LOCUS       sequence-332113-        8487 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces equimolar expression of ystagRFP and mCherry for use in 
            yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8487)
  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 8487)
  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: ystagRFP-T2A-mTq2 pdrf1-GW
FEATURES             Location/Qualifiers
     source          1..8487
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    89..110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(125..130,131..148,149..155)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..179
                     /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     168..190
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     187..203
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     187..203
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(233..688)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(370..391)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     582..601
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1496..1520
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1525..1534
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2269..2322
                     /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      2338..2347
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2344..2346,2347..2349,2350..3063)
                     /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(2389..2410)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2650..2669)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2997..3018
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3073..3097)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3159..3178)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3160..3178)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3326..3513
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3381..3405)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3525..3542)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3525..3541)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3534..3556)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3977..5323
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(5434..6237)
                     /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(6238..6453)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6478..6496)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6564..6668
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6669..6737,6738..7529)
                     /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(6887..6906)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7700..8288
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8189..8208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8442..8459
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
       61 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
      121 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      181 ttcacacagg aaacagctat gaccatgatt acgccaagct taccgcatca ggaaattgta
      241 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
      301 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
      361 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
      421 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
      481 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
      541 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
      601 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
      661 gcgcttaatg cgccgctaca gggcgcgtcc attcgccaag cttcctgaaa cggagaaaca
      721 taaacaggca ttgctgggat cacccataca tcactctgtt ttgcctgacc ttttccggta
      781 atttgaaaac aaacccggtc tcgaagcgga gatccggcga taattaccgc agaaataaac
      841 ccatacacga gacgtagaac cagccgcaca tggccggaga aactcctgcg agaatttcgt
      901 aaactcgcgc gcattgcatc tgtatttcct aatgcggcac ttccaggcct cgatcgagac
      961 cgtttatcca ttgctttttt gttgtctttt tccctcgttc acagaaagtc tgaagaagct
     1021 atagtagaac tatgagcttt ttttgtttct gttttccttt tttttttttt tacctctgtg
     1081 gaaattgtta ctctcacact ctttagttcg tttgtttgtt ttgtttattc caattatgac
     1141 cggtgacgaa acgtggtcga tggtgggtac cgcttatgct cccctccatt agtttcgatt
     1201 atataaaaag gccaaatatt gtattatttt caaatgtcct atcattatcg tctaacatct
     1261 aatttctctt aaattttttc tctttctttc ctataacacc aatagtgaaa atcttttttt
     1321 cttctatatc tacaaaaact ttttttttct atcaacctcg ttgataaatt ttttctttaa
     1381 caatcgttaa taattaatta attggaaaat aaccattttt tctctctttt atacacacat
     1441 tcaaaagaaa gaaaaaaaat ataccccagc ctcgactagt gaattcgcgg ccatcacaag
     1501 tttgtacaaa aaagcaggct gctagccacc atggtatcta aaggtgaaga gttgataaag
     1561 gaaaatatgc acatgaaact ctatatggaa ggcacagtga ataaccatca ttttaagtgt
     1621 acttcagaag gagaaggtaa gccttatgag gggacacaaa ctatgagaat taaagttgtt
     1681 gaaggaggtc cactaccatt cgcttttgac atcctggcaa cgagcttcat gtatggctcc
     1741 agaacattca taaaccatac acaaggtatt ccagatttct tcaaacaaag ttttccagaa
     1801 ggctttactt gggagagagt tactacctac gaggacgggg gtgttttaac tgcgacacag
     1861 gatacatcat tacaagatgg ctgtttgatc tacaacgtaa aaatcagagg tgtcaatttt
     1921 ccatctaacg gacctgttat gcagaaaaag accttgggtt gggaggctaa tactgaaatg
     1981 ctatacccag cagatggagg attggaaggt aggacagtca tggcattgaa acttgtagga
     2041 ggcggtcacc taatttgcaa ctttaagaca acctacagaa gcaaaaagcc tgctaaaaat
     2101 cttaaaatgc caggggtgta ctacgtggat catagacttg aacgtatcaa ggaagctgac
     2161 aaagagactt acgttgaaca acacgaagtc gccgtcgcca gatactgtga cttaccttca
     2221 aagttaggtc ataaactgaa tggtatggat gaattgtata agtccggaga gggcagagga
     2281 agtcttctaa catgcggtga cgtggaggag aatcccggcc ctggggatcc accggtcgcc
     2341 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2401 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2461 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2521 accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg
     2581 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2641 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2701 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2761 cacaagctgg agtacaacta ctttagcgac aacgtctata tcaccgccga caagcagaag
     2821 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
     2881 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2941 cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg
