LOCUS       sequence-431070-        6529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Activity reporter backbone with an empty protease cassette (BsaI) 
            under (lexA-box)3PminCYC1 promoter and an empty substrate cassette 
            (BsmBI) under pGAL1 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6529)
  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 6529)
  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
FEATURES             Location/Qualifiers
     source          1..6529
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(101..120)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_feature    854..1357
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     1398..1416
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1454..1476)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1576..1595
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1612..1892
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             1893..2567
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        3447..3888
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    3447..3564
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3859..3882
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             4208..4219
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             4382..4393
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4400..5113
                     /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      5125..5196
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5212..5239
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(5616..6204)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5696..5715)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6307..6411
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6412..6480,6481..6529,1..743)
                     /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"
ORIGIN
        1 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
       61 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
      121 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
      181 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
      241 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
      301 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
      361 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
      421 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
      481 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
      541 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
      601 cccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
      661 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
      721 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
      781 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatcgcg
      841 gccgctcggg acggatcgct tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg
      901 aataatttgg gaatttactc tgtgtttatt tatttttatg ttttgtattt ggattttaga
      961 aagtaaataa agaaggtaga agagttacgg aatgaagaaa aaaaaataaa caaaggttta
     1021 aaaaatttca acaaaaagcg tactttacat atatatttat tagacaagaa aagcagatta
     1081 aatagatata cattcgatta acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg
     1141 gttttctaca cagacaagat gaaacaattc ggcattaata cctgagagca ggaagagcaa
     1201 gataaaaggt agtatttgtt ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa
     1261 aactattttt tctttaattt ctttttttac tttctatttt taatttatat atttatatta
     1321 aaaaatttaa attataatta tttttatagc acgtgatact ctaagaaacc attattatca
     1381 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgactcgcg cgtttcggtg
     1441 atgacggtga aaacctctga cacatgcagc tcccggacac ggtcacagct tgtctgtaag
     1501 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     1561 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat aaacgacatt
     1621 actatatata taatatagga agcatttaat agacagcatc gtaatatatg tgtactttgc
     1681 agttatgacg ccagatggca gtagtggaag atattcttta ttgaaaaata gcttgtcacc
     1741 ttacgtacaa tcttgatccg gagcttttct ttttttgccg attaagaatt aattcggtcg
     1801 aaaaaagaaa aggagagggc caagagggag ggcattggtg actattgagc acgtgagtat
     1861 acgtgattaa gcacacaaag gcagcttgga gtatgtctgt tattaatttc acaggtagtt
     1921 ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga ggccgcagaa tgtgctctag
     1981 attctgatgc tgacttgctg ggtattatat gtgtgcccaa tagaaagaga acaattgacc
     2041 cggttattgc aaggaaaatt tcaagtcttg taaaagcata taaaaatagt tcaggcactc
     2101 cgaaatactt ggttggcgtg tttcgtaatc aacctaagga ggatgttttg gctctggtca
     2161 atgattacgg cattgatatc gtccaactgc atggagatga gtcgtggcaa gaataccaag
     2221 agttcctcgg tttgccagtt attaaaagac tcgtatttcc aaaagactgc aacatactac
     2281 tcagtgcagc ttcacagaaa cctcattcgt ttattccctt gtttgattca gaagcaggtg
     2341 ggacaggtga acttttggat tggaactcga tttctgactg ggttggaagg caagagagcc
     2401 ccgaaagctt acattttatg ttagctggtg gactgacgcc agaaaatgtt ggtgatgcgc
