LOCUS       sequence-437599-        8459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  equimolar expression of yeBFP2 and mCherry in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8459)
  AUTHORS   Botman D, de Groot DH, Wehrens M, van Rossum K, Yska S, van der 
            Kieft B, Albert V, Minghetti N, Goedhart J, Teusink B
  TITLE     Side-by-side systematic characterization of novel FPs in budding 
            yeast
  JOURNAL   bioRxiv 2025.06.27.661975; doi: 
            https://doi.org/10.1101/2025.06.27.661975
REFERENCE   2  (bases 1 to 8459)
  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: "bioRxiv 
            2025.06.27.661975; doi: https://doi.org/10.1101/2025.06.27.661975"
COMMENT     Sequence Label: pDRF1 yeBFP2-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..8459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             1554..1607
                     /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      1623..1632
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1629..2339
                     /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(1774..1792)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2042..2061
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2349..2373)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2435..2454)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2436..2454)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2602..2789
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2657..2681)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2801..2818)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2801..2817)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2810..2832)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3253..4599
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4710..5513)
                     /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(5514..5729)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5754..5772)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5840..5944
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5945..6013,6014..6805)
                     /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(6163..6182)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6976..7564
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7465..7484
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7718..7735
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7852..7873
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7888..7893,7894..7911,7912..7918)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7926..7942
                     /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     7931..7953
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7950..7966
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7950..7966
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7996..8451)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8133..8154)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8345..8364
                     /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 atggtttcca
      841 agggtgaaga attattcacc ggtgttgtcc ctattttggt cgaattggac ggtgatgtca
      901 acggtcacaa gttctccgtt agaggtgaag gtgaaggtga cgctaccaac ggtaagttga
      961 ccttgaagtt catctgtacc actggtaagt tgccagttcc ttggccaact ttggttacca
     1021 ctttgtctca cggtgttcaa tgtttcgccc gttacccaga tcacatgaag caacatgatt
     1081 tctttaaatc cgccatgcca gaaggttacg ttcaagaaag aactatcttc ttcaaggacg
     1141 atggtactta caaaactaga gccgaagtca agttcgaagg tgatacttta gtcaatagaa
     1201 tcgaattgaa gggtgttgat ttcaaggaag atggtaacat tttgggtcac aagttagaat
     1261 acaatttcaa ctcccataat atttacatta tggctgttaa gcaaaagaac ggtattaagg
     1321 tcaactttaa aatcagacac aacgttgaag acggttctgt ccaattagct gaccactacc
     1381 aacaaaacac tccaattggt gacggtccag tcttattacc agattctcat tacttgtcta
     1441 cccaatctgt cttgtctaag gacccaaatg aaaagagaga tcacatggtc ttgttagaat
     1501 ttcgtactgc tgctggtatc actttgggta tggacgaatt gtacaagtcc ggagagggca
     1561 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctggg gatccaccgg
     1621 tcgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     1681 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     1741 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     1801 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     1861 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     1921 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     1981 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     2041 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     2101 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     2161 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     2221 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     2281 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaaa
     2341 gcggccgcac ccagctttct tgtacaaagt ggtgtagtta attcatgatg gccgctgcag
     2401 gtcgacctcg agggggggcc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg
     2461 gatccagctt tggacttctt cgccagaggt ttggtcaagt ctccaatcaa ggttgtcggc
     2521 ttgtctacct tgccagaaat ttacgaaaag atggaaaagg gtcaaatcgt tggtagatac
     2581 gttgttgaca cttctaaata agcgaatttc ttatgattta tgatttttat tattaaataa
     2641 gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa
     2701 ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagcatgag
     2761 gtcgctctta ttgaccacac ctctaccggc atgccaattc actggccgtc gttttacaac
     2821 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     2881 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     2941 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3001 cacaccgcat aatcggatcg tacttgttac ccatcattga attttgaaca tccgaacctg
     3061 gaagttttcc ctgaaacaga tagtatattt gaacctgtat aataatatat agtctagcgc
     3121 tttacggaag acaatgtatg tatttcggtt cctggagaaa ctattgcatc tattgcatag
     3181 gtaatcttgc acgtcgcatc cccggttcat tttctgcgtt tccatcttgc acttcaatag
     3241 catatctttg ttaacgaagc atctgtgctt cattttgtag aacaaaaatg caacgcgaga
     3301 gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg
     3361 aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa aaatgcaacg
     3421 cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa cagaaatgca
     3481 acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc tacaaaaatg
     3541 catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt ctcctttgtg
     3601 cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa ggttagaaga
     3661 aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac ttcccgcgtt
     3721 tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc cccgattata
     3781 ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg ttgatgattc
     3841 ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata ctacgtatag
     3901 gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct tactacaatt
     3961 tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc gagtttagat
     4021 gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc acagagatat
     4081 atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat attttagtag
     4141 ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag cgcttttggt
     4201 tttcaaaagc gctctgaagt tcctatactt tctagctaga gaataggaac ttcggaatag
     4261 gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat
     4321 acagctcact gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga
     4381 gaagaacggc atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta
     4441 ggatgaaagg tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat
     4501 gcttccttca gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt
     4561 ctcatccttc aatgctatca tttcctttga tattggatcg atccgatgat aagctgtcaa
     4621 acatgagaat tgggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt
     4681 atacatgcat ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat
     4741 gcttcccagc ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca
     4801 aatagtcctc ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt
     4861 ctatactgtt gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc
     4921 aaccaatcgt aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca
     4981 aaatctttgt cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg
     5041 gagcccttgc atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct
     5101 tctgccgcct gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta
     5161 atgtctgccc attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta
     5221 ccaatgtcag caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc
     5281 tttagcggct taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt
     5341 agtaaacaaa ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga
     5401 acatccaatg aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca
     5461 gcaacaggac taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat
     5521 cttcgtttcc tgcatgtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat
     5581 gtttcttcaa cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt
     5641 tcttccttct gttcggagat taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa
     5701 aaagaataaa aaaaaaatga tgaattgaaa agctaattct tgaagacgaa agggcctcgt
     5761 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     5821 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5881 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5941 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6001 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6061 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6121 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6181 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6241 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6301 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6361 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6421 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6481 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6541 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6601 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6661 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6721 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6781 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6841 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6901 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6961 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     7021 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     7081 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     7141 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     7201 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     7261 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     7321 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7381 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7441 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7501 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7561 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7621 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7681 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     7741 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     7801 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     7861 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     7921 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     7981 gcttaccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     8041 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     8101 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     8161 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     8221 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     8281 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     8341 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     8401 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgc
//
