LOCUS       sequence-431077-        8066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MET-HYGRO plasmid (218133) 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 8066)
  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 8066)
  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-MET-IV
FEATURES             Location/Qualifiers
     source          1..8066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gene            complement(1352..2926)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1352..1549)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1554..2582)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWARGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        3907..4348
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    3907..4024
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     4319..4342
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             4668..4679
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             4842..4853
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4860..5573
                     /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      5585..5656
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5672..5699
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(7153..7741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7233..7252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(7955..7974)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(7966..8066,1..715)
                     /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             join(7966..8066,1..715)
                     /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(8039..8058)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
       61 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
      121 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
      181 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
      241 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
      301 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgcctcgctc aggcgcaatc
      361 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
      421 tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt
      481 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
      541 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
      601 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
      661 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
      721 attggttaat tggttgtaac acgcggccgc tcggatggaa agtagcatcc aaccaaatcc
      781 cgcgaagttt tcctgattta cataacaagc aaatactaaa aacgttaaca cacatgtgct
      841 tctttattgt aaagaaacct ccatcatcct ctattgtaac ttggtcctac aataaattta
      901 tccagtgtga cagctttata ggaggcgtaa agtagtctca tgaagtagat gagtcttgtt
      961 caccatattt ttcttctcga ctgcgaatta acactgttct ttgatgttag aacaatttag
     1021 gttcaaagta cgagtcacga catgtaacaa gggaaaaaaa ggatattcat ttcaataaag
     1081 ttcgttttat aaaagtatag tacttgtgag agaaagtagg tttatacata attttacaac
     1141 tcattacgca cactcatggt ttttggccag cgaaaacagt ttcaaaagat tgctggaagt
     1201 ctgcaataat gtcatcaata aattcgatac caacagagac acgaattaag tccttggtaa
     1261 caccagatgc caacttttct ttgtcattta attgtttgtg ggtagtgaag tatggagcaa
     1321 tgactaaggt cttggcatca ccaacatact ccagtatagc gaccagcatt cacatacgat
     1381 tgacgcatga tattactttc tgcgcactta acttcgcatc tgggcagatg atgtcgaggc
     1441 gaaaaaaaat ataaatcacg ctaacatttg attaaaatag aacaactaca atataaaaaa
     1501 actatacaaa tgacaagttc ttgaaaacaa gaatcttttt attgtcagtt actttattcc
     1561 tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac ttctacacag
     1621 ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac agtcccggct
     1681 ccggatcgga cgattgcgtc gcatcgaccc cgcgcccaag ctgcatcatc gaaattgccg
     1741 tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc ccggagccgc
     1801 ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa
     1861 gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc cccgaacatc
     1921 gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac attgttggag
     1981 ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca aagcatcagc
     2041 tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt ttgccagtga
     2101 tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt
     2161 ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc agcgatcgca
     2221 tccatggcct ccgcgaccgg ttgcagaaca gcgggcagtt cggtttcagg caggtcttgc
     2281 aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct aaattcccca
     2341 atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata aacataacga
     2401 tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg ccctcctaca
     2461 tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc ggacacgctg
     2521 tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg ctttttaccc
     2581 atttttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt ttaaaaggaa
     2641 gtatatgaaa gaagaacctc agtggcaaat cctaaccttt tatatttctc tacaggggcg
     2701 cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg caggtctgga
     2761 gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg gatgcaaatg
     2821 attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat gcccctgagc
     2881 tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtcgctg gccgggtgac
     2941 ccggcgggga cgaggcaagc taaactgtac tgcagtgcac tagttggact gttgtgatat
     3001 gccatatatg cagtggtgtt cgccggcgct ggttccggct gtcttgctta gttgtgagtc
     3061 gccaggcagt catcggtatg atctgtacat gattcgtcag tgtgagcacc actgacgagc
     3121 agatttccgt atcatgctca gcgaattgga gctcaattct cttaggattc gattcacatt
     3181 catctttttt tagctactaa taggataaat tataggaatt tataacaaat tgaaatatgg
     3241 caggcagcaa aattaaaatt gtcttagttt tttagtgtat agaagtgaat agctatataa
     3301 agtatgtgta aagttggtaa cggaacgaaa aatagaaaag gatattacat gggaaaacat
