LOCUS       sequence-240360-        8463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces equimolar expression of Clover and mTq2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8463)
  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 8463)
  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 Clover-T2A-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..8463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    139..163
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      180..189
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             888..941
                     /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      957..966
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(963..965,966..968,969..1682)
                     /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(1269..1288)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1692..1716)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1778..1797)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1779..1797)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      1945..2132
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2000..2024)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2144..2161)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2144..2160)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2153..2175)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2596..3942
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4053..4856)
                     /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(4857..5072)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5097..5115)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5183..5287
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5288..5356,5357..6148)
                     /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(5506..5525)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6319..6907
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6808..6827
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7061..7078
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7195..7216
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7231..7236,7237..7254,7255..7261)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7269..7285
                     /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     7274..7296
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7293..7309
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7293..7309
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7339..7794)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7476..7497)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7688..7707
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcgttgataa attttttctt taacaatcgt taataattaa ttaattggaa aataaccatt
       61 ttttctctct tttatacaca cattcaaaag aaagaaaaaa aatatacccc agcctcgact
      121 agtgaattcg cggccatcac aagtttgtac aaaaaagcag gctgctagcg ctaccggtcg
      181 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      241 tggacggcga cgtaaacggc cacaagttca gcgtccgcgg cgagggcgag ggcgatgcca
      301 ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
      361 ccaccctcgt gaccaccttc ggctacggcg tggcctgctt cagccgctac cccgaccaca
      421 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
      481 tctctttcaa ggacgacggt acctacaaga cccgcgccga ggtgaagttc gagggcgaca
      541 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
      601 ggcacaagct ggagtacaac ttcaacagcc acaacgtcta tatcacggcc gacaagcaga
      661 agaacggcat caaggctaac ttcaagatcc gccacaacgt tgaggacggc agcgtgcagc
      721 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
      781 accactacct gagccatcag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
      841 tggtcctgct ggagttcgtg accgccgccg gactgtacaa gtccggagag ggcagaggaa
      901 gtcttctaac atgcggtgac gtggaggaga atcccggccc tggggatcca ccggtcgcca
      961 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1021 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1081 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1141 ccctcgtgac caccctgtcc tggggcgtgc agtgcttcgc ccgctacccc gaccacatga
     1201 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1261 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1321 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1381 acaagctgga gtacaactac tttagcgaca acgtctatat caccgccgac aagcagaaga
     1441 acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg
     1501 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1561 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     1621 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1681 aaagcggccg cacccagctt tcttgtacaa agtggtgtag ttaattcatg atggccgctg
     1741 caggtcgacc tcgagggggg gcccggtacc caattcgccc tatagtgagt cgtattacgc
     1801 gcggatccag ctttggactt cttcgccaga ggtttggtca agtctccaat caaggttgtc
     1861 ggcttgtcta ccttgccaga aatttacgaa aagatggaaa agggtcaaat cgttggtaga
     1921 tacgttgttg acacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa
     1981 taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga
     2041 aaattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagcat
     2101 gaggtcgctc ttattgacca cacctctacc ggcatgccaa ttcactggcc gtcgttttac
     2161 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     2221 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     2281 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     2341 tttcacaccg cataatcgga tcgtacttgt tacccatcat tgaattttga acatccgaac
     2401 ctgggagttt tccctgaaac agatagtata tttgaacctg tataataata tatagtctag
     2461 cgctttacgg aagacaatgt atgtatttcg gttcctggag aaactattgc atctattgca
     2521 taggtaatct tgcacgtcgc atccccggtt cattttctgc gtttccatct tgcacttcaa
     2581 tagcatatct ttgttaacga agcatctgtg cttcattttg tagaacaaaa atgcaacgcg
     2641 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac
     2701 gcgaaagcgc tattttacca acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca
     2761 acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat
     2821 gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg ttctacaaaa
     2881 atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt tttctccttt
     2941 gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt taaggttaga
