LOCUS       sequence-223784-        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    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             1549..1602
                     /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      1618..1627
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1624..1626,1627..1629,1630..2343)
                     /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(1930..1949)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(2353..2377)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2439..2458)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2440..2458)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2606..2793
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2661..2685)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2805..2822)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2805..2821)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2814..2836)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3257..4603
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4714..5517)
                     /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(5518..5733)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5758..5776)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5844..5948
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5949..6017,6018..6809)
                     /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(6167..6186)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6980..7568
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7469..7488
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7722..7739
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7856..7877
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7892..7897,7898..7915,7916..7922)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7930..7946
                     /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     7935..7957
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7954..7970
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7954..7970
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(8000..8455)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8137..8158)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8349..8368
                     /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 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      901 ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg gcgagggcga gggcgatgcc
      961 accaacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1021 cccaccctcg tgaccacctt cggctacggc gtggcctgct tcagccgcta ccccgaccac
     1081 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1141 atctctttca aggacgacgg tacctacaag acccgcgccg aggtgaagtt cgagggcgac
     1201 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1261 gggcacaagc tggagtacaa cttcaacagc cacaacgtct atatcacggc cgacaagcag
     1321 aagaacggca tcaaggctaa cttcaagatc cgccacaacg ttgaggacgg cagcgtgcag
     1381 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1441 aaccactacc tgagccatca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1501 atggtcctgc tggagttcgt gaccgccgcc ggactgtaca agtccggaga gggcagagga
     1561 agtcttctaa catgcggtga cgtggaggag aatcccggcc ctggggatcc accggtcgcc
     1621 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1681 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1741 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1801 accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg
     1861 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1921 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1981 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2041 cacaagctgg agtacaacta ctttagcgac aacgtctata tcaccgccga caagcagaag
     2101 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
     2161 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2221 cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg
     2281 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2341 taaagcggcc gcacccagct ttcttgtaca aagtggtgta gttaattcat gatggccgct
     2401 gcaggtcgac ctcgaggggg ggcccggtac ccaattcgcc ctatagtgag tcgtattacg
     2461 cgcggatcca gctttggact tcttcgccag aggtttggtc aagtctccaa tcaaggttgt
     2521 cggcttgtct accttgccag aaatttacga aaagatggaa aagggtcaaa tcgttggtag
     2581 atacgttgtt gacacttcta aataagcgaa tttcttatga tttatgattt ttattattaa
     2641 ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg
     2701 anaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca
     2761 tgaggtcgct cttattgacc acacctctac cggcatgcca attcactggc cgtcgtttta
     2821 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     2881 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2941 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3001 atttcacacc gcataatcgg atcgtacttg ttacccatca ttgaattttg aacatccgaa
     3061 cctgggagtt ttccctgaaa cagatagtat atttgaacct gtataataat atatagtcta
     3121 gcgctttacg gaagacaatg tatgtatttc ggttcctgga gaaactattg catctattgc
     3181 ataggtaatc ttgcacgtcg catccccggt tcattttctg cgtttccatc ttgcacttca
     3241 atagcatatc tttgttaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc
     3301 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa
     3361 cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc
     3421 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
     3481 tgcaacgcga gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa
     3541 aatgcatccc gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt
     3601 tgtgcgctct ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag
     3661 aagaaggcta ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg
     3721 cgtttactga ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat
     3781 tatattctat accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg
     3841 attcttcatt ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt
     3901 ataggaaatg tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac
     3961 aatttttttg tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt
     4021 agatgcaagt tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag
     4081 atatatagca aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta
     4141 gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt
     4201 tggttttcaa aagcgctctg aagttcctat actttctagc tagagaatag gaacttcgga
     4261 ataggaactt caaagcgttt ccgaaaacga gcgcttccga aaatgcaacg cgagctgcgc
     4321 acatacagct cactgttcac gtcgcaccta tatctgcgtg ttgcctgtat atatatatac
     4381 atgagaagaa cggcatagtg cgtgtttatg cttaaatgcg tacttatatg cgtctattta
     4441 tgtaggatga aaggtagtct agtacctcct gtgatattat cccattccat gcggggtatc
     4501 gtatgcttcc ttcagcacta ccctttagct gttctatatg ctgccactcc tcaattggat
     4561 tagtctcatc cttcaatgct atcatttcct ttgatattgg atcgatccga tgataagctg
     4621 tcaaacatga gaattgggta ataactgata taattaaatt gaagctctaa tttgtgagtt
     4681 tagtatacat gcatttactt ataatacagt tttttagttt tgctggccgc atcttctcaa
     4741 atatgcttcc cagcctgctt ttctgtaacg ttcaccctct accttagcat cccttccctt
     4801 tgcaaatagt cctcttccaa caataataat gtcagatcct gtagagacca catcatccac
     4861 ggttctatac tgttgaccca atgcgtctcc cttgtcatct aaacccacac cgggtgtcat
     4921 aatcaaccaa tcgtaacctt catctcttcc acccatgtct ctttgagcaa taaagccgat
     4981 aacaaaatct ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga
     5041 tagggagccc ttgcatgaca attctgctaa catcaaaagg cctctaggtt cctttgttac
     5101 ttcttctgcc gcctgcttca aaccgctaac aatacctggg cccaccacac cgtgtgcatt
     5161 cgtaatgtct gcccattctg ctattctgta tacacccgca gagtactgca atttgactgt
     5221 attaccaatg tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact tggcggataa
     5281 tgcctttagc ggcttaactg tgccctccat ggaaaaatca gtcaagatat ccacatgtgt
     5341 ttttagtaaa caaattttgg gacctaatgc ttcaactaac tccagtaatt ccttggtggt
     5401 acgaacatcc aatgaagcac acaagtttgt ttgcttttcg tgcatgatat taaatagctt
     5461 ggcagcaaca ggactaggat gagtagcagc acgttcctta tatgtagctt tcgacatgat
     5521 ttatcttcgt ttcctgcatg tttttgttct gtgcagttgg gttaagaata ctgggcaatt
     5581 tcatgtttct tcaacactac atatgcgtat atataccaat ctaagtctgt gctccttcct
     5641 tcgttcttcc ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa accgaaatca
     5701 aaaaaaagaa taaaaaaaaa atgatgaatt gaaaagctaa ttcttgaaga cgaaagggcc
     5761 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     5821 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5881 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5941 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     6001 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     6061 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6121 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6181 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6241 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6301 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6361 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6421 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6481 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6541 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6601 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6661 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6721 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6781 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6841 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6901 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6961 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     7021 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     7081 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     7141 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     7201 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     7261 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     7321 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7381 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7441 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7501 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7561 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7621 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     7681 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     7741 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     7801 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     7861 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     7921 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     7981 ccaagcttac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     8041 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     8101 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     8161 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     8221 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     8281 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     8341 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     8401 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt
     8461 cgc
//
