LOCUS       sequence-356165-        7935 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual fluorescent reporter for 5' UTR activity in yeast YFP/mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7935)
  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 7935)
  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-YFP-mCherry
FEATURES             Location/Qualifiers
     source          1..7935
                     /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             3375..4091
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /translation="MRSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     terminator      complement(4309..4556)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     promoter        complement(5291..5934)
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     complement(5374..5397)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     complement(5954..5973)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5955..5973)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5980..5997)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5980..5996)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5989..6011)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6137..6592
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6224..6243)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6434..6455
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6723..7526)
                     /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(7527..7742)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7766..7785)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7885..7907
                     /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 tctaaaggtg aagaattatt cactggtgtt gtcccaattt
     3421 tggttgaatt agatggtgat gttaatggtc acaaattttc tgtctccggt gaaggtgaag
     3481 gtgatgctac ttacggtaaa ttgaccttaa aattaatttg tactactggt aaattgccag
     3541 ttccatggcc aaccttagtc actactttag gttatggttt aatgtgtttt gctagatacc
     3601 cagatcatat gaaacaacat gactttttca agtctgccat gccagaaggt tatgttcaag
     3661 aaagaactat ttttttcaaa gatgacggta actacaagac cagagctgaa gtcaagtttg
     3721 aaggtgatac cttagttaat agaatcgaat taaaaggtat tgattttaaa gaagatggta
     3781 acattttagg tcacaaattg gaatacaact ataactctca caatgtttac atcactgctg
     3841 acaaacaaaa gaatggtatc aaagctaact tcaaaattag acacaacatt gaagatggtg
     3901 gtgttcaatt agctgaccat tatcaacaaa atactccaat tggtgatggt ccagtcttgt
     3961 taccagacaa ccattactta tcctatcaat ctgccttatc caaagatcca aacgaaaaga
     4021 gagaccacat ggtcttgtta gaatttgtta ctgctgctgg tattacccat ggtatggatg
     4081 aattgtacaa atagttcctt tgtcgatatc atgtaattag ttatgtcacg cttacattca
     4141 cgccctcctc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag
     4201 gtccctattt atttttttta atagttatgt tagtattaag aacgttattt atatctcaaa
     4261 tttttctttt ttttctgtac aaacgcgtgt acgcatgtaa cagcggccgc aaattaaagc
     4321 cttcgagcgt cccaaaacct tctcaagcaa ggttttcagt ataatgttac atgcgtacac
     4381 gcgtctgtac agaaaaaaaa gaaaaatttg aaatataaat aacgttctta atactaacat
     4441 aactataaaa aaataaatag ggacctagac ttcaggttgt ctaactcctt ccttttcggt
     4501 tagagcggat gtggggggag ggcgtgaatg taagcgtgac ataactaatt acatgactcg
     4561 agttacttgt acaattcatc cataccaccg gtagagtgtc taccttcagc tctttcgtat
     4621 tgttcgacaa tggtgtaatc ttcgttgtga gaggtaatat ccaacttaat gttgacgttg
     4681 taagcacctg gcaattggac tggcttctta gccttgtagg tggtcttgac ttcagcatcg
     4741 tagtgaccac catccttcaa cttcaatctt tgcttaattt cacccttcaa agcaccatct
     4801 tctgggtaca ttctttcaga agaagcttcc caacccatgg tcttcttttg catgactgga
     4861 ccatcagatg ggaagttggt acctctcaac ttgaccttgt aaatgaattc accatcttgc
     4921 aaagaagaat cttgggtgac ggtgacgaca ccaccatctt cgaagttcat gactctttcc
     4981 cacttgaaac cttctgggaa agacaacttc aagtaatctg gaatatcagc tgggtgcttg
     5041 acgtaagcct tagaaccgta catgaattgt ggagacaaaa tatcccaagc gaatggcaat
     5101 ggaccaccct tggtgacctt caacttagcg gtttgggtac cttcgtatgg tctaccttca
     5161 ccttcacctt caatttcgaa ttcgtgaccg ttgacagaac cttccatgtg gaccttgaat
     5221 ctcatgaatt ccttaataat agccatattg tcttcttcgc cctttgaaac catactagtt
     5281 ctagaatccg tcgaaactaa gttctggtgt tttaaaacta aaaaaaagac taactataaa
     5341 agtagaattt aagaagttta agaaatagat ttacagaatt acaatcaata cctaccgtct
     5401 ttatatactt attagtcaag taggggaata atttcaggga actggtttca accttttttt
     5461 tcagcttttt ccaaatcaga gagagcagaa ggtaatagaa ggtgtaagaa aatgagatag
     5521 atacatgcgt gggtcaattg ccttgtgtca tcatttactc caggcaggtt gcatcactcc
     5581 attgaggttg tgcccgtttt ttgcctgttt gtgcccctgt tctctgtagt tgcgctaaga
     5641 gaatggacct atgaactgat ggttggtgaa gaaaacaata ttttggtgct gggattcttt
     5701 ttttttctgg atgccagctt aaaaagcggg ctccattata tttagtggat gccaggaata
     5761 aactgttcac ccagacacct acgatgttat atattctgtg taacccgccc cctattttgg
     5821 gcatgtacgg gttacagcag aattaaaagg ctaatttttt gactaaataa agttaggaaa
     5881 atcactacta ttaattattt acgtattctt tgaaatggcg agtattgata atgataaact
     5941 gagctccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg
     6001 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     6061 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     6121 cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6181 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6241 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6301 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6361 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     6421 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     6481 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     6541 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc
     6601 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catagggtaa taactgatat
     6661 aattaaattg aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt
     6721 ttttagtttt gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt
     6781 tcaccctcta ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg
     6841 tcagatcctg tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc
     6901 ttgtcatcta aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca
     6961 cccatgtctc tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca
     7021 acagtaccct tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac
     7081 atcaaaaggc ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca
     7141 atacctgggc ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat
     7201 acacccgcag agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg
     7261 aagagtaaaa aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg
     7321 gaaaaatcag tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct
     7381 tcaactaact ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt
     7441 tgcttttcgt gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca
     7501 cgttccttat atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg
     7561 tgcagttggg ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata
     7621 tataccaatc taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa
     7681 tcaaaaaaat ttcaaggaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg
     7741 aaaaggtggt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     7801 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     7861 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     7921 caccgaaacg cgcga
//
