LOCUS       sequence-223807-        8488 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Produces equimolar expression of ymYPET and mTq2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8488)
  AUTHORS   Botman D, de Groot DH, Schmidt P, Goedhart J, Teusink B
  TITLE     In vivo characterisation of fluorescent proteins in budding yeast.
  JOURNAL   Sci Rep. 2019 Feb 19;9(1):2234. doi: 10.1038/s41598-019-38913-z.
  PUBMED    30783202
REFERENCE   2  (bases 1 to 8488)
  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: "Sci Rep."; 
            date: "2019-02-19"; volume: "9(1):2234. doi"; pages: " 
            10.1038/s41598-019-38913-z"
COMMENT     Sequence Label: pDRF1-GW ymYPET-T2A-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..8488
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    800..824
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1574..1627
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      1643..1652
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1649..1651,1652..1654,1655..2368)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1694..1715)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1955..1974)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2302..2323
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2378..2402)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2464..2483)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2465..2483)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2631..2818
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2686..2710)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2830..2847)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2830..2846)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2839..2861)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3282..4628
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4739..5542)
                     /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(5543..5758)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5783..5801)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5869..5973
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5974..6042,6043..6834)
                     /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(6192..6211)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7005..7593
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7494..7513
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7747..7764
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7881..7902
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7917..7922,7923..7940,7941..7947)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7955..7971
                     /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     7960..7982
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7979..7995
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7979..7995
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(8025..8480)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8162..8183)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8374..8393
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 caagcttcct gaaacggaga aacataaaca ggcattgctg ggatcaccca tacatcactc
       61 tgttttgcct gaccttttcc ggtaatttga aaacaaaccc ggtctcgaag cggagatccg
      121 gcgataatta ccgcagaaat aaacccatac acgagacgta gaaccagccg cacatggccg
      181 gagaaactcc tgcgagaatt tcgtaaactc gcgcgcattg catctgtatt tcctaatgcg
      241 gcacttccag gcctcgatcg agaccgttta tccattgctt ttttgttgtc tttttccctc
      301 gttcacagaa agtctgaaga agctatagta gaactatgag ctttttttgt ttctgttttc
      361 cttttttttt tttttacctc tgtggaaatt gttactctca cactctttag ttcgtttgtt
      421 tgttttgttt attccaatta tgaccggtga cgaaacgtgg tcgatggtgg gtaccgctta
      481 tgctcccctc cattagtttc gattatataa aaaggccaaa tattgtatta ttttcaaatg
      541 tcctatcatt atcgtctaac atctaatttc tcttaaattt tttctctttc tttcctataa
      601 caccaatagt gaaaatcttt ttttcttcta tatctacaaa aacttttttt ttctatcaac
      661 ctcgttgata aattttttct ttaacaatcg ttaataatta attaattgga aaataaccat
      721 tttttctctc ttttatacac acattcaaaa gaaagaaaaa aaatataccc cagcctcgac
      781 tagtgaattc gcggccatca caagtttgta caaaaaagca ggctgctagc actagtaagc
      841 ttttaattaa atggtctcca agggcgaaga attgttcacc ggtgtggtcc caatcttggt
      901 cgagttggac ggtgacgtca acggtcacaa gttctctgtt tccggtgaag gtgaaggaga
      961 cgccacctac ggtaaattga ccttgaagtt gttgtgcacc accggcaagc tgccagttcc
     1021 atggccaaca ttggtcacca ctttaggtta tggtttacaa tgttttgcta gatatccaga
     1081 tcatatgaaa caacatgatt tcttcaaatc tgctatgcca gaaggttatg ttcaagaaag
     1141 aactatattt ttcaaagatg atggtaatta taaaactaga gctgaagtta aatttgaagg
     1201 agatacttta gttaatagaa ttgaattgaa aggtattgat tttaaagaag atggtaatat
     1261 tttaggtcat aaattagaat ataattataa ttctcataat gtttatatta cagctgataa
     1321 acaaaagaat ggtattaagg ctaattttaa aattagacat aatattgaag atggtggtgt
     1381 tcaattagct gatcattatc aacaaaatac tcctattggt gacggtccag ttttattacc
     1441 tgataatcat tatttgtcat atcaatctaa attgtcaaaa gatccaaatg aaaaaagaga
     1501 tcatatggtt ttgttagaat ttttgactgc agctggtatt actttaggta tggatgaatt
     1561 gtataaatcc ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc
     1621 cggccctggg gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     1681 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     1741 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     1801 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgtcctggg gcgtgcagtg
     1861 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1921 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     1981 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2041 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacttta gcgacaacgt
     2101 ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa
     2161 catcgaggac ggcggcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2221 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga
     2281 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     2341 tctcggcatg gacgagctgt acaagtaaag cggccgcacc cagctttctt gtacaaagtg
     2401 gtgtagttaa ttcatgatgg ccgctgcagg tcgacctcga gggggggccc ggtacccaat
     2461 tcgccctata gtgagtcgta ttacgcgcgg atccagcttt ggacttcttc gccagaggtt
     2521 tggtcaagtc tccaatcaag gttgtcggct tgtctacctt gccagaaatt tacgaaaaga
     2581 tggaaaaggg tcaaatcgtt ggtagatacg ttgttgacac ttctaaataa gcgaatttct