     2461 ttagattaaa tggcgttatt ggtgttgatg taagcggagg tgtggagaca aatggtgtaa
     2521 aagactctaa caaaatagca aatttcgtca aaaatgctaa gaaataggtt attactgagt
     2581 agtatttatt taagtattgt ttgtgcactt gcctgcggtg tgaaataccg cacagatgcg
     2641 taaggagaaa ataccgcatc aggtgtactg cagtgcacta gtgaattgga gctcaattct
     2701 cttaggattc gattcacatt catctttttt tagctactaa taggataaat tataggaatt
     2761 tataacaaat tgaaatatgg caggcagcaa aattaaaatt gtcttagttt tttagtgtat
     2821 agaagtgaat agctatataa agtatgtgta aagttggtaa cggaacgaaa aatagaaaag
     2881 gatattacat gggaaaacat gttgtttacg gagaaatgaa aagtatattg tattttgtac
     2941 gagctaaaag tacagtggga acaaagtcga ttttgttaca tctacactgt tgttatcaga
     3001 tctcgagcta ttactagccg cggtaccaag cttatgagac catgcatgct tagtcttaag
     3061 ctgcagggtc tcttgctaaa actgaagcaa taacagaaaa tattgaaaaa cagcgaagta
     3121 actgcatgga tccactagta tagaagtata gtaatttatg ctgcaaaggt cctaatgtat
     3181 aaggaaagaa gagaaaagaa gaaaacaaga gttttatata catacagagc acatgctcta
     3241 gaccctgggt gtatatacag ttatgctgtg agtatataca gcacctaggg agctcgaaaa
     3301 gaaatatagt cttcaaggtt atattataca ggtttatata ttattgcatg tttaaacgaa
     3361 ttctagcccc attatcttag cctaaaaaaa ccttctcttt ggaactttca gtaatacgct
     3421 taactgctca ttgctatatt gaagtacgga ttagaagccg ccgagcgggc gacagccctc
     3481 cgacggatga ctctcctccg tgcgtcctcg tcatcaccgg tcgcgttcct gaaacgcaga
     3541 tgtgcctcgc gccgcactgc tccgaacaat aaagattcta caatactagc ttttatggtt
     3601 atgaagagga aaaattggca gtaacctggc cccacaaacc ttcaaattaa cgaatcaaat
     3661 taacaaccat aggatgataa tgcgattagt tttttagcct tatttctggg gtaattaatc
     3721 agcgaagcga tgatttttga tctattaaca gatatataaa tggaaaagct gcataaccac
     3781 tttaactaat actttcaaca ttttcagttt gtattacttc ttattcaaat gtcataaaag
     3841 tatcaacaaa aaattgttaa tatacctcta tactttaacg tcaaggagaa aaaactatac
     3901 ccggattcta gagagctcac tagtatcgat gcagttactt cgctgttttt caatattttc
     3961 tgttattgct tcagttttag cacaggaact gacaactata tgcgagcaaa tcccctcacc
     4021 aactttagaa tcgacgccgt actctttgtc aacgactact attttggcca acgggaaggc
     4081 aatgcaagga gtttttgaat attacaaatc agtaacgttt gtcagtaatt gcggttctca
     4141 cccctcaaca actagcaaag gcagccccat aaacacacag tatgttttta aggacaatag
     4201 ctcgacgatt gaaggtagat cgagacggga tccgaaagtg aaacgtgatt tcatgcgtca
     4261 ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg caattcacat ttaatttatg
     4321 aatgttttct taacatcgcg gcaactcaag aaacggcagg ttcggatctt agctactaga
     4381 gaaagaggag aaatactaga tgcgtaaagg cgaagagctg ttcactggtg tcgtccctat
     4441 tctggtggaa ctggatggtg atgtcaacgg tcataagttt tccgtgcgtg gcgagggtga
     4501 aggtgacgca actaatggta aactgacgct gaagttcatc tgtactactg gtaaactgcc
     4561 ggttccttgg ccgactctgg taacgacgct gacttatggt gttcagtgct ttgctcgtta
     4621 tccggaccat atgaagcagc atgacttctt caagtccgcc atgccggaag gctatgtgca
     4681 ggaacgcacg atttccttta aggatgacgg cacgtacaaa acgcgtgcgg aagtgaaatt
     4741 tgaaggcgat accctggtaa accgcattga gctgaaaggc attgacttta aagaggacgg
     4801 caatatcctg ggccataagc tggaatacaa ttttaacagc cacaatgttt acatcaccgc
     4861 cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt cgccacaacg tggaggatgg
     4921 cagcgtgcag ctggctgatc actaccagca aaacactcca atcggtgatg gtcctgttct
     4981 gctgccagac aatcactatc tgagcacgca aagcgttctg tctaaagatc cgaacgagaa
     5041 acgcgatcat atggttctgc tggagttcgt aaccgcagcg ggcatcacgc atggtatgga
     5101 tgaactgtac aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     5161 tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct
     5221 tcgggtgggc ctttctgcgt ttatattaat taacgtctca caatccatgg tctatacatg
     5281 ggtgaccaaa agagcgggcg gatacacgcg tcaccacaag cagaataaaa ggtaaacctg
     5341 aaattgtttt aacataaaat gaaaaatgct tgtttgcaac cctatataga atcataaaac
     5401 attcgtgact ataaaatgaa taaactaaac tattctaaga aaatgaaata aatgacaaaa
     5461 aaacgtgttt tttggactag aaggcttaat caaaagctga attcaggggc ggccgcacgg
     5521 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5581 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5641 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5701 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5761 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5821 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5881 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5941 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6001 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6061 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6121 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6181 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6241 cagtggaacg aaaactcacg ttaagggatt ttggtcatga caggtggcac ttttcgggga
     6301 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     6361 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     6421 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     6481 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtg
//
