LOCUS       sequence-251803-        8218 bp    DNA     circular SYN 14-APR-2025
DEFINITION  A dual reporter vector modified from Sharon et al (Nature Biotech, 
            2012) containing Ura3 and Nat1 selectable markers, a constant 
            background RFP (TEF2::mCherry), and a variable YFP 
            (pTpA+MCS::yEVenus)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8218)
  AUTHORS   de Boer CG, Vaishnav ED, Sadeh R, Abeyta EL, Friedman N, Regev A
  TITLE     Deciphering eukaryotic gene-regulatory logic with 100 million random
            promoters.
  JOURNAL   Nat Biotechnol. 2020 Jan;38(1):56-65. doi: 
            10.1038/s41587-019-0315-8. Epub 2019 Dec 2.
  PUBMED    31792407
REFERENCE   2  (bases 1 to 8218)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41587-019-0315-8"; journalName: "Nat Biotechnol"; date: 
            "2020-01"; volume: "38"; issue: "1"; pages: "56-65"
COMMENT     Sequence Label: yeast_DualReporter
FEATURES             Location/Qualifiers
     source          1..8218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(93..115)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        315..333
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     315..332
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        407..622
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             623..1426
                     /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"
     primer_bind     complement(1694..1715)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             1960..2676
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2697..2884
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     gene            2959..4078
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        2959..3302
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3303..3875
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      3881..4078
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    4145..4648
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     complement(4704..4723)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4705..4723)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4810..4827)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4981..5569)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5061..5080)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5740..6531,6532..6600))
                     /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     6363..6382
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6601..6705)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(6770..6957)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(6984..7694)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(7262..7281)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7531..7549
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
ORIGIN
        1 gaagcgccta cgcttgacat ctactatatg taagtatacg gccccatata taggcccttt
       61 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
      121 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
      181 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
      241 gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt
      301 atcatacaca tacgatttag gtgacactat agaacgcggc cgccagctga agctttaact
      361 atgcggcatc agagcagatt gtactgagag tgcaccatac caccttttca attcatcatt
      421 ttttttttat tctttttttt gatttcggtt tccttgaaat ttttttgatt cggtaatctc
      481 cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata tacgcatatg
      541 tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca gaacaaaaac
      601 ctgcaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg ctgctactca
      661 tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa caaacttgtg
      721 tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag cattaggtcc
      781 caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca tggagggcac
      841 agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg aagacagaaa
      901 atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat acagaatagc
      961 agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg ttagcggttt
     1021 gaagcaggcg gcagaagaag taacaaagga acctagaggc cttttgatgt tagcagaatt
     1081 gtcatgcaag ggctccctat ctactggaga atatactaag ggtactgttg acattgcgaa
     1141 gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg gaagagatga
     1201 aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg gagacgcatt
     1261 gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca ttattattgt
     1321 tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac gttacagaaa
     1381 agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa ctgtattata
     1441 agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat atcagttatt
     1501 accctatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat
     1561 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     1621 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     1681 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     1741 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     1801 aagtgctagc aggaatgatg caaaaggttc ccgattcgaa ctgcattttt ttcacatctc
     1861 gaggttacgg ctgtttctta attaaaaaaa gatagaaaac attaggagtg taacacaaga
     1921 ctttcggatc ctgagcaggc aagataaacg aaggcaaaga tgtctaaagg tgaagaatta
     1981 ttcactggtg ttgtcccaat tttggttgaa ttagatggtg atgttaatgg tcacaaattt
     2041 tctgtctccg gtgaaggtga aggtgatgct acttacggta aattgacctt aaaattgatt
     2101 tgtactactg gtaaattgcc agttccatgg ccaaccttag tcactacttt aggttatggt
     2161 ttgcaatgtt ttgctagata cccagatcat atgaaacaac atgacttttt caagtctgcc
     2221 atgccagaag gttatgttca agaaagaact atttttttca aagatgacgg taactacaag
     2281 accagagctg aagtcaagtt tgaaggtgat accttagtta atagaatcga attaaaaggt
     2341 attgatttta aagaagatgg taacatttta ggtcacaaat tggaatacaa ctataactct
     2401 cacaatgttt acatcactgc tgacaaacaa aagaatggta tcaaagctaa cttcaaaatt
     2461 agacacaaca ttgaagatgg tggtgttcaa ttagctgacc attatcaaca aaatactcca
     2521 attggtgatg gtccagtctt gttaccagac aaccattact tatcctatca atctgcctta
     2581 tccaaagatc caaacgaaaa gagagaccac atggtcttgt tagaatttgt tactgctgct
     2641 ggtattaccc atggtatgga tgaattgtac aaataaggcg cgccacttct aaataagcga
     2701 atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca
