LOCUS       sequence-246507-        8479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces equimolar expression of yeast codon optimized mNeongreen 
            and mTurquoise2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8479)
  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 8479)
  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-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..8479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    415..439
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      721..730
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1180..1233
                     /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      1249..1258
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1255..1257,1258..1260,1261..1974)
                     /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(1300..1321)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1561..1580)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1908..1929
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1984..2008)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2070..2089)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2071..2089)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2237..2424
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2292..2316)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2436..2453)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2436..2452)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2445..2467)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2888..4234
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4345..5148)
                     /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(5149..5364)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5389..5407)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5475..5579
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5580..5648,5649..6440)
                     /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(5798..5817)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6611..7199
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7100..7119
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7353..7370
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7487..7508
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7523..7528,7529..7546,7547..7553)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7561..7577
                     /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     7566..7588
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7585..7601
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7585..7601
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7631..8086)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7768..7789)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7980..7999
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aaattgttac tctcacactc tttagttcgt ttgtttgttt tgtttattcc aattatgacc
       61 ggtgacgaaa cgtggtcgat ggtgggtacc gcttatgctc ccctccatta gtttcgatta
      121 tataaaaagg ccaaatattg tattattttc aaatgtccta tcattatcgt ctaacatcta
      181 atttctctta aattttttct ctttctttcc tataacacca atagtgaaaa tctttttttc
      241 ttctatatct acaaaaactt tttttttcta tcaacctcgt tgataaattt tttctttaac
      301 aatcgttaat aattaattaa ttggaaaata accatttttt ctctctttta tacacacatt
      361 caaaagaaag aaaaaaaata taccccagcc tcgactagtg aattcgcggc catcacaagt
      421 ttgtacaaaa aagcaggctg ctagcactag taagctttta attaaatggt ctctaagggt
      481 gaagaagata acatggcttc tttgccagcc acccacgagt tgcacatctt cggttccatt
      541 aacggtgtcg acttcgatat ggtcggtcaa ggtaccggta acccaaacga cggttacgaa
      601 gaattgaact tgaagtctac caagggcgac ttgcagttct ccccttggat tttggttcca
      661 cacatcggtt acggtttcca ccaatacttg ccatacccag acggtatgtc cccattccaa
      721 gccgccatgg ttgacggttc tggttaccaa gtccacagaa ccatgcaatt cgaagacggt
      781 gcttccttga ccgtgaacta cagatacacc tacgaaggtt ctcacatcaa gggcgaagcc
      841 caagttaagg gtaccggttt ccctgctgac ggtccagtca tgactaactc cttgaccgct
      901 gctgactggt gtcgttccaa gaagacctac ccaaacgata agaccatcat ttccactttc
      961 aagtggtcct acaccaccgg taacggcaag agataccgct ctaccgctcg taccacctac
     1021 accttcgcca agccaatggc tgccaactac ttgaagaacc agcctatgta cgtcttccgt
     1081 aagaccgagc tgaagcattc caagactgaa ttgaacttca aggaatggca aaaggctttc
     1141 accgacgtca tgggtatgga cgaattgtac aagtccggag agggcagagg aagtcttcta
     1201 acatgcggtg acgtggagga gaatcccggc cctggggatc caccggtcgc caccatggtg
     1261 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1321 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1381 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1441 accaccctgt cctggggcgt gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     1501 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1561 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1621 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1681 gagtacaact actttagcga caacgtctat atcaccgccg acaagcagaa gaacggcatc
     1741 aaggccaact tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgccgaccac
     1801 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1861 agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1921 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc
     1981 cgcacccagc tttcttgtac aaagtggtgt agttaattca tgatggccgc tgcaggtcga
     2041 cctcgagggg gggcccggta cccaattcgc cctatagtga gtcgtattac gcgcggatcc
     2101 agctttggac ttcttcgcca gaggtttggt caagtctcca atcaaggttg tcggcttgtc
     2161 taccttgcca gaaatttacg aaaagatgga aaagggtcaa atcgttggta gatacgttgt
     2221 tgacacttct aaataagcga atttcttatg atttatgatt tttattatta aataagttat
     2281 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
     2341 attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc
     2401 tcttattgac cacacctcta ccggcatgcc aattcactgg ccgtcgtttt acaacgtcgt
     2461 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2521 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2581 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2641 cgcataatcg gatcgtactt gttacccatc attgaatttt gaacatccga acctgggagt
     2701 tttccctgaa acagatagta tatttgaacc tgtataataa tatatagtct agcgctttac
     2761 ggaagacaat gtatgtattt cggttcctgg agaaactatt gcatctattg cataggtaat
     2821 cttgcacgtc gcatccccgg ttcattttct gcgtttccat cttgcacttc aatagcatat
