LOCUS       sequence-240363-        8472 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces equimolar expression of tagRFP 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 tagRFP-T2A-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..8472
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    69..93
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      110..119
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             827..880
                     /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      896..905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(902..904,905..907,908..1621)
                     /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(947..968)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1208..1227)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1555..1576
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1631..1655)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1717..1736)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1718..1736)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      1884..2071
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1939..1963)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2083..2100)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2083..2099)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2092..2114)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2535..3881
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(3992..4795)
                     /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(4796..5011)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5036..5054)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5122..5226
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5227..5295,5296..6087)
                     /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(5445..5464)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6258..6846
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6747..6766
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7000..7017
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7134..7155
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7170..7175,7176..7193,7194..7200)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7208..7224
                     /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     7213..7235
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7232..7248
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7232..7248
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7278..7733)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7415..7436)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7627..7646
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tttatacaca cattcaaaag aaagaaaaaa aatatacccc agcctcgact agtgaattcg
       61 cggccatcac aagtttgtac aaaaaagcag gctgctagcg ctaccggtcg ccaccatggt
      121 gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctctaca tggagggcac
      181 cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
      241 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
      301 ggctaccagc ttcatgtacg gcagcagaac cttcatcaac cacacccagg gcatccccga
      361 cttctttaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
      421 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
      481 cgtcaagatc agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact
      541 cggctgggag gccaacaccg agatgctgta ccccgctgac ggcggcctgg aaggcagaag
      601 cgacatggcc ctgaagctcg tgggcggggg ccacctgatc tgcaacttca agaccacata
      661 cagatccaag aaacccgcta agaacctcaa gatgcccggc gtctactatg tggaccacag
      721 actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt
      781 ggccagatac tgcgacctcc ctagcaaact gctgtacaag tccggagagg gcagaggaag
      841 tcttctaaca tgcggtgacg tggaggagaa tcccggccct ggggatccac cggtcgccac
      901 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      961 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1021 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1081 cctcgtgacc accctgtcct ggggcgtgca gtgcttcgcc cgctaccccg accacatgaa
     1141 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1201 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1261 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1321 caagctggag tacaactact ttagcgacaa cgtctatatc accgccgaca agcagaagaa
     1381 cggcatcaag gccaacttca agatccgcca caacatcgag gacggcggcg tgcagctcgc
     1441 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1501 ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
     1561 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     1621 aagcggccgc acccagcttt cttgtacaaa gtggtgtagt taattcatga tggccgctgc
     1681 aggtcgacct cgaggggggg cccggtaccc aattcgccct atagtgagtc gtattacgcg
     1741 cggatccagc tttggacttc ttcgccagag gtttggtcaa gtctccaatc aaggttgtcg
     1801 gcttgtctac cttgccagaa atttacgaaa agatggaaaa gggtcaaatc gttggtagat
     1861 acgttgttga cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat
     1921 aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa
     1981 aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg
     2041 aggtcgctct tattgaccac acctctaccg gcatgccaat tcactggccg tcgttttaca
     2101 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2161 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2221 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2281 ttcacaccgc ataatcggat cgtacttgtt acccatcatt gaattttgaa catccgaacc
     2341 tgggagtttt ccctgaaaca gatagtatat ttgaacctgt ataataatat atagtctagc
     2401 gctttacgga agacaatgta tgtatttcgg ttcctggaga aactattgca tctattgcat
     2461 aggtaatctt gcacgtcgca tccccggttc attttctgcg tttccatctt gcacttcaat
     2521 agcatatctt tgttaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga
     2581 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg
     2641 cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa
     2701 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg
     2761 caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa
     2821 tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg
     2881 tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa
     2941 gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg
     3001 tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta
     3061 tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat
     3121 tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat
     3181 aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa
     3241 tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag
     3301 atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat
     3361 atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt
     3421 agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg
     3481 gttttcaaaa gcgctctgaa gttcctatac tttctagcta gagaatagga acttcggaat
     3541 aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg agctgcgcac
     3601 atacagctca ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat atatatacat
     3661 gagaagaacg gcatagtgcg tgtttatgct taaatgcgta cttatatgcg tctatttatg
     3721 taggatgaaa ggtagtctag tacctcctgt gatattatcc cattccatgc ggggtatcgt
     3781 atgcttcctt cagcactacc ctttagctgt tctatatgct gccactcctc aattggatta
     3841 gtctcatcct tcaatgctat catttccttt gatattggat cgatccgatg ataagctgtc
     3901 aaacatgaga attgggtaat aactgatata attaaattga agctctaatt tgtgagttta
     3961 gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat cttctcaaat
     4021 atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc cttccctttg
     4081 caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca tcatccacgg
     4141 ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg ggtgtcataa
     4201 tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata aagccgataa
     4261 caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct ccagtagata
     4321 gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc tttgttactt
     4381 cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg tgtgcattcg
     4441 taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat ttgactgtat
     4501 taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg gcggataatg
     4561 cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc acatgtgttt
     4621 ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc ttggtggtac
     4681 gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta aatagcttgg
     4741 cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc gacatgattt
     4801 atcttcgttt cctgcatgtt tttgttctgt gcagttgggt taagaatact gggcaatttc
     4861 atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc tccttccttc
     4921 gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa
     4981 aaaaagaata aaaaaaaaat gatgaattga aaagctaatt cttgaagacg aaagggcctc
     5041 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5101 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5161 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5221 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5281 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5341 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5401 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5461 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5521 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5581 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5641 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5701 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5761 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5821 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     5881 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     5941 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6001 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6061 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6121 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6181 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6241 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6301 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6361 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6421 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6481 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6541 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6601 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6661 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6721 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6781 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6841 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6901 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     6961 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     7021 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     7081 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     7141 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     7201 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     7261 aagcttaccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     7321 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     7381 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     7441 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     7501 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     7561 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     7621 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     7681 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg
     7741 ccaagcttcc tgaaacggag aaacataaac aggcattgct gggatcaccc atacatcact
     7801 ctgttttgcc tgaccttttc cggtaatttg aaaacaaacc cggtctcgaa gcggagatcc
     7861 ggcgataatt accgcagaaa taaacccata cacgagacgt agaaccagcc gcacatggcc
     7921 ggagaaactc ctgcgagaat ttcgtaaact cgcgcgcatt gcatctgtat ttcctaatgc
     7981 ggcacttcca ggcctcgatc gagaccgttt atccattgct tttttgttgt ctttttccct
     8041 cgttcacaga aagtctgaag aagctatagt agaactatga gctttttttg tttctgtttt
     8101 cctttttttt ttttttacct ctgtggaaat tgttactctc acactcttta gttcgtttgt
     8161 ttgttttgtt tattccaatt atgaccggtg acgaaacgtg gtcgatggtg ggtaccgctt
     8221 atgctcccct ccattagttt cgattatata aaaaggccaa atattgtatt attttcaaat
     8281 gtcctatcat tatcgtctaa catctaattt ctcttaaatt ttttctcttt ctttcctata
     8341 acaccaatag tgaaaatctt tttttcttct atatctacaa aaactttttt tttctatcaa
     8401 cctcgttgat aaattttttc tttaacaatc gttaataatt aattaattgg aaaataacca
     8461 ttttttctct ct
//
