LOCUS       sequence-356188-        7938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual fluorescent reporter for 5' UTR activity in yeast mCherry/YFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7938)
  AUTHORS   May GE, Akirtava C, Agar-Johnson M, Micic J, Woolford J, McManus J
  TITLE     Unraveling the influences of sequence and position on yeast uORF 
            activity using massively parallel reporter systems and machine 
            learning.
  JOURNAL   Elife. 2023 May 25;12:e69611. doi: 10.7554/eLife.69611.
  PUBMED    37227054
REFERENCE   2  (bases 1 to 7938)
  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: "Elife."; 
            date: "2023-05-25"; volume: "12:e69611. doi"; pages: " 
            10.7554/eLife.69611"
COMMENT     Sequence Label: pGM-ENO2-mCherry-YFP
FEATURES             Location/Qualifiers
     source          1..7938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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"
     primer_bind     complement(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(4563..5276)
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        complement(5294..5937)
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     complement(5377..5400)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     complement(5957..5976)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5958..5976)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5983..6000)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5983..5999)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5992..6014)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6140..6595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6227..6246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6437..6458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6726..7529)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(7530..7745)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7769..7788)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7888..7910
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcgaaatta
     2761 accctcacta aagggaacaa aagctggtac cgggcccccc ctcgaggtcg accgctcagc
     2821 atctgcttct tcccaaagat gaacgcggcg ttatgtcact aacgacgtgc accaacttgc
     2881 ggaaagtgga atcccgttcc aaaactggca tccactaatt gatacatcta cacaccgcac
     2941 gccttttttc tgaagcccac tttcgtggac tttgccatat gcaaaattca tgaagtgtga
     3001 taccaagtca gcatacacct cactagggta gtttctttgg ttgtattgat catttggttc
     3061 atcgtggttc attaattttt tttctccatt gctttctggc tttgatctta ctatcatttg
     3121 gatttttgtc gaaggttgta gaattgtatg tgacaagtgg caccaagcat atataaaaaa
     3181 aaaaagcatt atcttcctac cagagttgat tgttaaaaac gtatttatag caaacgcaat
     3241 tgtaattaat tcttattttg tatcttttct tccctaggct caatctttta tttttatttt
     3301 atttttcttt tcttagtttc tttcataaca ccaagcaact aatacccggg aataaacaca
     3361 cataaacaaa caaaatgaga tctgtttcaa agggcgaaga agacaatatg gctattatta
     3421 aggaattcat gagattcaag gtccacatgg aaggttctgt caacggtcac gaattcgaaa
     3481 ttgaaggtga aggtgaaggt agaccatacg aaggtaccca aaccgctaag ttgaaggtca
     3541 ccaagggtgg tccattgcca ttcgcttggg atattttgtc tccacaattc atgtacggtt
     3601 ctaaggctta cgtcaagcac ccagctgata ttccagatta cttgaagttg tctttcccag
     3661 aaggtttcaa gtgggaaaga gtcatgaact tcgaagatgg tggtgtcgtc accgtcaccc
     3721 aagattcttc tttgcaagat ggtgaattca tttacaaggt caagttgaga ggtaccaact
     3781 tcccatctga tggtccagtc atgcaaaaga agaccatggg ttgggaagct tcttctgaaa
     3841 gaatgtaccc agaagatggt gctttgaagg gtgaaattaa gcaaagattg aagttgaagg
     3901 atggtggtca ctacgatgct gaagtcaaga ccacctacaa ggctaagaag ccagtccaat
     3961 tgccaggtgc ttacaacgtc aacattaagt tggatattac ctctcacaac gaagattaca
     4021 ccattgtcga acaatacgaa agagctgaag gtagacactc taccggtggt atggatgaat
     4081 tgtacaagta actcgagtca tgtaattagt tatgtcacgc ttacattcac gccctccccc
     4141 cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta
     4201 tttttttata gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt
     4261 tctgtacaga cgcgtgtacg catgtgcggc cgcaaattaa agccttcgag cgtcccaaaa
     4321 ccttctcaag caaggttttc agtataatgt ttgttacatg cgtacacgcg tttgtacaga
     4381 aaaaaaagaa aaatttgaga tataaataac gttcttaata ctaacataac tattaaaaaa
     4441 aataaatagg gacctagact tcaggttgtc taactccttc cttttcggtt agagcggatg
