LOCUS       sequence-431074-        6470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Activity reporter backbone with an empty protease cassette (BsaI) 
            and an empty substrate cassette (BsmBI), both under a divergent 
            pGAL1-10 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6470)
  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 6470)
  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
FEATURES             Location/Qualifiers
     source          1..6470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(108..127)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_feature    861..1364
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     1405..1423
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1461..1483)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1583..1602
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1619..1899
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             1900..2574
                     /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"
     primer_bind     complement(3141..3160)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3142..3160)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3171..3835
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     primer_bind     complement(3184..3207)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    3502..3619
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     CDS             4156..4167
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             4330..4341
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4348..5061
                     /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      5073..5144
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5160..5187
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(5564..6152)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5644..5663)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6255..6359
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6360..6428,6429..6470,1..750)
                     /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 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
       61 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
      121 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
      181 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
      241 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
      301 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
      361 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
      421 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
      481 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
      541 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
      601 cgtgggtccc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
      661 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
      721 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
      781 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
      841 aatcgcggcc gctcgggacg gatcgcttgc ctgtaactta cacgcgcctc gtatctttta
      901 atgatggaat aatttgggaa tttactctgt gtttatttat ttttatgttt tgtatttgga
      961 ttttagaaag taaataaaga aggtagaaga gttacggaat gaagaaaaaa aaataaacaa
     1021 aggtttaaaa aatttcaaca aaaagcgtac tttacatata tatttattag acaagaaaag
     1081 cagattaaat agatatacat tcgattaacg ataagtaaaa tgtaaaatca caggattttc
     1141 gtgtgtggtt ttctacacag acaagatgaa acaattcggc attaatacct gagagcagga
     1201 agagcaagat aaaaggtagt atttgttggc gatcccccta gagtctttta catcttcgga
     1261 aaacaaaaac tattttttct ttaatttctt tttttacttt ctatttttaa tttatatatt
     1321 tatattaaaa aatttaaatt ataattattt ttatagcacg tgatactcta agaaaccatt
     1381 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg actcgcgcgt
     1441 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggacacggt cacagcttgt
     1501 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     1561 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccataaa
     1621 cgacattact atatatataa tataggaagc atttaataga cagcatcgta atatatgtgt
     1681 actttgcagt tatgacgcca gatggcagta gtggaagata ttctttattg aaaaatagct
     1741 tgtcacctta cgtacaatct tgatccggag cttttctttt tttgccgatt aagaattaat
     1801 tcggtcgaaa aaagaaaagg agagggccaa gagggagggc attggtgact attgagcacg
     1861 tgagtatacg tgattaagca cacaaaggca gcttggagta tgtctgttat taatttcaca
     1921 ggtagttctg gtccattggt gaaagtttgc ggcttgcaga gcacagaggc cgcagaatgt
     1981 gctctagatt ctgatgctga cttgctgggt attatatgtg tgcccaatag aaagagaaca
     2041 attgacccgg ttattgcaag gaaaatttca agtcttgtaa aagcatataa aaatagttca
     2101 ggcactccga aatacttggt tggcgtgttt cgtaatcaac ctaaggagga tgttttggct
     2161 ctggtcaatg attacggcat tgatatcgtc caactgcatg gagatgagtc gtggcaagaa
     2221 taccaagagt tcctcggttt gccagttatt aaaagactcg tatttccaaa agactgcaac
     2281 atactactca gtgcagcttc acagaaacct cattcgttta ttcccttgtt tgattcagaa
