LOCUS       sequence-407617-        8449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of yoTagRFP657-T2A-mtq2 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8449)
  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 8449)
  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 yoTagRFP657-T2A-mtq2
FEATURES             Location/Qualifiers
     source          1..8449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    799..823
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1535..1588
                     /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      1604..1613
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1610..1612,1613..1615,1616..2329)
                     /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(1655..1676)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1916..1935)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2263..2284
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2339..2363)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2425..2444)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2426..2444)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2592..2779
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2647..2671)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2791..2808)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2791..2807)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2800..2822)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3243..4589
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4700..5503)
                     /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(5504..5719)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5744..5762)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5830..5934
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5935..6003,6004..6795)
                     /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(6153..6172)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6966..7554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7455..7474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7708..7725
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7842..7863
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7878..7883,7884..7901,7902..7908)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7916..7932
                     /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     7921..7943
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7940..7956
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7940..7956
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7986..8441)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8123..8144)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8335..8354
                     /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 ttttacctct gtggaaattg ttactctcac actctttagt tcgtttgttt
      421 gttttgttta ttccaattat gaccggtgac gaaacgtggt cgatggtggg taccgcttat
      481 gctcccctcc attagtttcg attatataaa aaggccaaat attgtattat tttcaaatgt
      541 cctatcatta tcgtctaaca tctaatttct cttaaatttt ttctctttct ttcctataac
      601 accaatagtg aaaatctttt tttcttctat atctacaaaa actttttttt tctatcaacc
      661 tcgttgataa attttttctt taacaatcgt taataattaa ttaattggaa aataaccatt
      721 ttttctctct tttatacaca cattcaaaag aaagaaaaaa aatatacccc agcctcgact
      781 agtgaattcg cggccatcac aagtttgtac aaaaaagcag gctgctagca tgagcgaatt
      841 gatcacagaa aatatgcaca tgaagttgta tatggaggga accgtcaata accatcatgt
      901 caaatgcact tctgaaggtg agggcaaacc ttacgaaggg acccaaacac agagaattaa
      961 ggtagtagaa gggggtccac tgccatttgc ctttgacatc ctagcaacat ccttcatgta
     1021 cggttcccac acattcataa accacactca aggaatacca gacttttgga agcagagttt
     1081 tccagaaggt tttacgtggg aaagagttac tacttacgag gacggtggtg tattaacagc
     1141 cacacaagat acatcattac aagatggttg tctgatctac aacgttaaaa tcagaggtgt
     1201 taactttcct tcaaatgggc cagttatgca aaagaaaact ttaggctggg aggcccatac
     1261 tgaaatgctt tacccagcag atggcggctt ggaaggcagg acggctttgg ctttaaagct
     1321 agttggtgga ggacatttga tttgtaattt caaaacaact tacagaagca aaaagcctgc
     1381 taaaaatctt aagatgccag gagtgtacta tgtcgattac agattggaac gtattaaaga
     1441 ggctgataag gaaacctatg tggaacaaca tgaagtggca gtcgcgagat attgtgatct
     1501 tccttctaaa ctaggtcata aactcaattc cggagagggc agaggaagtc ttctaacatg
     1561 cggtgacgtg gaggagaatc ccggccctgg ggatccaccg gtcgccacca tggtgagcaa
     1621 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1681 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1741 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1801 cctgtcctgg ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     1861 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     1921 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1981 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2041 caactacttt agcgacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc
     2101 caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca
     2161 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     2221 ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2281 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcac
     2341 ccagctttct tgtacaaagt ggtgtagtta attcatgatg gccgctgcag gtcgacctcg
     2401 agggggggcc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg gatccagctt
     2461 tggacttctt cgccagaggt ttggtcaagt ctccaatcaa ggttgtcggc ttgtctacct
     2521 tgccagaaat ttacgaaaag atggaaaagg gtcaaatcgt tggtagatac gttgttgaca
     2581 cttctaaata agcgaatttc ttatgattta tgatttttat tattaaataa gttataaaaa
     2641 aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa ttcttattct
     2701 tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagcatgag gtcgctctta
     2761 ttgaccacac ctctaccggc atgccaattc actggccgtc gttttacaac gtcgtgactg
     2821 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     2881 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     2941 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3001 aatcggatcg tacttgttac ccatcattga attttgaaca tccgaacctg ggagttttcc