     2881 ctttgttaac gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct
     2941 aatttttcaa acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc
     3001 gctattttac caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga
     3061 gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg
     3121 agagcgctat tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc
     3181 cgagagcgct atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc
     3241 tataatgcag tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct
     3301 actttggtgt ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg
     3361 attactagcg aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta
     3421 taccgatgtg gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat
     3481 tggtcagaaa attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat
     3541 gtttacattt tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt
     3601 gtctaaagag taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag
     3661 ttcaaggagc gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc
     3721 aaagagatac ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt
     3781 tacagtccgg tgcgtttttg gttttttgaa agtgcgtctt cagagcgctt ttggttttca
     3841 aaagcgctct gaagttccta tactttctag ctagagaata ggaacttcgg aataggaact
     3901 tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg cacatacagc
     3961 tcactgttca cgtcgcacct atatctgcgt gttgcctgta tatatatata catgagaaga
     4021 acggcatagt gcgtgtttat gcttaaatgc gtacttatat gcgtctattt atgtaggatg
     4081 aaaggtagtc tagtacctcc tgtgatatta tcccattcca tgcggggtat cgtatgcttc
     4141 cttcagcact accctttagc tgttctatat gctgccactc ctcaattgga ttagtctcat
     4201 ccttcaatgc tatcatttcc tttgatattg gatcgatccg atgataagct gtcaaacatg
     4261 agaattgggt aataactgat ataattaaat tgaagctcta atttgtgagt ttagtataca
     4321 tgcatttact tataatacag ttttttagtt ttgctggccg catcttctca aatatgcttc
     4381 ccagcctgct tttctgtaac gttcaccctc taccttagca tcccttccct ttgcaaatag
     4441 tcctcttcca acaataataa tgtcagatcc tgtagagacc acatcatcca cggttctata
     4501 ctgttgaccc aatgcgtctc ccttgtcatc taaacccaca ccgggtgtca taatcaacca
     4561 atcgtaacct tcatctcttc cacccatgtc tctttgagca ataaagccga taacaaaatc
     4621 tttgtcgctc ttcgcaatgt caacagtacc cttagtatat tctccagtag atagggagcc
     4681 cttgcatgac aattctgcta acatcaaaag gcctctaggt tcctttgtta cttcttctgc
     4741 cgcctgcttc aaaccgctaa caatacctgg gcccaccaca ccgtgtgcat tcgtaatgtc
     4801 tgcccattct gctattctgt atacacccgc agagtactgc aatttgactg tattaccaat
     4861 gtcagcaaat tttctgtctt cgaagagtaa aaaattgtac ttggcggata atgcctttag
     4921 cggcttaact gtgccctcca tggaaaaatc agtcaagata tccacatgtg tttttagtaa
     4981 acaaattttg ggacctaatg cttcaactaa ctccagtaat tccttggtgg tacgaacatc
     5041 caatgaagca cacaagtttg tttgcttttc gtgcatgata ttaaatagct tggcagcaac
     5101 aggactagga tgagtagcag cacgttcctt atatgtagct ttcgacatga tttatcttcg
     5161 tttcctgcat gtttttgttc tgtgcagttg ggttaagaat actgggcaat ttcatgtttc
     5221 ttcaacacta catatgcgta tatataccaa tctaagtctg tgctccttcc ttcgttcttc
     5281 cttctgttcg gagattaccg aatcaaaaaa atttcaagga aaccgaaatc aaaaaaaaga
     5341 ataaaaaaaa aatgatgaat tgaaaagcta attcttgaag acgaaagggc ctcgtgatac
     5401 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     5461 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5521 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5581 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5641 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5701 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5761 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5821 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5881 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5941 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     6001 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6061 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6121 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6181 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6241 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6301 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6361 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6421 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6481 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6541 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6601 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6661 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6721 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6781 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6841 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6901 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6961 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     7021 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7081 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7141 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7201 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7261 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     7321 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     7381 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     7441 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     7501 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     7561 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta
     7621 ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     7681 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     7741 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     7801 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     7861 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     7921 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     7981 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     8041 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccaagct
     8101 tcctgaaacg gagaaacata aacaggcatt gctgggatca cccatacatc actctgtttt
     8161 gcctgacctt ttccggtaat ttgaaaacaa acccggtctc gaagcggaga tccggcgata
     8221 attaccgcag aaataaaccc atacacgaga cgtagaacca gccgcacatg gccggagaaa
     8281 ctcctgcgag aatttcgtaa actcgcgcgc attgcatctg tatttcctaa tgcggcactt
     8341 ccaggcctcg atcgagaccg tttatccatt gcttttttgt tgtctttttc cctcgttcac
     8401 agaaagtctg aagaagctat agtagaacta tgagcttttt ttgtttctgt tttccttttt
     8461 ttttttttta cctctgtgg
//