     2341 gcaggtggga caggtgaact tttggattgg aactcgattt ctgactgggt tggaaggcaa
     2401 gagagccccg aaagcttaca ttttatgtta gctggtggac tgacgccaga aaatgttggt
     2461 gatgcgctta gattaaatgg cgttattggt gttgatgtaa gcggaggtgt ggagacaaat
     2521 ggtgtaaaag actctaacaa aatagcaaat ttcgtcaaaa atgctaagaa ataggttatt
     2581 actgagtagt atttatttaa gtattgtttg tgcacttgcc tgcggtgtga aataccgcac
     2641 agatgcgtaa ggagaaaata ccgcatcagg tgtactgcag tgcactagtg aattggagct
     2701 caattctctt aggattcgat tcacattcat ctttttttag ctactaatag gataaattat
     2761 aggaatttat aacaaattga aatatggcag gcagcaaaat taaaattgtc ttagtttttt
     2821 agtgtataga agtgaatagc tatataaagt atgtgtaaag ttggtaacgg aacgaaaaat
     2881 agaaaaggat attacatggg aaaacatgtt gtttacggag aaatgaaaag tatattgtat
     2941 tttgtacgag ctaaaagtac agtgggaaca aagtcgattt tgttacatct acactgttgt
     3001 tatcagatct cgagctatta ctagccgcgg taccaagctt atgagaccat gcatgcttag
     3061 tcttaagctg cagggtctct tgctaaaact gaagcaataa cagaaaatat tgaaaaacag
     3121 cgaagtaact gcatgctagc ccctatagtg agtcgtatta cggatccggg gttttttctc
     3181 cttgacgtta aagtatagag gtatattaac aattttttgt tgatactttt attacatttg
     3241 aataagaagt aatacaaacc gaaaatgttg aaagtattag ttaaagtggt tatgcagttt
     3301 ttgcatttat atatctgtta atagatcaaa aatcatcgct tcgctgatta attaccccag
     3361 aaataaggct aaaaaactaa tcgcattatc atcctatggt tgttaatttg attcgttcat
     3421 ttgaaggttt gtggggccag gttactgcca atttttcctc ttcataacca taaaagctag
     3481 tattgtagaa tctttattgt tcggagcagt gcggcgcgag gcacatctgc gtttcaggaa
     3541 cgcgaccggt gaagacgagg acgcacggag gagagtcttc cttcggaggg ctgtcacccg
     3601 ctcggcggct tctaatccgt acttcaatat agcaatgagc agttaagcgt attactgaaa
     3661 gttccaaaga gaaggttttt ttaggctaag ataatggggc tctttacatt tccacaacat
     3721 ataagtaaga ttagatatgg atatgtatat ggatatgtat atggtggtaa tgccatgtaa
     3781 tatgattatt aaacttcttt gcgtccatcc aaaaaaaaag taagaatttt tgaaaattcg
     3841 aattcaaccc tcactaaagt ctgactaata tcatcgatgc agttacttcg ctgtttttca
     3901 atattttctg ttattgcttc agttttagca caggaactga caactatatg cgagcaaatc
     3961 ccctcaccaa ctttagaatc gacgccgtac tctttgtcaa cgactactat tttggccaac
     4021 gggaaggcaa tgcaaggagt ttttgaatat tacaaatcag taacgtttgt cagtaattgc
     4081 ggttctcacc cctcaacaac tagcaaaggc agccccataa acacacagta tgtttttaag
     4141 gacaatagct cgacgattga aggtagatcg agacgggatc cgaaagtgaa acgtgatttc
     4201 atgcgtcatt ttgaacattt tgtaaatctt atttaataat gtgtgcggca attcacattt
     4261 aatttatgaa tgttttctta acatcgcggc aactcaagaa acggcaggtt cggatcttag
     4321 ctactagaga aagaggagaa atactagatg cgtaaaggcg aagagctgtt cactggtgtc
     4381 gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc
     4441 gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg tactactggt
     4501 aaactgccgg ttccttggcc gactctggta acgacgctga cttatggtgt tcagtgcttt
     4561 gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat gccggaaggc
     4621 tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac gcgtgcggaa
     4681 gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat tgactttaaa
     4741 gaggacggca atatcctggg ccataagctg gaatacaatt ttaacagcca caatgtttac
     4801 atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg ccacaacgtg
     4861 gaggatggca gcgtgcagct ggctgatcac taccagcaaa acactccaat cggtgatggt
     4921 cctgttctgc tgccagacaa tcactatctg agcacgcaaa gcgttctgtc taaagatccg
     4981 aacgagaaac gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg catcacgcat
     5041 ggtatggatg aactgtacaa atgaccaggc atcaaataaa acgaaaggct cagtcgaaag
     5101 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg
     5161 gctcaccttc gggtgggcct ttctgcgttt atattaatta acgtctcaca atccatggtc
     5221 tatacatggg tgaccaaaag agcgggcgga tacacgcgtc accacaagca gaataaaagg
     5281 taaacctgaa attgttttaa cataaaatga aaaatgcttg tttgcaaccc tatatagaat
     5341 cataaaacat tcgtgactat aaaatgaata aactaaacta ttctaagaaa atgaaataaa
     5401 tgacaaaaaa acgtgttttt tggactagaa ggcttaatca aaagctgaat tcaggggcgg
     5461 ccgcacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5521 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5581 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5641 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5701 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5761 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5821 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5881 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5941 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6001 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6061 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6121 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6181 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaca ggtggcactt
     6241 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     6301 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     6361 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     6421 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
//