     2641 tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta tacaaatttt
     2701 aaagtgactc ttaggtttta aaacganaat tcttattctt gagtaactct ttcctgtagg
     2761 tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc tctaccggca
     2821 tgccaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     2881 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     2941 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     3001 ttttctcctt acgcatctgt gcggtatttc acaccgcata atcggatcgt acttgttacc
     3061 catcattgaa ttttgaacat ccgaacctgg gagttttccc tgaaacagat agtatatttg
     3121 aacctgtata ataatatata gtctagcgct ttacggaaga caatgtatgt atttcggttc
     3181 ctggagaaac tattgcatct attgcatagg taatcttgca cgtcgcatcc ccggttcatt
     3241 ttctgcgttt ccatcttgca cttcaatagc atatctttgt taacgaagca tctgtgcttc
     3301 attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     3361 gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg
     3421 cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct
     3481 gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat
     3541 ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc
     3601 atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt
     3661 tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat
     3721 aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt
     3781 ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg
     3841 tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct
     3901 tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat
     3961 tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa
     4021 cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta
     4081 ggttatatag ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg
     4141 tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt
     4201 tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt
     4261 ctagctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct
     4321 tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct
     4381 gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa
     4441 atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat
     4501 attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct
     4561 atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat
     4621 attggatcga tccgatgata agctgtcaaa catgagaatt gggtaataac tgatataatt
     4681 aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt
     4741 agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac
     4801 cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag
     4861 atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt
     4921 catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca
     4981 tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag
     5041 tacccttagt atattctcca gtagataggg agcccttgca tgacaattct gctaacatca
     5101 aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac
     5161 ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac
     5221 ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga
     5281 gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa
     5341 aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa
     5401 ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct
     5461 tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt
     5521 ccttatatgt agctttcgac atgatttatc ttcgtttcct gcatgttttt gttctgtgca
     5581 gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata
     5641 ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa
     5701 aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa
     5761 gctaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5821 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5881 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5941 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6001 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6061 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6121 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6181 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6241 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6301 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6361 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6421 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6481 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6541 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6601 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6661 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6721 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6781 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6841 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6901 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6961 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7021 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7081 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7141 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7201 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7261 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7321 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7381 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7441 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7501 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7561 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7621 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     7681 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     7741 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     7801 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     7861 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     7921 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     7981 ggaaacagct atgaccatga ttacgccaag cttaccgcat caggaaattg taagcgttaa
     8041 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     8101 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     8161 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     8221 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     8281 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     8341 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     8401 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     8461 tgcgccgcta cagggcgcgt ccattcgc
//
