LOCUS       sequence-407586-        8438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of yomTagBFP2-T2A-mCherry in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8438)
  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 8438)
  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-GW yomTagBFP2-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..8438
                     /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             1533..1586
                     /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      1602..1611
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1608..2318
                     /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(1753..1771)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2021..2040
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2328..2352)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2414..2433)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2415..2433)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2581..2768
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2636..2660)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2780..2797)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2780..2796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2789..2811)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3232..4578
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4689..5492)
                     /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(5493..5708)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5733..5751)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5819..5923
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5924..5992,5993..6784)
                     /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(6142..6161)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6955..7543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7444..7463
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7697..7714
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7831..7852
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7867..7872,7873..7890,7891..7897)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7905..7921
                     /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     7910..7932
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7929..7945
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7929..7945
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7975..8430)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8112..8133)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8324..8343
                     /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 atgatgagtg
      841 agttgattaa ggaaaacatg cacatgaagt tgtacatgga aggaacagta gataatcatc
      901 atttcaaatg tacgtcagaa ggtgagggca aaccttatga aggaactcaa actatgagaa
      961 ttaaagtcgt tgagggtggc ccactaccat ttgcattcga tatcctcgca acatcttttc
     1021 tgtatggttc taaaaccttt atcaaccata cacaaggcat accagatttc tttaagcaat
     1081 catttcctga aggtttcaca tgggaaagag taactaccta tgaagatggt ggtgtcctta
     1141 cagctactca agacacgtct ttgcaggatg gttgtttaat ctacaatgtt aagataagag
     1201 gagtgaactt tacatccaat ggaccagtca tgcaaaagaa aactctaggt tgggaggcat
     1261 tcacagaaac tttgtaccca gccgatgggg gcttagaagg gagaaatgac atggctttaa
     1321 aactggttgg cggtagccat ttgattgcga acgctaaaac aacctacaga agcaaaaagc
     1381 cagctaaaaa tcttaagatg cctgtatact acgtggatta cagattagaa aggaaggaag
     1441 ctaataatga aacttacgtt gaacagcacg aggtggccgt tgcccgttat tgcgaccttc
     1501 cttcaaaatt gggacacaaa ctaaactccg gagagggcag aggaagtctt ctaacatgcg
     1561 gtgacgtgga ggagaatccc ggccctgggg atccaccggt cgccaccatg gtgagcaagg
     1621 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     1681 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     1741 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     1801 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     1861 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     1921 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     1981 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     2041 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     2101 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     2161 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     2221 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     2281 gccactccac cggcggcatg gacgagctgt acaagtaaag cggccgcacc cagctttctt
     2341 gtacaaagtg gtgtagttaa ttcatgatgg ccgctgcagg tcgacctcga gggggggccc
     2401 ggtacccaat tcgccctata gtgagtcgta ttacgcgcgg atccagcttt ggacttcttc
     2461 gccagaggtt tggtcaagtc tccaatcaag gttgtcggct tgtctacctt gccagaaatt
     2521 tacgaaaaga tggaaaaggg tcaaatcgtt ggtagatacg ttgttgacac ttctaaataa
     2581 gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta
     2641 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct
     2701 ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc
     2761 tctaccggca tgccaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     2821 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     2881 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
     2941 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata atcggatcgt
     3001 acttgttacc catcattgaa ttttgaacat ccgaacctgg aagttttccc tgaaacagat
     3061 agtatatttg aacctgtata ataatatata gtctagcgct ttacggaaga caatgtatgt
     3121 atttcggttc ctggagaaac tattgcatct attgcatagg taatcttgca cgtcgcatcc
     3181 ccggttcatt ttctgcgttt ccatcttgca cttcaatagc atatctttgt taacgaagca
     3241 tctgtgcttc attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag
     3301 aatctgagct gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga
     3361 agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa
     3421 caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc
     3481 aacaaagaat ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt
     3541 ctaacaaagc atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt
     3601 gataactttt tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt
     3661 tctcttccat aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg
     3721 cgggtgcatt ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc
     3781 gcatactttg tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg
     3841 aacggtttct tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat
     3901 tgttttcgat tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac
     3961 tagagataaa cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg
     4021 tggatgggta ggttatatag ggatatagca cagagatata tagcaaagag atacttttga
     4081 gcaatgtttg tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt
     4141 tttggttttt tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt
     4201 cctatacttt ctagctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa
     4261 aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc
     4321 acctatatct gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt
     4381 ttatgcttaa atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac
     4441 ctcctgtgat attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt
     4501 tagctgttct atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat
     4561 ttcctttgat attggatcga tccgatgata agctgtcaaa catgagaatt gggtaataac
     4621 tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat
     4681 acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg
     4741 taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata
     4801 ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg
     4861 tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct
     4921 cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca
     4981 atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca tgacaattct
     5041 gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg
     5101 ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt
     5161 ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg
     5221 tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc
     5281 tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct
     5341 aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag
     5401 tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta
     5461 gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct gcatgttttt
     5521 gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg
     5581 cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt
     5641 accgaatcaa aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat
     5701 gaattgaaaa gctaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt
     5761 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5821 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5881 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5941 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6001 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6061 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6121 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     6181 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6241 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6301 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6361 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6421 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6481 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6541 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6601 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6661 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6721 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6781 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6841 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6901 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6961 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7021 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7081 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7141 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7201 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7261 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7321 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7381 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7441 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7501 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7561 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7621 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7681 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     7741 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     7801 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     7861 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     7921 atttcacaca ggaaacagct atgaccatga ttacgccaag cttaccgcat caggaaattg
     7981 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     8041 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     8101 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     8161 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     8221 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     8281 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     8341 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     8401 ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgc
//
