LOCUS       sequence-223800-        8472 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces equimolar expression of tagRFPT and mTq2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8472)
  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 8472)
  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 tagRFPT-T2A-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..8472
                     /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      841..850
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1558..1611
                     /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      1627..1636
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1633..1635,1636..1638,1639..2352)
                     /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(1678..1699)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1939..1958)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2286..2307
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2362..2386)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2448..2467)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2449..2467)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2615..2802
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2670..2694)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2814..2831)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2814..2830)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2823..2845)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3266..4612
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4723..5526)
                     /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(5527..5742)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5767..5785)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5853..5957
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5958..6026,6027..6818)
                     /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(6176..6195)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6989..7577
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7478..7497
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7731..7748
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7865..7886
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7901..7906,7907..7924,7925..7931)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7939..7955
                     /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     7944..7966
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7963..7979
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7963..7979
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(8009..8464)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8146..8167)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8358..8377
                     /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 gctaccggtc
      841 gccaccatgg tgtctaaggg cgaagagctg attaaggaga acatgcacat gaagctctac
      901 atggagggca ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga aggcaagccc
      961 tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc
     1021 ttcgacatcc tggctaccag cttcatgtac ggcagcagaa ccttcatcaa ccacacccag
     1081 ggcatccccg acttctttaa gcagtccttc cctgagggct tcacatggga gagagtcacc
     1141 acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc
     1201 ctcatctaca acgtcaagat cagaggggtg aacttcccat ccaacggccc tgtgatgcag
     1261 aagaaaacac tcggctggga ggccaacacc gagatgctgt accccgctga cggcggcctg
     1321 gaaggcagaa ccgacatggc cctgaagctc gtgggcgggg gccacctgat ctgcaacttc
     1381 aagaccacat acagatccaa gaaacccgct aagaacctca agatgcccgg cgtctactat
     1441 gtggaccaca gactggaaag aatcaaggag gccgacaaag agacctacgt cgagcagcac
     1501 gaggtggctg tggccagata ctgcgacctc cctagcaaac tgctgtacaa gtccggagag
     1561 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc tggggatcca
     1621 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1681 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1741 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1801 ccctggccca ccctcgtgac caccctgtcc tggggcgtgc agtgcttcgc ccgctacccc
     1861 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1921 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1981 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     2041 atcctggggc acaagctgga gtacaactac tttagcgaca acgtctatat caccgccgac
     2101 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc
     2161 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     2221 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     2281 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2341 ctgtacaagt aaagcggccg cacccagctt tcttgtacaa agtggtgtag ttaattcatg
     2401 atggccgctg caggtcgacc tcgagggggg gcccggtacc caattcgccc tatagtgagt
     2461 cgtattacgc gcggatccag ctttggactt cttcgccaga ggtttggtca agtctccaat
     2521 caaggttgtc ggcttgtcta ccttgccaga aatttacgaa aagatggaaa agggtcaaat
     2581 cgttggtaga tacgttgttg acacttctaa ataagcgaat ttcttatgat ttatgatttt
     2641 tattattaaa taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt
     2701 tttaaaacga naattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctca
     2761 ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccaa ttcactggcc
     2821 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     2881 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     2941 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat
     3001 ctgtgcggta tttcacaccg cataatcgga tcgtacttgt tacccatcat tgaattttga
     3061 acatccgaac ctgggagttt tccctgaaac agatagtata tttgaacctg tataataata
     3121 tatagtctag cgctttacgg aagacaatgt atgtatttcg gttcctggag aaactattgc
     3181 atctattgca taggtaatct tgcacgtcgc atccccggtt cattttctgc gtttccatct
     3241 tgcacttcaa tagcatatct ttgttaacga agcatctgtg cttcattttg tagaacaaaa
     3301 atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca
     3361 gaaatgcaac gcgaaagcgc tattttacca acgaagaatc tgtgcttcat ttttgtaaaa
     3421 caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca
     3481 gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg
     3541 ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt
     3601 tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt
     3661 taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc
     3721 cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc
     3781 atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata
     3841 gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat
     3901 atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt
     3961 tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag
     4021 gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat
     4081 agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc
     4141 aatattttag tagctcgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca
     4201 gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagct agagaatagg
     4261 aacttcggaa taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc
     4321 gagctgcgca catacagctc actgttcacg tcgcacctat atctgcgtgt tgcctgtata
     4381 tatatataca tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt acttatatgc
     4441 gtctatttat gtaggatgaa aggtagtcta gtacctcctg tgatattatc ccattccatg
     4501 cggggtatcg tatgcttcct tcagcactac cctttagctg ttctatatgc tgccactcct
     4561 caattggatt agtctcatcc ttcaatgcta tcatttcctt tgatattgga tcgatccgat
     4621 gataagctgt caaacatgag aattgggtaa taactgatat aattaaattg aagctctaat
     4681 ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt gctggccgca
     4741 tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc
     4801 ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg tagagaccac
     4861 atcatccacg gttctatact gttgacccaa tgcgtctccc ttgtcatcta aacccacacc
     4921 gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat
     4981 aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc
     5041 tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc
     5101 ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc
     5161 gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa
     5221 tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt
     5281 ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc
     5341 cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc
     5401 cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt gcatgatatt
     5461 aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat atgtagcttt
     5521 cgacatgatt tatcttcgtt tcctgcatgt ttttgttctg tgcagttggg ttaagaatac
     5581 tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc taagtctgtg
     5641 ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat ttcaaggaaa
     5701 ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aaaagctaat tcttgaagac
     5761 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     5821 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5881 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5941 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     6001 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     6061 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     6121 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     6181 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     6241 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     6301 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6361 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6421 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6481 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6541 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6601 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6661 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6721 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6781 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6841 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6901 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6961 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     7021 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     7081 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     7141 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     7201 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     7261 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     7321 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7381 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7441 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7501 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7561 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7621 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7681 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     7741 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     7801 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     7861 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     7921 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     7981 atgattacgc caagcttacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc
     8041 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     8101 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     8161 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     8221 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     8281 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     8341 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     8401 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     8461 gcgtccattc gc
//
