LOCUS       sequence-431079-        8071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LYS-Leu2 plasmid (218114) with the functional cassettes of pY3 
            (218116) inserted at the sfGFP dropout locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8071)
  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 8071)
  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: pY3-LYS-IV
FEATURES             Location/Qualifiers
     source          1..8071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    76..193
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     CDS             730..741
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             904..915
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             922..1635
                     /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"
     terminator      1647..1718
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1734..1761
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(2930..3518)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3010..3029)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3732..3751)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3743..4558
                     /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             3743..4558
                     /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(3816..3835)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(5415..6509)
                     /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(6510..6917)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(7786..7805)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7787..7805)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(7816..8071,1..409)
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     primer_bind     complement(7829..7852)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
ORIGIN
        1 gtttgtgggg ccaggttact gccaattttt cctcttcata accataaaag ctagtattgt
       61 agaatcttta ttgttcggag cagtgcggcg cgaggcacat ctgcgtttca ggaacgcgac
      121 cggtgaagac gaggacgcac ggaggagagt cttccttcgg agggctgtca cccgctcggc
      181 ggcttctaat ccgtacttca atatagcaat gagcagttaa gcgtattact gaaagttcca
      241 aagagaaggt ttttttaggc taagataatg gggctcttta catttccaca acatataagt
      301 aagattagat atggatatgt atatggatat gtatatggtg gtaatgccat gtaatatgat
      361 tattaaactt ctttgcgtcc atccaaaaaa aaagtaagaa tttttgaaaa ttcgaattca
      421 accctcacta aagtctgact aatatcatcg atgcagttac ttcgctgttt ttcaatattt
      481 tctgttattg cttcagtttt agcacaggaa ctgacaacta tatgcgagca aatcccctca
      541 ccaactttag aatcgacgcc gtactctttg tcaacgacta ctattttggc caacgggaag
      601 gcaatgcaag gagtttttga atattacaaa tcagtaacgt ttgtcagtaa ttgcggttct
      661 cacccctcaa caactagcaa aggcagcccc ataaacacac agtatgtttt taaggacaat
      721 agctcgacga ttgaaggtag atcgagacgg gatccgaaag tgaaacgtga tttcatgcgt
      781 cattttgaac attttgtaaa tcttatttaa taatgtgtgc ggcaattcac atttaattta
      841 tgaatgtttt cttaacatcg cggcaactca agaaacggca ggttcggatc ttagctacta
      901 gagaaagagg agaaatacta gatgcgtaaa ggcgaagagc tgttcactgg tgtcgtccct
      961 attctggtgg aactggatgg tgatgtcaac ggtcataagt tttccgtgcg tggcgagggt
     1021 gaaggtgacg caactaatgg taaactgacg ctgaagttca tctgtactac tggtaaactg
     1081 ccggttcctt ggccgactct ggtaacgacg ctgacttatg gtgttcagtg ctttgctcgt
     1141 tatccggacc atatgaagca gcatgacttc ttcaagtccg ccatgccgga aggctatgtg
     1201 caggaacgca cgatttcctt taaggatgac ggcacgtaca aaacgcgtgc ggaagtgaaa
     1261 tttgaaggcg ataccctggt aaaccgcatt gagctgaaag gcattgactt taaagaggac
     1321 ggcaatatcc tgggccataa gctggaatac aattttaaca gccacaatgt ttacatcacc
     1381 gccgataaac aaaaaaatgg cattaaagcg aattttaaaa ttcgccacaa cgtggaggat
     1441 ggcagcgtgc agctggctga tcactaccag caaaacactc caatcggtga tggtcctgtt
     1501 ctgctgccag acaatcacta tctgagcacg caaagcgttc tgtctaaaga tccgaacgag
     1561 aaacgcgatc atatggttct gctggagttc gtaaccgcag cgggcatcac gcatggtatg