     4501 tgggaggagg gcgtgaatgt aagcgtgaca taactaatta catgatatcg acaaaggaac
     4561 tatttgtaca attcatccat accatgggta ataccagcag cagtaacaaa ttctaacaag
     4621 accatgtggt ctctcttttc gtttggatct ttggataagg cagattgata ggataagtaa
     4681 tggttgtctg gtaacaagac tggaccatca ccaattggag tattttgttg ataatggtca
     4741 gctaattgaa caccaccatc ttcaatgttg tgtctaattt tgaagttagc tttgatacca
     4801 ttcttttgtt tgtcagcagt gatgtaaaca ttgtgagagt tatagttgta ttccaatttg
     4861 tgacctaaaa tgttaccatc ttctttaaaa tcaatacctt ttaattcgat tctattaact
     4921 aaggtatcac cttcaaactt gacttcagct ctggtcttgt agttaccgtc atctttgaaa
     4981 aaaatagttc tttcttgaac ataaccttct ggcatggcag acttgaaaaa gtcatgttgt
     5041 ttcatatgat ctgggtatct agcaaaacac attaaaccat aacctaaagt agtgactaag
     5101 gttggccatg gaactggcaa tttaccagta gtacaaatta attttaaggt caatttaccg
     5161 taagtagcat caccttcacc ttcaccggag acagaaaatt tgtgaccatt aacatcacca
     5221 tctaattcaa ccaaaattgg gacaacacca gtgaataatt cttcaccttt agacatacta
     5281 gttctagaat ccgtcgaaac taagttctgg tgttttaaaa ctaaaaaaaa gactaactat
     5341 aaaagtagaa tttaagaagt ttaagaaata gatttacaga attacaatca atacctaccg
     5401 tctttatata cttattagtc aagtagggga ataatttcag ggaactggtt tcaacctttt
     5461 ttttcagctt tttccaaatc agagagagca gaaggtaata gaaggtgtaa gaaaatgaga
     5521 tagatacatg cgtgggtcaa ttgccttgtg tcatcattta ctccaggcag gttgcatcac
     5581 tccattgagg ttgtgcccgt tttttgcctg tttgtgcccc tgttctctgt agttgcgcta
     5641 agagaatgga cctatgaact gatggttggt gaagaaaaca atattttggt gctgggattc
     5701 tttttttttc tggatgccag cttaaaaagc gggctccatt atatttagtg gatgccagga
     5761 ataaactgtt cacccagaca cctacgatgt tatatattct gtgtaacccg ccccctattt
     5821 tgggcatgta cgggttacag cagaattaaa aggctaattt tttgactaaa taaagttagg
     5881 aaaatcacta ctattaatta tttacgtatt ctttgaaatg gcgagtattg ataatgataa
     5941 actgagctcc aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca
     6001 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     6061 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     6121 cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     6181 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     6241 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     6301 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     6361 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     6421 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     6481 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     6541 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttcctga
     6601 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataggg taataactga
     6661 tataattaaa ttgaagctct aatttgtgag tttagtatac atgcatttac ttataataca
     6721 gttttttagt tttgctggcc gcatcttctc aaatatgctt cccagcctgc ttttctgtaa
     6781 cgttcaccct ctaccttagc atcccttccc tttgcaaata gtcctcttcc aacaataata
     6841 atgtcagatc ctgtagagac cacatcatcc acggttctat actgttgacc caatgcgtct
     6901 cccttgtcat ctaaacccac accgggtgtc ataatcaacc aatcgtaacc ttcatctctt
     6961 ccacccatgt ctctttgagc aataaagccg ataacaaaat ctttgtcgct cttcgcaatg
     7021 tcaacagtac ccttagtata ttctccagta gatagggagc ccttgcatga caattctgct
     7081 aacatcaaaa ggcctctagg ttcctttgtt acttcttctg ccgcctgctt caaaccgcta
     7141 acaatacctg ggcccaccac accgtgtgca ttcgtaatgt ctgcccattc tgctattctg
     7201 tatacacccg cagagtactg caatttgact gtattaccaa tgtcagcaaa ttttctgtct
     7261 tcgaagagta aaaaattgta cttggcggat aatgccttta gcggcttaac tgtgccctcc
     7321 atggaaaaat cagtcaagat atccacatgt gtttttagta aacaaatttt gggacctaat
     7381 gcttcaacta actccagtaa ttccttggtg gtacgaacat ccaatgaagc acacaagttt
     7441 gtttgctttt cgtgcatgat attaaatagc ttggcagcaa caggactagg atgagtagca
     7501 gcacgttcct tatatgtagc tttcgacatg atttatcttc gtttcctgca ggtttttgtt
     7561 ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt cttcaacact acatatgcgt
     7621 atatatacca atctaagtct gtgctccttc cttcgttctt ccttctgttc ggagattacc
     7681 gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag aataaaaaaa aaatgatgaa
     7741 ttgaaaaggt ggtatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7801 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     7861 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     7921 catcaccgaa acgcgcga
//