     3001 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3061 taaagcggcc gcacccagct ttcttgtaca aagtggtgta gttaattcat gatggccgct
     3121 gcaggtcgac ctcgaggggg ggcccggtac ccaattcgcc ctatagtgag tcgtattacg
     3181 cgcggatcca gctttggact tcttcgccag aggtttggtc aagtctccaa tcaaggttgt
     3241 cggcttgtct accttgccag aaatttacga aaagatggaa aagggtcaaa tcgttggtag
     3301 atacgttgtt gacacttcta aataagcgaa tttcttatga tttatgattt ttattattaa
     3361 ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg
     3421 aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca
     3481 tgaggtcgct cttattgacc acacctctac cggcatgcca attcactggc cgtcgtttta
     3541 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     3601 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     3661 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3721 atttcacacc gcataatcgg atcgtacttg ttacccatca ttgaattttg aacatccgaa
     3781 cctgggagtt ttccctgaaa cagatagtat atttgaacct gtataataat atatagtcta
     3841 gcgctttacg gaagacaatg tatgtatttc ggttcctgga gaaactattg catctattgc
     3901 ataggtaatc ttgcacgtcg catccccggt tcattttctg cgtttccatc ttgcacttca
     3961 atagcatatc tttgttaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc
     4021 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa
     4081 cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc
     4141 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
     4201 tgcaacgcga gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa
     4261 aatgcatccc gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt
     4321 tgtgcgctct ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag
     4381 aagaaggcta ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg
     4441 cgtttactga ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat
     4501 tatattctat accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg
     4561 attcttcatt ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt
     4621 ataggaaatg tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac
     4681 aatttttttg tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt
     4741 agatgcaagt tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag
     4801 atatatagca aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta
     4861 gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt
     4921 tggttttcaa aagcgctctg aagttcctat actttctagc tagagaatag gaacttcgga
     4981 ataggaactt caaagcgttt ccgaaaacga gcgcttccga aaatgcaacg cgagctgcgc
     5041 acatacagct cactgttcac gtcgcaccta tatctgcgtg ttgcctgtat atatatatac
     5101 atgagaagaa cggcatagtg cgtgtttatg cttaaatgcg tacttatatg cgtctattta
     5161 tgtaggatga aaggtagtct agtacctcct gtgatattat cccattccat gcggggtatc
     5221 gtatgcttcc ttcagcacta ccctttagct gttctatatg ctgccactcc tcaattggat
     5281 tagtctcatc cttcaatgct atcatttcct ttgatattgg atcgatccga tgataagctg
     5341 tcaaacatga gaattgggta ataactgata taattaaatt gaagctctaa tttgtgagtt
     5401 tagtatacat gcatttactt ataatacagt tttttagttt tgctggccgc atcttctcaa
     5461 atatgcttcc cagcctgctt ttctgtaacg ttcaccctct accttagcat cccttccctt
     5521 tgcaaatagt cctcttccaa caataataat gtcagatcct gtagagacca catcatccac
     5581 ggttctatac tgttgaccca atgcgtctcc cttgtcatct aaacccacac cgggtgtcat
     5641 aatcaaccaa tcgtaacctt catctcttcc acccatgtct ctttgagcaa taaagccgat
     5701 aacaaaatct ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga
     5761 tagggagccc ttgcatgaca attctgctaa catcaaaagg cctctaggtt cctttgttac
     5821 ttcttctgcc gcctgcttca aaccgctaac aatacctggg cccaccacac cgtgtgcatt
     5881 cgtaatgtct gcccattctg ctattctgta tacacccgca gagtactgca atttgactgt
     5941 attaccaatg tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact tggcggataa
     6001 tgcctttagc ggcttaactg tgccctccat ggaaaaatca gtcaagatat ccacatgtgt
     6061 ttttagtaaa caaattttgg gacctaatgc ttcaactaac tccagtaatt ccttggtggt
     6121 acgaacatcc aatgaagcac acaagtttgt ttgcttttcg tgcatgatat taaatagctt
     6181 ggcagcaaca ggactaggat gagtagcagc acgttcctta tatgtagctt tcgacatgat
     6241 ttatcttcgt ttcctgcatg tttttgttct gtgcagttgg gttaagaata ctgggcaatt
     6301 tcatgtttct tcaacactac atatgcgtat atataccaat ctaagtctgt gctccttcct
     6361 tcgttcttcc ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa accgaaatca
     6421 aaaaaaagaa taaaaaaaaa atgatgaatt gaaaagctaa ttcttgaaga cgaaagggcc
     6481 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     6541 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     6601 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     6661 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     6721 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     6781 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6841 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6901 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6961 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     7021 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     7081 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     7141 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     7201 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     7261 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     7321 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     7381 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     7441 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     7501 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     7561 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     7621 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     7681 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     7741 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     7801 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     7861 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     7921 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     7981 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     8041 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     8101 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     8161 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     8221 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     8281 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     8341 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     8401 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     8461 aagcggaaga gcgcccaata cgcaaac
//
