LOCUS       sequence-437577-        7829 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of Cyterm-ymStayGoldd in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7829)
  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 7829)
  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 Cyterm-ymStayGold
FEATURES             Location/Qualifiers
     source          1..7829
                     /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"
     regulatory      1024..1033
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1030..1707
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     protein_bind    complement(1719..1743)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1805..1824)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1806..1824)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      1972..2159
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2027..2051)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2171..2188)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2171..2187)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2180..2202)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2623..3969
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4080..4883)
                     /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(4884..5099)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5124..5142)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5210..5314
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5315..5383,5384..6175)
                     /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(5533..5552)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6346..6934
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6835..6854
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7088..7105
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7222..7243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7258..7263,7264..7281,7282..7288)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7296..7312
                     /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     7301..7323
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7320..7336
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7320..7336
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7366..7821)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7503..7524)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7715..7734
                     /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 gctaccggac
      841 tcagatctgg taccccttgc agcttccagt tgtcaggaag agaaggctcc catggatcct
      901 gtggtggtgc tggggctctg tctctcctgt ttgcttctcc tttcactctg gaaacagagc
      961 tatgggggag ggaagcttcg aattctgcag tcgacggtac cgcgggcccg ggatccaccg
     1021 gtcgccacca tggtttctac tggtgaagaa ttgtttactg gtgttgttcc attcaagttc
     1081 caattaaagg gtactattaa cggtaagtct ttcactgttg aaggtgaagg tgaaggtaac
     1141 tctcatgaag gttctcataa gggtaagtac gtttgtactt ctggtaagtt gccaatgtct
     1201 tgggctgctt taggtacttc tttcggttac ggtatgaagt actacactaa gtacccatct
     1261 ggtttgaaga actggttcca tgaagttatg cctgaaggtt tcacttacga tagacatatt
     1321 caatacaagg gtgatggttc tattcatgct aagcatcaac atttcatgaa gaacggtact
     1381 taccataaca ttgttgaatt cactggtcaa gatttcaagg aaaactcccc agttttgact
     1441 ggtgatatgg atgtttcttt gccaaacgaa gttcaacata ttccaattga tgatggtgtt
     1501 gaatgtactg ttactttgca atacccattg ttgtctgatg aatctaagtg tgttgaagcc
     1561 taccaaaata ctattattaa gccattgcat aaccaaccag ctccagatgt tccattccac
     1621 tggattagaa aacaatacac tcaatctaag gatgatactg aagaaagaga tcatattatt
     1681 caatctgaaa ctttggaagc tcatttgtaa gcggccgcac ccagctttct tgtacaaagt
     1741 ggtgtagtta attcatgatg gccgctgcag gtcgacctcg agggggggcc cggtacccaa
     1801 ttcgccctat agtgagtcgt attacgcgcg gatccagctt tggacttctt cgccagaggt
     1861 ttggtcaagt ctccaatcaa ggttgtcggc ttgtctacct tgccagaaat ttacgaaaag
     1921 atggaaaagg gtcaaatcgt tggtagatac gttgttgaca cttctaaata agcgaatttc
     1981 ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt atacaaattt
     2041 taaagtgact cttaggtttt aaaacgaaaa ttcttattct tgagtaactc tttcctgtag
     2101 gtcaggttgc tttctcaggt atagcatgag gtcgctctta ttgaccacac ctctaccggc
     2161 atgccaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     2221 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     2281 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     2341 attttctcct tacgcatctg tgcggtattt cacaccgcat aatcggatcg tacttgttac
     2401 ccatcattga attttgaaca tccgaacctg ggagttttcc ctgaaacaga tagtatattt
     2461 gaacctgtat aataatatat agtctagcgc tttacggaag acaatgtatg tatttcggtt
     2521 cctggagaaa ctattgcatc tattgcatag gtaatcttgc acgtcgcatc cccggttcat
     2581 tttctgcgtt tccatcttgc acttcaatag catatctttg ttaacgaagc atctgtgctt
     2641 cattttgtag aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc
     2701 tgcattttta cagaacagaa atgcaacgcg aaagcgctat tttaccaacg aagaatctgt
     2761 gcttcatttt tgtaaaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc
     2821 tgagctgcat ttttacagaa cagaaatgca acgcgagagc gctattttac caacaaagaa
     2881 tctatacttc ttttttgttc tacaaaaatg catcccgaga gcgctatttt tctaacaaag
     2941 catcttagat tacttttttt ctcctttgtg cgctctataa tgcagtctct tgataacttt
     3001 ttgcactgta ggtccgttaa ggttagaaga aggctacttt ggtgtctatt ttctcttcca