     2761 aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc
     2821 tgtaggtcag gttgctttct caggtatagt atgaggtcgc tcttattgac cacacctcta
     2881 ccggcagatc cgctagggat aacagggtaa tatagatctg tttagcttgc ctcgtccccg
     2941 ccgggtcacc cggccagcga catggaggcc cagaataccc tccttgacag tcttgacgtg
     3001 cgcagctcag gggcatgatg tgactgtcgc ccgtacattt agcccataca tccccatgta
     3061 taatcatttg catccataca ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc
     3121 tcgctgcaga cctgcgagca gggaaacgct cccctcacag acgcgttgaa ttgtccccac
     3181 gccgcgcccc tgtagagaaa tataaaaggt taggatttgc cactgaggtt cttctttcat
     3241 atacttcctt ttaaaatctt gctaggatac agttctcaca tcacatccga acataaacaa
     3301 ccatgggtac cactcttgac gacacggctt accggtaccg caccagtgtc ccgggggacg
     3361 ccgaggccat cgaggcactg gatgggtcct tcaccaccga caccgtcttc cgcgtcaccg
     3421 ccaccgggga cggcttcacc ctgcgggagg tgccggtgga cccgcccctg accaaggtgt
     3481 tccccgacga cgaatcggac gacgaatcgg acgacgggga ggacggcgac ccggactccc
     3541 ggacgttcgt cgcgtacggg gacgacggcg acctggcggg cttcgtggtc gtctcgtact
     3601 ccggctggaa ccgccggctg accgtcgagg acatcgaggt cgccccggag caccgggggc
     3661 acggggtcgg gcgcgcgttg atggggctcg cgacggagtt cgcccgcgag cggggcgccg
     3721 ggcacctctg gctggaggtc accaacgtca acgcaccggc gatccacgcg taccggcgga
     3781 tggggttcac cctctgcggc ctggacaccg ccctgtacga cggcaccgcc tcggacggcg
     3841 agcaggcgct ctacatgagc atgccctgcc cctaatcagt actgacaata aaaagattct
     3901 tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc tattttaatc
     3961 aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgcccag atgcgaagtt
     4021 aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg ctatactgct
     4081 gtcgattcga tactaacgcc gccatccagt gtcgaaaacg agctcgaatt cctgggtcct
     4141 tttcatcacg tgctataaaa ataattataa tttaaatttt ttaatataaa tatataaatt
     4201 aaaaatagaa agtaaaaaaa gaaattaaag aaaaaatagt ttttgttttc cgaagatgta
     4261 aaagactcta gggggatcgc caacaaatac taccttttat cttgctcttc ctgctctcag
     4321 gtattaatgc cgaattgttt catcttgtct gtgtagaaga ccacacacga aaatcctgtg
     4381 attttacatt ttacttatcg ttaatcgaat gtatatctat ttaatctgct tttcttgtct
     4441 aataaatata tatgtaaagt acgctttttg ttgaaatttt ttaaaccttt gtttattttt
     4501 ttttcttcat tccgtaactc ttctaccttc tttatttact ttctaaaatc caaatacaaa
     4561 acataaaaat aaataaacac agagtaaatt cccaaattat tccatcatta aaagatacga
     4621 ggcgcgtgta agttacaggc aagcgatccg tccgatatca tcagatccac tagtggccta
     4681 tgcggccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt
     4741 taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4801 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4861 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4921 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4981 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5041 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5101 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5161 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5221 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5281 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5341 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5401 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5461 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5521 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5581 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5641 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5701 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5761 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5821 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5881 agacccacgt tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5941 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6001 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6061 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6121 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6181 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6241 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6301 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6361 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6421 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6481 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6541 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6601 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6661 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6721 agtgccacct gacgtcatct atattaccct gttatcccta gcggatctgc cggtagaggt
     6781 gtggtcaata agagcgacct catactatac ctgagaaagc aacctgacct acaggaaaga
     6841 gttactcaag aataagaatt ttcgttttaa aacctaagag tcactttaaa atttgtatac
     6901 acttattttt tttataactt atttaataat aaaaatcata aatcataaga aattcgctta
     6961 tttagaagtg gcgcgccggt ccgttacttg tacagctcgt ccatgccgcc ggtggagtgg
     7021 cggccctcgg cgcgttcgta ctgttccacg atggtgtagt cctcgttgtg ggaggtgatg
     7081 tccaacttga tgttgacgtt gtaggcgccg ggcagctgca cgggcttctt ggccttgtag
     7141 gtggtcttga cctcagcgtc gtagtggccg ccgtccttca gcttcagcct ctgcttgatc
     7201 tcgcccttca gggcgccgtc ctcggggtac atccgctcgg aggaggcctc ccagcccatg
     7261 gtcttcttct gcattacggg gccgtcggag gggaagttgg tgccgcgcag cttcaccttg
     7321 tagatgaact cgccgtcctg cagggaggag tcctgggtca cggtcaccac gccgccgtcc
     7381 tcgaagttca tcacgcgctc ccacttgaag ccctcgggga aggacagctt caagtagtcg
     7441 gggatgtcgg cggggtgctt cacgtaggcc ttggagccgt acatgaactg aggggacagg
     7501 atgtcccagg cgaagggcag ggggccaccc ttggtcacct tcagcttggc ggtctgggtg
     7561 ccctcgtagg ggcggccctc gccctcgccc tcgatctcga actcgtggcc gttcacggag
     7621 ccctccatgt gcaccttgaa gcgcatgaac tccttgatga tggccatgtt atcctcctcg
     7681 cccttgctca ccatggtact agtgtttagt taattatagt tcgttgaccg tatattctaa
     7741 aaacaagtac tccttaaaaa aaaaccttga agggaataaa caagtagaat agatagagag
     7801 aaaaatagaa aatgcaagag aatttatata ttagaaagag agaaagaaaa atggaaaaaa
     7861 aaaaatagga aaagccagaa atagcactag aaggagcgac accagaaaag aaggtgatgg
     7921 aaccaattta gctatatata gttaactacc ggctcgatca tctctgcctc cagcatagtc
     7981 gaagaagaat tttttttttc ttgaggcttc tgtcagcaac tcgtattttt tctttctttt
     8041 ttggtgagcc taaaaagttc ccacgttctc ttgtacgacg ccgtcacaaa caaccttatg
     8101 ggtaatttgt cgcggtctgg gtgtataaat gtgtgggtgc aacatgaatg tacggaggta
     8161 gtttgctgat tggcggtcta tagatacctt ggttatggcg ccctcacagc cggcaggg
//
