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2715..3530
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2715..3530
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2788..2807)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(4387..5481)
                     /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(5482..5889)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     terminator      complement(6566..6593)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(6609..6680)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6692..7405)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     RBS             7412..7423
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             complement(7586..7597)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        complement(join(7917..8130,1..228))
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     primer_bind     complement(7923..7946)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
ORIGIN
        1 tcctatggtt gttaatttga ttcgttaatt tgaaggtttg tggggccagg ttactgccaa
       61 tttttcctct tcataaccat aaaagctagt attgtagaat ctttattgtt cggagcagtg
      121 cggcgcgagg cacatctgcg tttcaggaac gcgaccggtg atgacgagga cgcacggagg
      181 agagtcatcc gtcggagggc tgtcgcccgc tcggcggctt ctaatccgta cttcaatata
      241 gcaatgagca gttaagcgta ttactgaaag ttccaaagag aaggtttttt taggctaaga
      301 taatggggct agaattcgtt taaacatgca ataatatata aacctgtata atataacctt
      361 gaagactata tttcttttcg agctccctag gtgctgtata tactcacagc ataactgtat
      421 atacacccag ggtctagagc atgtgctctg tatgtatata aaactcttgt tttcttcttt
      481 tctcttcttt ccttatacat taggaccttt gcagcataaa ttactatact tctatactag
      541 tggatccatg cagttacttc gctgtttttc aatattttct gttattgctt cagttttagc
      601 aagagaccct gcagcttaag actaagcatg catggtctca taagcttggt accgcggcta
      661 gtaatagctc gagatctgat aacaacagtg tagatgtaac aaaatcgact ttgttcccac
      721 tgtactttta gctcgtacaa aatacaatat acttttcatt tctccgtaaa caacatgttt
      781 tcccatgtaa tatccttttc tatttttcgt tccgttacca actttacaca tactttatat
      841 agctattcac ttctatacac taaaaaacta agacaatttt aattttgctg cctgccatat
      901 ttcaatttgt tataaattcc tataatttat cctattagta gctaaaaaaa gatgaatgtg
      961 aatcgaatcc taagagaatt gagctccaat tccgttcgtc tgcaattatc ggccagttca
     1021 gtgagatggt attgcgacga attgccacgt tgtcgcagtg tctgtactgt gggttaatgt
     1081 ctgtgatcgc cgtcgtgagt tcgtgtcatc cgcgcatgat tcagatggtc ctggagatcg
     1141 ttgcttgtac cggctgtttg tggctctacc atctagatgc gaattcaggg agcagtcaaa
     1201 tattgagtaa attggtccgc aacaatggtt actctttcat tcgaatacag taagctgtta
     1261 taaattaagt tcaaaacatg cgcattatta ctggtatcca aattcaaagc aaagtccact
     1321 aaatgatgct tgaactcgtc taatttgaaa tcttggtttt ccaaaaaggc caaacggaac
     1381 aactgagggg tcctttccag atcttggcaa ctttgaattt tttcagctag ctcgtcaaag
     1441 gaaaaattgg cctcaataga attgagcttg ttcaactcat tgttaattgt agaatacagc
     1501 tcattaaatg accacgttgg ttgaatattg aatcttaaga ttttgttatt cgcaatataa
     1561 agaacaatat catcgtcacc ggttactcta tatatcaatg cagcccatac actcaaagcg
     1621 acagcgtatt tgttagaaaa actatcatga ggcacatcga gctgaggtag ctgtaacgaa
     1681 tatgtagctt gtttcgtata aggttcttgt tgtgggcgta aaaagtcatg tggtaacact
     1741 gaaagagttg gattatccaa cttctctatc cagacctttt cgttagtcat attggcggcc
     1801 gcacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1861 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1921 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1981 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2041 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2101 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2161 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2221 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2281 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2341 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2401 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2461 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2521 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgactga tccttcaact