     3061 taaaaaaagc ctgactccac ttcccgcgtt tactgattac tagcgaagct gcgggtgcat
     3121 tttttcaaga taaaggcatc cccgattata ttctataccg atgtggattg cgcatacttt
     3181 gtgaacagaa agtgatagcg ttgatgattc ttcattggtc agaaaattat gaacggtttc
     3241 ttctattttg tctctatata ctacgtatag gaaatgttta cattttcgta ttgttttcga
     3301 ttcactctat gaatagttct tactacaatt tttttgtcta aagagtaata ctagagataa
     3361 acataaaaaa tgtagaggtc gagtttagat gcaagttcaa ggagcgaaag gtggatgggt
     3421 aggttatata gggatatagc acagagatat atagcaaaga gatacttttg agcaatgttt
     3481 gtggaagcgg tattcgcaat attttagtag ctcgttacag tccggtgcgt ttttggtttt
     3541 ttgaaagtgc gtcttcagag cgcttttggt tttcaaaagc gctctgaagt tcctatactt
     3601 tctagctaga gaataggaac ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc
     3661 ttccgaaaat gcaacgcgag ctgcgcacat acagctcact gttcacgtcg cacctatatc
     3721 tgcgtgttgc ctgtatatat atatacatga gaagaacggc atagtgcgtg tttatgctta
     3781 aatgcgtact tatatgcgtc tatttatgta ggatgaaagg tagtctagta cctcctgtga
     3841 tattatccca ttccatgcgg ggtatcgtat gcttccttca gcactaccct ttagctgttc
     3901 tatatgctgc cactcctcaa ttggattagt ctcatccttc aatgctatca tttcctttga
     3961 tattggatcg atccgatgat aagctgtcaa acatgagaat tgggtaataa ctgatataat
     4021 taaattgaag ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt
     4081 tagttttgct ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca
     4141 ccctctacct tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca
     4201 gatcctgtag agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg
     4261 tcatctaaac ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc
     4321 atgtctcttt gagcaataaa gccgataaca aaatctttgt cgctcttcgc aatgtcaaca
     4381 gtacccttag tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc
     4441 aaaaggcctc taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata
     4501 cctgggccca ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca
     4561 cccgcagagt actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag
     4621 agtaaaaaat tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa
     4681 aaatcagtca agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca
     4741 actaactcca gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc
     4801 ttttcgtgca tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt
     4861 tccttatatg tagctttcga catgatttat cttcgtttcc tgcatgtttt tgttctgtgc
     4921 agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat
     4981 accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca
     5041 aaaaaatttc aaggaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaaa
     5101 agctaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5161 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5221 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5281 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5341 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5401 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5461 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5521 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5581 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5641 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5701 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5761 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5821 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5881 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5941 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6001 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6061 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6121 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6181 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6241 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6301 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6361 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6421 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6481 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6541 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6601 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6661 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6721 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6781 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6841 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6901 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6961 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     7021 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     7081 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     7141 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     7201 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     7261 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     7321 aggaaacagc tatgaccatg attacgccaa gcttaccgca tcaggaaatt gtaagcgtta
     7381 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     7441 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     7501 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     7561 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     7621 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     7681 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     7741 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     7801 atgcgccgct acagggcgcg tccattcgc
//
