LOCUS       sequence-245964-        8470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces equimolar expression of yeast codon optimized mNeongreen 
            and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8470)
  AUTHORS   Botman D, de Groot DH, Schmidt P, Goedhart J, Teusink B
  TITLE     In vivo characterisation of fluorescent proteins in budding yeast.
  JOURNAL   Sci Rep. 2019 Feb 19;9(1):2234. doi: 10.1038/s41598-019-38913-z.
  PUBMED    30783202
REFERENCE   2  (bases 1 to 8470)
  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: "Sci Rep."; 
            date: "2019-02-19"; volume: "9(1):2234. doi"; pages: " 
            10.1038/s41598-019-38913-z"
COMMENT     Sequence Label: pDRF1-GW ymNeongreen-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..8470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    252..276
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      558..567
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1017..1070
                     /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      1086..1095
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1092..1802
                     /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(1237..1255)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1505..1524
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1812..1836)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1898..1917)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1899..1917)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2065..2252
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2120..2144)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2264..2281)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2264..2280)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2273..2295)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2716..4062
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4173..4976)
                     /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(4977..5192)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5217..5235)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5303..5407
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5408..5476,5477..6268)
                     /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(5626..5645)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6439..7027
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6928..6947
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7181..7198
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7315..7336
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7351..7356,7357..7374,7375..7381)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7389..7405
                     /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     7394..7416
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7413..7429
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7413..7429
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7459..7914)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7596..7617)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7808..7827
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttatcgtcta acatctaatt tctcttaaat tttttctctt tctttcctat aacaccaata
       61 gtgaaaatct ttttttcttc tatatctaca aaaacttttt ttttctatca acctcgttga
      121 taaatttttt ctttaacaat cgttaataat taattaattg gaaaataacc attttttctc
      181 tcttttatac acacattcaa aagaaagaaa aaaaatatac cccagcctcg actagtgaat
      241 tcgcggccat cacaagtttg tacaaaaaag caggctgcta gcactagtaa gcttttaatt
      301 aaatggtctc taagggtgaa gaagataaca tggcttcttt gccagccacc cacgagttgc
      361 acatcttcgg ttccattaac ggtgtcgact tcgatatggt cggtcaaggt accggtaacc
      421 caaacgacgg ttacgaagaa ttgaacttga agtctaccaa gggcgacttg cagttctccc
      481 cttggatttt ggttccacac atcggttacg gtttccacca atacttgcca tacccagacg
      541 gtatgtcccc attccaagcc gccatggttg acggttctgg ttaccaagtc cacagaacca
      601 tgcaattcga agacggtgct tccttgaccg tgaactacag atacacctac gaaggttctc
      661 acatcaaggg cgaagcccaa gttaagggta ccggtttccc tgctgacggt ccagtcatga
      721 ctaactcctt gaccgctgct gactggtgtc gttccaagaa gacctaccca aacgataaga
      781 ccatcatttc cactttcaag tggtcctaca ccaccggtaa cggcaagaga taccgctcta
      841 ccgctcgtac cacctacacc ttcgccaagc caatggctgc caactacttg aagaaccagc
      901 ctatgtacgt cttccgtaag accgagctga agcattccaa gactgaattg aacttcaagg
      961 aatggcaaaa ggctttcacc gacgtcatgg gtatggacga attgtacaag tccggagagg
     1021 gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct ggggatccac
     1081 cggtcgccac catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     1141 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1201 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     1261 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     1321 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     1381 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     1441 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     1501 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     1561 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     1621 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     1681 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     1741 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     1801 aaagcggccg cacccagctt tcttgtacaa agtggtgtag ttaattcatg atggccgctg
     1861 caggtcgacc tcgagggggg gcccggtacc caattcgccc tatagtgagt cgtattacgc
     1921 gcggatccag ctttggactt cttcgccaga ggtttggtca agtctccaat caaggttgtc
     1981 ggcttgtcta ccttgccaga aatttacgaa aagatggaaa agggtcaaat cgttggtaga
     2041 tacgttgttg acacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa
     2101 taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga
     2161 aaattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagcat
     2221 gaggtcgctc ttattgacca cacctctacc ggcatgccaa ttcactggcc gtcgttttac
     2281 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     2341 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     2401 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     2461 tttcacaccg cataatcgga tcgtacttgt tacccatcat tgaattttga acatccgaac
     2521 ctgggagttt tccctgaaac agatagtata tttgaacctg tataataata tatagtctag
     2581 cgctttacgg aagacaatgt atgtatttcg gttcctggag aaactattgc atctattgca
     2641 taggtaatct tgcacgtcgc atccccggtt cattttctgc gtttccatct tgcacttcaa
     2701 tagcatatct ttgttaacga agcatctgtg cttcattttg tagaacaaaa atgcaacgcg
     2761 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac
     2821 gcgaaagcgc tattttacca acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca
     2881 acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat
     2941 gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg ttctacaaaa
     3001 atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt tttctccttt
     3061 gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt taaggttaga
     3121 agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc cacttcccgc
     3181 gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc atccccgatt
     3241 atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga
     3301 ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat atactacgta
     3361 taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt tcttactaca
     3421 atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag gtcgagttta
     3481 gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat agcacagaga
     3541 tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc aatattttag
     3601 tagctcgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca gagcgctttt
     3661 ggttttcaaa agcgctctga agttcctata ctttctagct agagaatagg aacttcggaa
     3721 taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca
     3781 catacagctc actgttcacg tcgcacctat atctgcgtgt tgcctgtata tatatataca
     3841 tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt acttatatgc gtctatttat
     3901 gtaggatgaa aggtagtcta gtacctcctg tgatattatc ccattccatg cggggtatcg
     3961 tatgcttcct tcagcactac cctttagctg ttctatatgc tgccactcct caattggatt
     4021 agtctcatcc ttcaatgcta tcatttcctt tgatattgga tcgatccgat gataagctgt
     4081 caaacatgag aattgggtaa taactgatat aattaaattg aagctctaat ttgtgagttt
     4141 agtatacatg catttactta taatacagtt ttttagtttt gctggccgca tcttctcaaa
     4201 tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc ccttcccttt
     4261 gcaaatagtc ctcttccaac aataataatg tcagatcctg tagagaccac atcatccacg
     4321 gttctatact gttgacccaa tgcgtctccc ttgtcatcta aacccacacc gggtgtcata
     4381 atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat aaagccgata
     4441 acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc tccagtagat
     4501 agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc ctttgttact
     4561 tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc gtgtgcattc
     4621 gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa tttgactgta
     4681 ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt ggcggataat
     4741 gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc cacatgtgtt
     4801 tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc cttggtggta
     4861 cgaacatcca atgaagcaca caagtttgtt tgcttttcgt gcatgatatt aaatagcttg
     4921 gcagcaacag gactaggatg agtagcagca cgttccttat atgtagcttt cgacatgatt
     4981 tatcttcgtt tcctgcatgt ttttgttctg tgcagttggg ttaagaatac tgggcaattt
     5041 catgtttctt caacactaca tatgcgtata tataccaatc taagtctgtg ctccttcctt
     5101 cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat ttcaaggaaa ccgaaatcaa
     5161 aaaaaagaat aaaaaaaaaa tgatgaattg aaaagctaat tcttgaagac gaaagggcct
     5221 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5281 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5341 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5401 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5461 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5521 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5581 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5641 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5701 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5761 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5821 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5881 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5941 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6001 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6061 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6121 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6181 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6241 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6301 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6361 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6421 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6481 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6541 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6601 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6661 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6721 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6781 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6841 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6901 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6961 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7021 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7081 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     7141 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     7201 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     7261 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     7321 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     7381 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     7441 caagcttacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     7501 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     7561 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     7621 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     7681 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     7741 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     7801 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     7861 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc
     7921 gccaagcttc ctgaaacgga gaaacataaa caggcattgc tgggatcacc catacatcac
     7981 tctgttttgc ctgacctttt ccggtaattt gaaaacaaac ccggtctcga agcggagatc
     8041 cggcgataat taccgcagaa ataaacccat acacgagacg tagaaccagc cgcacatggc
     8101 cggagaaact cctgcgagaa tttcgtaaac tcgcgcgcat tgcatctgta tttcctaatg
     8161 cggcacttcc aggcctcgat cgagaccgtt tatccattgc ttttttgttg tctttttccc
     8221 tcgttcacag aaagtctgaa gaagctatag tagaactatg agcttttttt gtttctgttt
     8281 tccttttttt tttttttacc tctgtggaaa ttgttactct cacactcttt agttcgtttg
     8341 tttgttttgt ttattccaat tatgaccggt gacgaaacgt ggtcgatggt gggtaccgct
     8401 tatgctcccc tccattagtt tcgattatat aaaaaggcca aatattgtat tattttcaaa
     8461 tgtcctatca
//