     1621 gatgaactgt acaaatgacc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     1681 cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac
     1741 cttcgggtgg gcctttctgc gtttatatta attaacgtct cacaatccat ggtctataca
     1801 tgggtgacca aaagagcggg cggatacacg cgtcaccaca agcagaataa aaggtaaacc
     1861 tgaaattgtt ttaacataaa atgaaaaatg cttgtttgca accctatata gaatcataaa
     1921 acattcgtga ctataaaatg aataaactaa actattctaa gaaaatgaaa taaatgacaa
     1981 aaaaacgtgt tttttggact agaaggctta atcaaaagct cgttcgtctg caattatcgg
     2041 ccagttcagt gagatggtat tgcgacgaat tgccacgttg tcgcagtgtc tgtactgtgg
     2101 gttaatgtct gtgatcgccg tcgtgagttc gtgtcatccg cgcatgattc agatggtcct
     2161 ggagatcgtt gcttgtaccg gctgtttgtg gctctaccat ctagatgcga attcagggag
     2221 cagtcaaata ttgagtaaat tggtccgcaa caatggttac tctttcattc gaatacagta
     2281 agctgttata aattaagttc aaaacatgcg cattattact ggtatccaaa ttcaaagcaa
     2341 agtccactaa atgatgcttg aactcgtcta atttgaaatc ttggttttcc aaaaaggcca
     2401 aacggaacaa ctgaggggtc ctttccagat cttggcaact ttgaattttt tcagctagct
     2461 cgtcaaagga aaaattggcc tcaatagaat tgagcttgtt caactcattg ttaattgtag
     2521 aatacagctc attaaatgac cacgttggtt gaatattgaa tcttaagatt ttgttattcg
     2581 caatataaag aacaatatca tcgtcaccgg ttactctata tatcaatgca gcccatacac
     2641 tcaaagcgac agcgtatttg ttagaaaaac tatcatgagg cacatcgagc tgaggtagct
     2701 gtaacgaata tgtagcttgt ttcgtataag gttcttgttg tgggcgtaaa aagtcatgtg
     2761 gtaacactga aagagttgga ttatccaact tctctatcca gaccttttcg ttagtcatat
     2821 tggcggccgc acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2881 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2941 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3001 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3061 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3121 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3181 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3241 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3301 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3361 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3421 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3481 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3541 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgactgatc
     3601 cttcaactca gcaaaagttc gatttattca acaaagccac gttgtgtctc aaaatctctg
     3661 atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat
     3721 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctccaggcc
     3781 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     3841 cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt
     3901 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     3961 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     4021 tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata
     4081 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     4141 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgcctcg ctcaggcgca
     4201 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     4261 gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac cggattcagt
     4321 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     4381 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     4441 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     4501 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     4561 agaattggtt aattggttgt aacacgcggc cgctcggtta agctgctgcg gagcttccac
     4621 gagcaccagc acctgaagca actagactta tttgcgcttg agttagttct atacttggca
     4681 gtggaagttt gtcattatga gttggtggag gtaaaaatcc aaccttgttt aaaaatgcaa
     4741 tatatatacc aacctcttct ggagtaacac ctattccatt agaccaatca acacctgtcc
     4801 atgcggtgtc tttctttaaa gatgccacgg cgttcctatc gtctagttcc ggagctttgg
     4861 tactttcagg taagttgtct aagaccatgt gtagcaaagg atacaacgca ttttcttcat
     4921 tcctgtcaat aacagacgcc tccaatgatt tcttccattt agaatagctt tcgatttcga