     3361 gttgtttacg gagaaatgaa aagtatattg tattttgtac gagctaaaag tacagtggga
     3421 acaaagtcga ttttgttaca tctacactgt tgttatcaga tctcgagcta ttactagccg
     3481 cggtaccaag cttatgagac catgcatgct tagtcttaag ctgcagggtc tcttgctaaa
     3541 actgaagcaa taacagaaaa tattgaaaaa cagcgaagta actgcatgga tccactagta
     3601 tagaagtata gtaatttatg ctgcaaaggt cctaatgtat aaggaaagaa gagaaaagaa
     3661 gaaaacaaga gttttatata catacagagc acatgctcta gaccctgggt gtatatacag
     3721 ttatgctgtg agtatataca gcacctaggg agctcgaaaa gaaatatagt cttcaaggtt
     3781 atattataca ggtttatata ttattgcatg tttaaacgaa ttctagcccc attatcttag
     3841 cctaaaaaaa ccttctcttt ggaactttca gtaatacgct taactgctca ttgctatatt
     3901 gaagtacgga ttagaagccg ccgagcgggc gacagccctc cgacggatga ctctcctccg
     3961 tgcgtcctcg tcatcaccgg tcgcgttcct gaaacgcaga tgtgcctcgc gccgcactgc
     4021 tccgaacaat aaagattcta caatactagc ttttatggtt atgaagagga aaaattggca
     4081 gtaacctggc cccacaaacc ttcaaattaa cgaatcaaat taacaaccat aggatgataa
     4141 tgcgattagt tttttagcct tatttctggg gtaattaatc agcgaagcga tgatttttga
     4201 tctattaaca gatatataaa tggaaaagct gcataaccac tttaactaat actttcaaca
     4261 ttttcagttt gtattacttc ttattcaaat gtcataaaag tatcaacaaa aaattgttaa
     4321 tatacctcta tactttaacg tcaaggagaa aaaactatac ccggattcta gagagctcac
     4381 tagtatcgat gcagttactt cgctgttttt caatattttc tgttattgct tcagttttag
     4441 cacaggaact gacaactata tgcgagcaaa tcccctcacc aactttagaa tcgacgccgt
     4501 actctttgtc aacgactact attttggcca acgggaaggc aatgcaagga gtttttgaat
     4561 attacaaatc agtaacgttt gtcagtaatt gcggttctca cccctcaaca actagcaaag
     4621 gcagccccat aaacacacag tatgttttta aggacaatag ctcgacgatt gaaggtagat
     4681 cgagacggga tccgaaagtg aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc
     4741 ttatttaata atgtgtgcgg caattcacat ttaatttatg aatgttttct taacatcgcg
     4801 gcaactcaag aaacggcagg ttcggatctt agctactaga gaaagaggag aaatactaga
     4861 tgcgtaaagg cgaagagctg ttcactggtg tcgtccctat tctggtggaa ctggatggtg
     4921 atgtcaacgg tcataagttt tccgtgcgtg gcgagggtga aggtgacgca actaatggta
     4981 aactgacgct gaagttcatc tgtactactg gtaaactgcc ggttccttgg ccgactctgg
     5041 taacgacgct gacttatggt gttcagtgct ttgctcgtta tccggaccat atgaagcagc
     5101 atgacttctt caagtccgcc atgccggaag gctatgtgca ggaacgcacg atttccttta
     5161 aggatgacgg cacgtacaaa acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa
     5221 accgcattga gctgaaaggc attgacttta aagaggacgg caatatcctg ggccataagc
     5281 tggaatacaa ttttaacagc cacaatgttt acatcaccgc cgataaacaa aaaaatggca
     5341 ttaaagcgaa ttttaaaatt cgccacaacg tggaggatgg cagcgtgcag ctggctgatc
     5401 actaccagca aaacactcca atcggtgatg gtcctgttct gctgccagac aatcactatc
     5461 tgagcacgca aagcgttctg tctaaagatc cgaacgagaa acgcgatcat atggttctgc
     5521 tggagttcgt aaccgcagcg ggcatcacgc atggtatgga tgaactgtac aaatgaccag
     5581 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     5641 gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt
     5701 ttatattaat taacgtctca caatccatgg tctatacatg ggtgaccaaa agagcgggcg
     5761 gatacacgcg tcaccacaag cagaataaaa ggtaaacctg aaattgtttt aacataaaat
     5821 gaaaaatgct tgtttgcaac cctatataga atcataaaac attcgtgact ataaaatgaa
     5881 taaactaaac tattctaaga aaatgaaata aatgacaaaa aaacgtgttt tttggactag
     5941 aaggcttaat caaaagctcg ttcgtctgca attatcggcc agttcagtga gaaggtattg
     6001 cgacgaattg ccacgttgtc gcagtgtctg tactgtgggt taatgtctgt gatcgccgtc
     6061 gtgagttcgt gtcatccgcg catgattcag atggtcctgg agatcgttgc ttgtaccggc
     6121 tgtttgtggc tctaccatct agatgcgaat tcagggtgga tgggggtaat agaattgtat
     6181 ctatgtatct gacgaccctg tattacacga aggaaatagt agatgaaaag acaagagagc
     6241 aagaaaaagg aaaaacttcc tttctaacag acgctttact taaccttata tatatcttat
     6301 ttttttcatc gagcgtgttc aattggacaa ggtgccacct tttcgacact tcggttatta
     6361 tgttacacag ttttcatgag gatggcgcct tgactaactt aattagtcat ttgccaacca
     6421 ccacagttcc ccaatatcga cagcttcacg tgccatttgc cattttgcga tcatgtgacc
     6481 tcaagagaaa aagcaaaaaa taatgagagc taagggattc gaacccttgc atccgaagat
     6541 atcagagatt ttagaggtta aatccaccta aatctgacgc cttaaaccac tcggccaaac
     6601 tcccattgta tgttggaaga attggattta aatgtctatt atagcattaa atatctttaa
     6661 atacattcaa gaaatggcga aattatttct tagtgttgga ataaaaatca actatcatct
     6721 actaactagt atttacgtta ctagtatatt atcatatacg gtgttagaag atgacgcaaa
     6781 tgatgagaaa tagtcatctt gttggaataa aaatcaacta tcatctacta actagtattt
     6841 acgttactag tatattatca tatacggtgt tagaagatga cgcaaatgat gagaaatagt
     6901 catctaaatt agtggaagct gaaacgcaag gattgataat gtaataggat caatgaatat
     6961 taacatataa aatgatgata ataatattta tagaattgtg tagaattgca gattcccttt
     7021 tatggattcc taaatcctcg aattggcggc cgcacggtta tccacagaat caggggataa
     7081 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     7141 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     7201 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     7261 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     7321 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     7381 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     7441 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     7501 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     7561 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     7621 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     7681 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     7741 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     7801 agggattttg gtcatgactg atccttcaac tcagcaaaag ttcgatttat tcaacaaagc
     7861 cacgttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     7921 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     7981 cgggaaacgt cttgctccag gccgcgatta aattccaaca tggatgctga tttatatggg
     8041 tataaatggg ctcgcgataa tgtcgg
//
