LOCUS       sequence-246506-        8460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces equimolar expression of yeast codon-optimized sfGFP and 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8460)
  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 8460)
  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 yosfGFP-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..8460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    197..221
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             952..1005
                     /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      1021..1030
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1027..1737
                     /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(1172..1190)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1440..1459
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1747..1771)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1833..1852)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1834..1852)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2000..2187
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2055..2079)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2199..2216)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2199..2215)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2208..2230)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2651..3997
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4108..4911)
                     /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(4912..5127)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5152..5170)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5238..5342
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5343..5411,5412..6203)
                     /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(5561..5580)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6374..6962
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6863..6882
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7116..7133
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7250..7271
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7286..7291,7292..7309,7310..7316)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7324..7340
                     /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     7329..7351
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7348..7364
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7348..7364
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7394..7849)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7531..7552)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7743..7762
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 caatagtgaa aatctttttt tcttctatat ctacaaaaac tttttttttc tatcaacctc
       61 gttgataaat tttttcttta acaatcgtta ataattaatt aattggaaaa taaccatttt
      121 ttctctcttt tatacacaca ttcaaaagaa agaaaaaaaa tataccccag cctcgactag
      181 tgaattcgcg gccatcacaa gtttgtacaa aaaagcaggc tgctagccac catgtctaaa
      241 ggcgaggaat tgtttacagg tgtagttcct attttggtcg aactagatgg agatgtcaac
      301 ggtcataagt tttctgtacg tggtgaaggc gaaggtgatg ctactaatgg caaactgact
      361 ctgaagttca tctgtacgac cggaaaattg ccagttccat ggcctacatt agtaacaaca
      421 ttaacctacg gagttcaatg cttttcaaga tacccagatc acatgaagca gcatgacttt
      481 ttcaaaagtg ctatgccaga aggttatgtg caggagagaa ctatctcctt caaagatgat
      541 ggtacttata agacaagagc agaggtaaag tttgaaggcg atacattggt taatagaatt
      601 gagcttaaag gcattgattt taaggaagat gggaatatcc tcggacataa acttgaatac
      661 aatttcaatt cacataatgt ttatatcaca gcagataaac aaaagaatgg tataaaagcg
      721 aactttaaga ttagacataa cgtggaagat ggtagcgtcc aattagccga ccattaccaa
      781 caaaacactc ctatcgggga cgggccagtt ctccttccag acaaccacta cctatcaacc
      841 caatctgttc tatctaaaga ccctaatgaa aaacgagatc acatggtctt gttagagttc
      901 gtgactgccg ctggtataac attaggaatg gacgaattgt acaaatccgg agagggcaga
      961 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctgggga tccaccggtc
     1021 gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     1081 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1141 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     1201 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     1261 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     1321 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     1381 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1441 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     1501 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     1561 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     1621 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     1681 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaaagc
     1741 ggccgcaccc agctttcttg tacaaagtgg tgtagttaat tcatgatggc cgctgcaggt
     1801 cgacctcgag ggggggcccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgga
     1861 tccagctttg gacttcttcg ccagaggttt ggtcaagtct ccaatcaagg ttgtcggctt
     1921 gtctaccttg ccagaaattt acgaaaagat ggaaaagggt caaatcgttg gtagatacgt
     1981 tgttgacact tctaaataag cgaatttctt atgatttatg atttttatta ttaaataagt
     2041 tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt
     2101 cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat agcatgaggt
     2161 cgctcttatt gaccacacct ctaccggcat gccaattcac tggccgtcgt tttacaacgt
     2221 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     2281 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     2341 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     2401 caccgcataa tcggatcgta cttgttaccc atcattgaat tttgaacatc cgaacctggg
     2461 agttttccct gaaacagata gtatatttga acctgtataa taatatatag tctagcgctt
     2521 tacggaagac aatgtatgta tttcggttcc tggagaaact attgcatcta ttgcataggt
     2581 aatcttgcac gtcgcatccc cggttcattt tctgcgtttc catcttgcac ttcaatagca
     2641 tatctttgtt aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc
     2701 gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgaa
     2761 agcgctattt taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg
     2821 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac
     2881 gcgagagcgc tattttacca acaaagaatc tatacttctt ttttgttcta caaaaatgca