     2581 cagcaaaagt tcgatttatt caacaaagcc acgttgtgtc tcaaaatctc tgatgttaca
     2641 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     2701 atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctccagg ccgcgattaa
     2761 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     2821 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
     2881 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     2941 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     3001 tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt
     3061 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     3121 tttgtaattg tccttttaac agcgatcgcg tatttcgcct cgctcaggcg caatcacgaa
     3181 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     3241 aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca gtcgtcactc
     3301 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     3361 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     3421 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     3481 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg
     3541 ttaattggtt gtaacacgcg gccgctcggt taagctgctg cggagcttcc acgagcacca
     3601 gcacctgaag caactagact tatttgcgct tgagttagtt ctatacttgg cagtggaagt
     3661 ttgtcattat gagttggtgg aggtaaaaat ccaaccttgt ttaaaaatgc aatatatata
     3721 ccaacctctt ctggagtaac acctattcca ttagaccaat caacacctgt ccatgcggtg
     3781 tctttcttta aagatgccac ggcgttccta tcgtctagtt ccggagcttt ggtactttca
     3841 ggtaagttgt ctaagaccat gtgtagcaaa ggatacaacg cattttcttc attcctgtca
     3901 ataacagacg cctccaatga tttcttccat ttagaatagc tttcgatttc gacatcgtaa
     3961 ccataatcgt gtaaagtata caagtagtct ttgaataata ttcttgggtg acccgttact
     4021 tgagcaacgg ccaattcatt ttctttggga ggattcaaag acgtagcaac aacaacacga
     4081 gcaacatgat ctactggaac catattcacg gaattttcga tatctggaat cttacctaat
     4141 tggactgaac ctttcaaaaa tctcaataag aaatcatctg tgtttgaaga accattggca
     4201 gaggcaccac tcctgcctat ttaacgccaa cgttgcgaga tatacactcg aaatgtgtga
     4261 tgtagatgta aacagacggc tatatgaatg ccactagccg gttattttac tcctagttaa
     4321 tacatatttc ctagaatttt ttacgtgcat aagagtagaa tagtctaaag agaagggtcc
     4381 tccaggttaa gcaaggattt tcttaacttc ttcggcgaca gcatcaccga cttcggtggt
     4441 actgttggaa ccacctaaat caccagttct gatacctgca tccaaaacct ttttaactgc
     4501 atcttcaatg gccttacctt cttcaggcaa gttcaatgac aatttcaaca tcattgcagc
     4561 agacaagata gtggcgatag ggttgacctt attctttggc aaatctggag cagaaccgtg
     4621 gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc aaagaggcca aggacgcaga
     4681 tggcaacaaa cccaaggaac ctgggataac ggaggcttca tcggagatga tatcaccaaa
     4741 catgttgctg gtgattataa taccatttag gtgggttggg ttcttaacta ggatcatggc
     4801 ggcagaatca atcaattgat gttgaacctt caatgtagga aattcgttct tgatggtttc
     4861 ctccacagtt tttctccata atcttgaaga ggccaaaaca ttagctttat ccaaggacca
     4921 aataggcaat ggtggctcat gttgtagggc catgaaagcg gccattcttg tgattctttg
     4981 cacttctgga acggtgtatt gttcactatc ccaagcgaca ccatcaccat cgtcctcctt
     5041 tctcttacca aagtaaatac ctcccactaa ttctctgaca acaacgaagt cagtaccttt
     5101 agcaaattgt ggcttgattg gagataagtc taaaagagag tcggatgcaa agttacatgg
     5161 tcttaagttg gcgtacaatt gaagttcttt acggattttt agtaaacctt gttcaggtct
     5221 aacactaccg gttccccatt taggaccacc cacagcacct aacaaaacgg catcagcctt