     3061 ctgaaacaga tagtatattt gaacctgtat aataatatat agtctagcgc tttacggaag
     3121 acaatgtatg tatttcggtt cctggagaaa ctattgcatc tattgcatag gtaatcttgc
     3181 acgtcgcatc cccggttcat tttctgcgtt tccatcttgc acttcaatag catatctttg
     3241 ttaacgaagc atctgtgctt cattttgtag aacaaaaatg caacgcgaga gcgctaattt
     3301 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg aaagcgctat
     3361 tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa aaatgcaacg cgagagcgct
     3421 aatttttcaa acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgagagc
     3481 gctattttac caacaaagaa tctatacttc ttttttgttc tacaaaaatg catcccgaga
     3541 gcgctatttt tctaacaaag catcttagat tacttttttt ctcctttgtg cgctctataa
     3601 tgcagtctct tgataacttt ttgcactgta ggtccgttaa ggttagaaga aggctacttt
     3661 ggtgtctatt ttctcttcca taaaaaaagc ctgactccac ttcccgcgtt tactgattac
     3721 tagcgaagct gcgggtgcat tttttcaaga taaaggcatc cccgattata ttctataccg
     3781 atgtggattg cgcatacttt gtgaacagaa agtgatagcg ttgatgattc ttcattggtc
     3841 agaaaattat gaacggtttc ttctattttg tctctatata ctacgtatag gaaatgttta
     3901 cattttcgta ttgttttcga ttcactctat gaatagttct tactacaatt tttttgtcta
     3961 aagagtaata ctagagataa acataaaaaa tgtagaggtc gagtttagat gcaagttcaa
     4021 ggagcgaaag gtggatgggt aggttatata gggatatagc acagagatat atagcaaaga
     4081 gatacttttg agcaatgttt gtggaagcgg tattcgcaat attttagtag ctcgttacag
     4141 tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag cgcttttggt tttcaaaagc
     4201 gctctgaagt tcctatactt tctagctaga gaataggaac ttcggaatag gaacttcaaa
     4261 gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat acagctcact
     4321 gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga gaagaacggc
     4381 atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta ggatgaaagg
     4441 tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat gcttccttca
     4501 gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt ctcatccttc
     4561 aatgctatca tttcctttga tattggatcg atccgatgat aagctgtcaa acatgagaat
     4621 tgggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat
     4681 ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc
     4741 ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca aatagtcctc
     4801 ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt
     4861 gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt
     4921 aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt
     4981 cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc
     5041 atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct
     5101 gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta atgtctgccc
     5161 attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag
     5221 caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct
     5281 taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt agtaaacaaa
     5341 ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg
     5401 aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac
     5461 taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc
     5521 tgcatgtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa
     5581 cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct
     5641 gttcggagat taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa aaagaataaa
     5701 aaaaaaatga tgaattgaaa agctaattct tgaagacgaa agggcctcgt gatacgccta
     5761 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5821 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5881 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5941 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6001 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6061 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6121 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6181 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6241 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6301 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6361 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6421 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6481 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6541 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6601 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6661 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6721 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6781 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6841 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6901 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6961 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7021 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7081 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7141 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7201 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7261 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7321 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7381 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7441 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7501 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7561 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7621 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7681 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7741 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     7801 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     7861 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     7921 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcttaccgca
     7981 tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     8041 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     8101 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     8161 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     8221 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     8281 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     8341 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     8401 caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgc
//