     2941 tcccgagagc gctatttttc taacaaagca tcttagatta ctttttttct cctttgtgcg
     3001 ctctataatg cagtctcttg ataacttttt gcactgtagg tccgttaagg ttagaagaag
     3061 gctactttgg tgtctatttt ctcttccata aaaaaagcct gactccactt cccgcgttta
     3121 ctgattacta gcgaagctgc gggtgcattt tttcaagata aaggcatccc cgattatatt
     3181 ctataccgat gtggattgcg catactttgt gaacagaaag tgatagcgtt gatgattctt
     3241 cattggtcag aaaattatga acggtttctt ctattttgtc tctatatact acgtatagga
     3301 aatgtttaca ttttcgtatt gttttcgatt cactctatga atagttctta ctacaatttt
     3361 tttgtctaaa gagtaatact agagataaac ataaaaaatg tagaggtcga gtttagatgc
     3421 aagttcaagg agcgaaaggt ggatgggtag gttatatagg gatatagcac agagatatat
     3481 agcaaagaga tacttttgag caatgtttgt ggaagcggta ttcgcaatat tttagtagct
     3541 cgttacagtc cggtgcgttt ttggtttttt gaaagtgcgt cttcagagcg cttttggttt
     3601 tcaaaagcgc tctgaagttc ctatactttc tagctagaga ataggaactt cggaatagga
     3661 acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac
     3721 agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga
     3781 agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg
     3841 atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc
     3901 ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct
     3961 catccttcaa tgctatcatt tcctttgata ttggatcgat ccgatgataa gctgtcaaac
     4021 atgagaattg ggtaataact gatataatta aattgaagct ctaatttgtg agtttagtat
     4081 acatgcattt acttataata cagtttttta gttttgctgg ccgcatcttc tcaaatatgc
     4141 ttcccagcct gcttttctgt aacgttcacc ctctacctta gcatcccttc cctttgcaaa
     4201 tagtcctctt ccaacaataa taatgtcaga tcctgtagag accacatcat ccacggttct
     4261 atactgttga cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg tcataatcaa
     4321 ccaatcgtaa ccttcatctc ttccacccat gtctctttga gcaataaagc cgataacaaa
     4381 atctttgtcg ctcttcgcaa tgtcaacagt acccttagta tattctccag tagataggga
     4441 gcccttgcat gacaattctg ctaacatcaa aaggcctcta ggttcctttg ttacttcttc
     4501 tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg cattcgtaat
     4561 gtctgcccat tctgctattc tgtatacacc cgcagagtac tgcaatttga ctgtattacc
     4621 aatgtcagca aattttctgt cttcgaagag taaaaaattg tacttggcgg ataatgcctt
     4681 tagcggctta actgtgccct ccatggaaaa atcagtcaag atatccacat gtgtttttag
     4741 taaacaaatt ttgggaccta atgcttcaac taactccagt aattccttgg tggtacgaac
     4801 atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg atattaaata gcttggcagc
     4861 aacaggacta ggatgagtag cagcacgttc cttatatgta gctttcgaca tgatttatct
     4921 tcgtttcctg catgtttttg ttctgtgcag ttgggttaag aatactgggc aatttcatgt
     4981 ttcttcaaca ctacatatgc gtatatatac caatctaagt ctgtgctcct tccttcgttc
     5041 ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa
     5101 agaataaaaa aaaaatgatg aattgaaaag ctaattcttg aagacgaaag ggcctcgtga
     5161 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     5221 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5281 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5341 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5401 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5461 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5521 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5581 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5641 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5701 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5761 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5821 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5881 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5941 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6001 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6061 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6121 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6181 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6241 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6301 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6361 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6421 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6481 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6541 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6601 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6661 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6721 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6781 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6841 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6901 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6961 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     7021 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     7081 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     7141 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     7201 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     7261 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     7321 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     7381 ttaccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     7441 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     7501 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     7561 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     7621 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     7681 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     7741 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     7801 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgccaa
     7861 gcttcctgaa acggagaaac ataaacaggc attgctggga tcacccatac atcactctgt
     7921 tttgcctgac cttttccggt aatttgaaaa caaacccggt ctcgaagcgg agatccggcg
     7981 ataattaccg cagaaataaa cccatacacg agacgtagaa ccagccgcac atggccggag
     8041 aaactcctgc gagaatttcg taaactcgcg cgcattgcat ctgtatttcc taatgcggca
     8101 cttccaggcc tcgatcgaga ccgtttatcc attgcttttt tgttgtcttt ttccctcgtt
     8161 cacagaaagt ctgaagaagc tatagtagaa ctatgagctt tttttgtttc tgttttcctt
     8221 tttttttttt ttacctctgt ggaaattgtt actctcacac tctttagttc gtttgtttgt
     8281 tttgtttatt ccaattatga ccggtgacga aacgtggtcg atggtgggta ccgcttatgc
     8341 tcccctccat tagtttcgat tatataaaaa ggccaaatat tgtattattt tcaaatgtcc
     8401 tatcattatc gtctaacatc taatttctct taaatttttt ctctttcttt cctataacac
//