     5281 cttggaggct tccagcgcct catctggaag tggaacacct gtagcatcga tagcagcacc
     5341 accaattaaa tgattttcga aatcgaactt gacattggaa cgaacatcag aaatagcttt
     5401 aagaacctta atggcttcgg ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac
     5461 gatcttctta ggggcagaca ttagaatggt atatccttga aatatatata tatatattgc
     5521 tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat tgctaaccac ctattggaaa
     5581 aaacaatagg tccttaaata atattgtcaa cttcaagtat tgtgatgcaa gcatttagtc
     5641 atgaacgctt ctctattcta tatgaaaagc cggttccggc gctctcacct ttcctttttc
     5701 tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga cctctgaaat taacaaaaaa
     5761 tttccagtca tcgaatttga ttctgtgcga tagcgcccct gtgtgttctc gttatgttga
     5821 ggaaaaaaat aatggttgct aagagattcg aactcttgca tcttacgata cctgagtatt
     5881 cccacagtta actgcggtca agatatttct tgaatcaggc gccttagacc gctcggccaa
     5941 acaaccaatt acttgttgag aaatagagta taattatcct ataaatataa cgtttttgaa
     6001 cacacatgaa caaggaagta caggacaatt gattttgaag agaatgtgga ttttgatgta
     6061 attgttggga ttccattttt aataaggcaa taatattagg tatgtagata tactagaagt
     6121 tctcctcgat gtactgcagt gcactagttg gactgttgtg atatgccata tatgcagtgg
     6181 tgttcgccgg cgctggttcc ggctgtcttg cttagttgtg agtcgccagg cagtcatcgg
     6241 tatgatctgt acatgattcg tcagtgtgag caccactgac gagcagattt ccgtcgcatg
     6301 ctcagcagct tttgattaag ccttctagtc caaaaaacac gtttttttgt catttatttc
     6361 attttcttag aatagtttag tttattcatt ttatagtcac gaatgtttta tgattctata
     6421 tagggttgca aacaagcatt tttcatttta tgttaaaaca atttcaggtt taccttttat
     6481 tctgcttgtg gtgacgcgtg tatccgcccg ctcttttggt cacccatgta tagaccatgg
     6541 attgtgagac gttaattaat ataaacgcag aaaggcccac ccgaaggtga gccagtgtga
     6601 ctctagtaga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
     6661 tgagcctttc gttttatttg atgcctggtc atttgtacag ttcatccata ccatgcgtga
     6721 tgcccgctgc ggttacgaac tccagcagaa ccatatgatc gcgtttctcg ttcggatctt
     6781 tagacagaac gctttgcgtg ctcagatagt gattgtctgg cagcagaaca ggaccatcac
     6841 cgattggagt gttttgctgg tagtgatcag ccagctgcac gctgccatcc tccacgttgt
     6901 ggcgaatttt aaaattcgct ttaatgccat ttttttgttt atcggcggtg atgtaaacat
     6961 tgtggctgtt aaaattgtat tccagcttat ggcccaggat attgccgtcc tctttaaagt
     7021 caatgccttt cagctcaatg cggtttacca gggtatcgcc ttcaaatttc acttccgcac
     7081 gcgttttgta cgtgccgtca tccttaaagg aaatcgtgcg ttcctgcaca tagccttccg
     7141 gcatggcgga cttgaagaag tcatgctgct tcatatggtc cggataacga gcaaagcact
     7201 gaacaccata agtcagcgtc gttaccagag tcggccaagg aaccggcagt ttaccagtag
     7261 tacagatgaa cttcagcgtc agtttaccat tagttgcgtc accttcaccc tcgccacgca
     7321 cggaaaactt atgaccgttg acatcaccat ccagttccac cagaataggg acgacaccag
     7381 tgaacagctc ttcgccttta cgcatctagt atttctcctc tttctctagt agctaagatc
     7441 cgaacctgcc gtttcttgag ttgccgcgat gttaagaaaa cattcataaa ttaaatgtga
     7501 attgccgcac acattattaa ataagattta caaaatgttc aaaatgacgc atgaaatcac
     7561 gtttcacttt cggatcccgt ctcgatctac cttcaatcgt cgagctattg tccttaaaaa
     7621 catactgtgt gtttatgggg ctgcctttgc tagttgttga ggggtgagaa ccgcaattac
     7681 tgacaaacgt tactgatttg taatattcaa aaactccttg cattgccttc ccgttggcca
     7741 aaatagtagt cgttgacaaa gagtacggcg tcgattctaa agttggtgag gggatttgct
     7801 cgcatatagt tgtcagttcc tgtgctaaaa ctgaagcaat aacagaaaat attgaaaaac
     7861 agcgaagtaa ctgcatcgat actagtgagc tctctagaat ccgggtatag ttttttctcc
     7921 ttgacgttaa agtatagagg tatattaaca attttttgtt gatactttta tgacatttga
     7981 ataagaagta atacaaactg aaaatgttga aagtattagt taaagtggtt atgcagcttt
     8041 tccatttata tatctgttaa tagatcaaaa atcatcgctt cgctgattaa ttaccccaga
     8101 aataaggcta aaaaactaat cgcattatca
//
