LOCUS       sequence-437590-        8411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  equimolar expression of yStayGold and mCherry in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8411)
  AUTHORS   Teusink B
  TITLE     Expression of FPs in budding yeast
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8411)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pDRF1 yStayGold-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..8411
                     /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             849..1499
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ivorra-Molla et al., 2023)"
                     /label=mStayGold (E138D)
                     /note="contains the monomerizing E138D mutation"
                     /translation="MASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKL
                     PMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFM
                     KNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKS
                     KCVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
     CDS             1506..1559
                     /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      1575..1584
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1581..2291
                     /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(1726..1744)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1994..2013
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2301..2325)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2387..2406)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2388..2406)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2554..2741
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2609..2633)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2753..2770)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2753..2769)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2762..2784)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3205..4551
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4662..5465)
                     /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(5466..5681)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5706..5724)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5792..5896
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5897..5965,5966..6757)
                     /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(6115..6134)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6928..7516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7417..7436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7670..7687
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7804..7825
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7840..7845,7846..7863,7864..7870)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7878..7894
                     /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     7883..7905
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7902..7918
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7902..7918
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7948..8403)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8085..8106)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8297..8316
                     /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 cccgggacta
      841 gtaagcttat ggcttctact ccattcaagt tccaattgaa gggtactatt aacggtaagt
      901 ctttcactgt tgaaggtgaa ggtgaaggta actctcatga aggttctcat aagggtaagt
      961 atgtttgtac ttctggtaag ttgccaatgt cttgggctgc tttgggtacc tctttcggtt
     1021 acggtatgaa gtactacact aagtatccat ctggtttaaa gaactggttc catgaagtta
     1081 tgccagaagg tttcacttac gatagacata ttcaatacaa gggtgatggt tctattcatg
     1141 ctaagcatca acatttcatg aagaacggta cttaccataa cattgttgaa ttcactggtc
     1201 aagattttaa ggaaaactct ccagttttga ctggtgatat gaatgtttct ttgccaaacg
     1261 atgttcaaca tattccaaga gatgatggtg ttgaatgtcc agttactttg ttgtacccat
     1321 tgttgtctga taagtctaag tgtgttgaag ctcatcaaaa cactatttgt aagccattgc
     1381 ataaccaacc agctccagat gttccatatc attggattag aaagcaatac actcaatcta
     1441 aggatgatac tgaagaaaga gatcatattt gtcaatctga aactttggaa gctcatttgt
     1501 ccggagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat cccggccctg
     1561 gggatccacc ggtcgccacc atggtgagca agggcgagga ggataacatg gccatcatca
     1621 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     1681 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     1741 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     1801 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     1861 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     1921 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     1981 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
     2041 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     2101 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     2161 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     2221 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     2281 tgtacaagta aagcggccgc acccagcttt cttgtacaaa gtggtgtagt taattcatga
     2341 tggccgctgc aggtcgacct cgaggggggg cccggtaccc aattcgccct atagtgagtc
     2401 gtattacgcg cggatccagc tttggacttc ttcgccagag gtttggtcaa gtctccaatc
     2461 aaggttgtcg gcttgtctac cttgccagaa atttacgaaa agatggaaaa gggtcaaatc
     2521 gttggtagat acgttgttga cacttctaaa taagcgaatt tcttatgatt tatgattttt
     2581 attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt
     2641 ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag
     2701 gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgccaat tcactggccg
     2761 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2821 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     2881 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     2941 tgtgcggtat ttcacaccgc ataatcggat cgtacttgtt acccatcatt gaattttgaa
     3001 catccgaacc tggaagtttt ccctgaaaca gatagtatat ttgaacctgt ataataatat
     3061 atagtctagc gctttacgga agacaatgta tgtatttcgg ttcctggaga aactattgca
     3121 tctattgcat aggtaatctt gcacgtcgca tccccggttc attttctgcg tttccatctt
     3181 gcacttcaat agcatatctt tgttaacgaa gcatctgtgc ttcattttgt agaacaaaaa
     3241 tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag
     3301 aaatgcaacg cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac
     3361 aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag
     3421 aacagaaatg caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt
     3481 tctacaaaaa tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt
     3541 ttctcctttg tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt
     3601 aaggttagaa gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc
     3661 acttcccgcg tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca
     3721 tccccgatta tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag
     3781 cgttgatgat tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata
     3841 tactacgtat aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt
     3901 cttactacaa tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg
     3961 tcgagtttag atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata
     4021 gcacagagat atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca
     4081 atattttagt agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag
     4141 agcgcttttg gttttcaaaa gcgctctgaa gttcctatac tttctagcta gagaatagga
     4201 acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg
     4261 agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat
     4321 atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta cttatatgcg
     4381 tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc cattccatgc
     4441 ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct gccactcctc
     4501 aattggatta gtctcatcct tcaatgctat catttccttt gatattggat cgatccgatg
     4561 ataagctgtc aaacatgaga attgggtaat aactgatata attaaattga agctctaatt
     4621 tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat
     4681 cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc
     4741 cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca
     4801 tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg
     4861 ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata
     4921 aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct
     4981 ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc
     5041 tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg
     5101 tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat
     5161 ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg
     5221 gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc
     5281 acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc
     5341 ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta
     5401 aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc
     5461 gacatgattt atcttcgttt cctgcatgtt tttgttctgt gcagttgggt taagaatact
     5521 gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc
     5581 tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac
     5641 cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga aaagctaatt cttgaagacg
     5701 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     5761 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5821 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5881 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5941 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6001 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6061 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6121 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6181 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6241 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6301 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6361 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6421 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6481 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6541 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6601 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6661 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6721 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6781 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6841 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6901 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6961 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7021 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7081 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7141 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7201 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7261 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7321 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7381 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7441 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7501 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7561 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     7621 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     7681 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     7741 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     7801 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     7861 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     7921 tgattacgcc aagcttaccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg
     7981 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     8041 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     8101 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     8161 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     8221 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     8281 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     8341 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     8401 cgtccattcg c
//