     3001 agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc cacttcccgc
     3061 gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc atccccgatt
     3121 atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga
     3181 ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat atactacgta
     3241 taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt tcttactaca
     3301 atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag gtcgagttta
     3361 gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat agcacagaga
     3421 tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc aatattttag
     3481 tagctcgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca gagcgctttt
     3541 ggttttcaaa agcgctctga agttcctata ctttctagct agagaatagg aacttcggaa
     3601 taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca
     3661 catacagctc actgttcacg tcgcacctat atctgcgtgt tgcctgtata tatatataca
     3721 tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt acttatatgc gtctatttat
     3781 gtaggatgaa aggtagtcta gtacctcctg tgatattatc ccattccatg cggggtatcg
     3841 tatgcttcct tcagcactac cctttagctg ttctatatgc tgccactcct caattggatt
     3901 agtctcatcc ttcaatgcta tcatttcctt tgatattgga tcgatccgat gataagctgt
     3961 caaacatgag aattgggtaa taactgatat aattaaattg aagctctaat ttgtgagttt
     4021 agtatacatg catttactta taatacagtt ttttagtttt gctggccgca tcttctcaaa
     4081 tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc ccttcccttt
     4141 gcaaatagtc ctcttccaac aataataatg tcagatcctg tagagaccac atcatccacg
     4201 gttctatact gttgacccaa tgcgtctccc ttgtcatcta aacccacacc gggtgtcata
     4261 atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat aaagccgata
     4321 acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc tccagtagat
     4381 agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc ctttgttact
     4441 tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc gtgtgcattc
     4501 gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa tttgactgta
     4561 ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt ggcggataat
     4621 gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc cacatgtgtt
     4681 tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc cttggtggta
     4741 cgaacatcca atgaagcaca caagtttgtt tgcttttcgt gcatgatatt aaatagcttg
     4801 gcagcaacag gactaggatg agtagcagca cgttccttat atgtagcttt cgacatgatt
     4861 tatcttcgtt tcctgcatgt ttttgttctg tgcagttggg ttaagaatac tgggcaattt
     4921 catgtttctt caacactaca tatgcgtata tataccaatc taagtctgtg ctccttcctt
     4981 cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat ttcaaggaaa ccgaaatcaa
     5041 aaaaaagaat aaaaaaaaaa tgatgaattg aaaagctaat tcttgaagac gaaagggcct
     5101 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5161 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5221 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5281 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5341 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5401 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5461 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5521 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5581 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5641 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5701 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5761 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5821 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5881 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5941 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6001 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6061 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6121 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6181 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6241 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6301 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6361 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6421 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6481 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6541 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6601 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6661 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6721 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6781 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6841 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6901 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6961 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     7021 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     7081 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     7141 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     7201 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     7261 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     7321 caagcttacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     7381 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     7441 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     7501 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     7561 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     7621 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     7681 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     7741 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc
     7801 gccaagcttc ctgaaacgga gaaacataaa caggcattgc tgggatcacc catacatcac
     7861 tctgttttgc ctgacctttt ccggtaattt gaaaacaaac ccggtctcga agcggagatc
     7921 cggcgataat taccgcagaa ataaacccat acacgagacg tagaaccagc cgcacatggc
     7981 cggagaaact cctgcgagaa tttcgtaaac tcgcgcgcat tgcatctgta tttcctaatg
     8041 cggcacttcc aggcctcgat cgagaccgtt tatccattgc ttttttgttg tctttttccc
     8101 tcgttcacag aaagtctgaa gaagctatag tagaactatg agcttttttt gtttctgttt
     8161 tccttttttt tttttttacc tctgtggaaa ttgttactct cacactcttt agttcgtttg
     8221 tttgttttgt ttattccaat tatgaccggt gacgaaacgt ggtcgatggt gggtaccgct
     8281 tatgctcccc tccattagtt tcgattatat aaaaaggcca aatattgtat tattttcaaa
     8341 tgtcctatca ttatcgtcta acatctaatt tctcttaaat tttttctctt tctttcctat
     8401 aacaccaata gtgaaaatct ttttttcttc tatatctaca aaaacttttt ttttctatca
     8461 acc
//