     4981 catcgtaacc ataatcgtgt aaagtataca agtagtcttt gaataatatt cttgggtgac
     5041 ccgttacttg agcaacggcc aattcatttt ctttgggagg attcaaagac gtagcaacaa
     5101 caacacgagc aacatgatct actggaacca tattcacgga attttcgata tctggaatct
     5161 tacctaattg gactgaacct ttcaaaaatc tcaataagaa atcatctgtg tttgaagaac
     5221 cattggcaga ggcaccactc ctgcctattt aacgccaacg ttgcgagata tacactcgaa
     5281 atgtgtgatg tagatgtaaa cagacggcta tatgaatgcc actagccggt tattttactc
     5341 ctagttaata catatttcct agaatttttt acgtgcataa gagtagaata gtctaaagag
     5401 aagggtcctc caggttaagc aaggattttc ttaacttctt cggcgacagc atcaccgact
     5461 tcggtggtac tgttggaacc acctaaatca ccagttctga tacctgcatc caaaaccttt
     5521 ttaactgcat cttcaatggc cttaccttct tcaggcaagt tcaatgacaa tttcaacatc
     5581 attgcagcag acaagatagt ggcgataggg ttgaccttat tctttggcaa atctggagca
     5641 gaaccgtggc atggttcgta caaaccaaat gcggtgttct tgtctggcaa agaggccaag
     5701 gacgcagatg gcaacaaacc caaggaacct gggataacgg aggcttcatc ggagatgata
     5761 tcaccaaaca tgttgctggt gattataata ccatttaggt gggttgggtt cttaactagg
     5821 atcatggcgg cagaatcaat caattgatgt tgaaccttca atgtaggaaa ttcgttcttg
     5881 atggtttcct ccacagtttt tctccataat cttgaagagg ccaaaacatt agctttatcc
     5941 aaggaccaaa taggcaatgg tggctcatgt tgtagggcca tgaaagcggc cattcttgtg
     6001 attctttgca cttctggaac ggtgtattgt tcactatccc aagcgacacc atcaccatcg
     6061 tcctcctttc tcttaccaaa gtaaatacct cccactaatt ctctgacaac aacgaagtca
     6121 gtacctttag caaattgtgg cttgattgga gataagtcta aaagagagtc ggatgcaaag
     6181 ttacatggtc ttaagttggc gtacaattga agttctttac ggatttttag taaaccttgt
     6241 tcaggtctaa cactaccggt tccccattta ggaccaccca cagcacctaa caaaacggca
     6301 tcagccttct tggaggcttc cagcgcctca tctggaagtg gaacacctgt agcatcgata
     6361 gcagcaccac caattaaatg attttcgaaa tcgaacttga cattggaacg aacatcagaa
     6421 atagctttaa gaaccttaat ggcttcggct gtgatttctt gaccaacgtg gtcacctggc
     6481 aaaacgacga tcttcttagg ggcagacatt agaatggtat atccttgaaa tatatatata
     6541 tatattgctg aaatgtaaaa ggtaagaaaa gttagaaagt aagacgattg ctaaccacct
     6601 attggaaaaa acaataggtc cttaaataat attgtcaact tcaagtattg tgatgcaagc
     6661 atttagtcat gaacgcttct ctattctata tgaaaagccg gttccggcgc tctcaccttt
     6721 cctttttctc ccaatttttc agttgaaaaa ggtatatgcg tcaggcgacc tctgaaatta
     6781 acaaaaaatt tccagtcatc gaatttgatt ctgtgcgata gcgcccctgt gtgttctcgt
     6841 tatgttgagg aaaaaaataa tggttgctaa gagattcgaa ctcttgcatc ttacgatacc
     6901 tgagtattcc cacagttaac tgcggtcaag atatttcttg aatcaggcgc cttagaccgc
     6961 tcggccaaac aaccaattac ttgttgagaa atagagtata attatcctat aaatataacg
     7021 tttttgaaca cacatgaaca aggaagtaca ggacaattga ttttgaagag aatgtggatt
     7081 ttgatgtaat tgttgggatt ccatttttaa taaggcaata atattaggta tgtagatata
     7141 ctagaagttc tcctcgatgt actgcagtgc actagttgga ctgttgtgat atgccatata
     7201 tgcagtggtg ttcgccggcg ctggttccgg ctgtcttgct tagttgtgag tcgccaggca
     7261 gtcatcggta tgatctgtac atgattcgtc agtgtgagca ccactgacga gcagatttcc
     7321 gtcgcatgct cagcgaattg gagctcaatt ctcttaggat tcgattcaca ttcatctttt
     7381 tttagctact aataggataa attataggaa tttataacaa attgaaatat ggcaggcagc
     7441 aaaattaaaa ttgtcttagt tttttagtgt atagaagtga atagctatat aaagtatgtg
     7501 taaagttggt aacggaacga aaaatagaaa aggatattac atgggaaaac atgttgttta
     7561 cggagaaatg aaaagtatat tgtattttgt acgagctaaa agtacagtgg gaacaaagtc
     7621 gattttgtta catctacact gttgttatca gatctcgagc tattactagc cgcggtacca
     7681 agcttatgag accatgcatg cttagtctta agctgcaggg tctcttgcta aaactgaagc
     7741 aataacagaa aatattgaaa aacagcgaag taactgcatg ctagccccta tagtgagtcg
     7801 tattacggat ccggggtttt ttctccttga cgttaaagta tagaggtata ttaacaattt
     7861 tttgttgata cttttattac atttgaataa gaagtaatac aaaccgaaaa tgttgaaagt
     7921 attagttaaa gtggttatgc agtttttgca tttatatatc tgttaataga tcaaaaatca
     7981 tcgcttcgct gattaattac cccagaaata aggctaaaaa actaatcgca ttatcatcct
     8041 atggttgtta atttgattcg ttcatttgaa g
//
