LOCUS       sequence-437587-        8699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  equimolar expression of yemiRFP670 and mTurquoise2 in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8699)
  AUTHORS   Teusink B
  TITLE     Expression of FPs in budding yeast
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8699)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pDRF1 yemiRFP670-T2A-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..8699
                     /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"
     CDS             1785..1838
                     /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      1854..1863
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1860..1862,1863..1865,1866..2579)
                     /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(1905..1926)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2166..2185)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2513..2534
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2589..2613)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2675..2694)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2676..2694)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2842..3029
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2897..2921)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3041..3058)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3041..3057)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3050..3072)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3493..4839
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4950..5753)
                     /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(5754..5969)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5994..6012)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6080..6184
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6185..6253,6254..7045)
                     /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(6403..6422)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7216..7804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7705..7724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7958..7975
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8092..8113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8128..8133,8134..8151,8152..8158)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8166..8182
                     /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     8171..8193
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8190..8206
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8190..8206
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(8236..8691)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8373..8394)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8585..8604
                     /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 cccgggacta
      841 gtaagcttat ggctgaaggt tctgttgcta gacaaccaga tttgttgact tgtgaacatg
      901 aagaaattca tttggctggt tctattcaac cacatggtgc tttgttggtc gtttctgaac
      961 atgaccatag agttattcaa gcctctgcta atgccgctga attcttaaac ttgggttctg
     1021 ttttaggtgt tccattagct gaaattgatg gtgatttgtt aattaagatt ttaccacatt
     1081 tagatccaac cgctgaaggt atgccagttg ctgttagatg tagaatcggt aatccttcca
     1141 ctgaatactg tggtttgatg catagaccac cagaaggtgg tttgattatt gaattggaaa
     1201 gagctggtcc atctattgat ttatccggta ctttggctcc agctttggaa agaattagaa
     1261 ctgctggttc tttaagagct ttgtgtgatg acactgtttt gttattccaa caatgtactg
     1321 gttatgatag agttatggtt tacagattcg atgaacaagg tcatggttta gttttctctg
     1381 aatgtcatgt ccctggtttg gaatcttact tcggtaatag atacccatcc tctactgttc
     1441 cacaaatggc tagacaatta tacgttagac aaagagttag agttttggtc gatgttactt
     1501 accaaccagt tccattggaa ccaagattat ccccattgac tggtagagat ttagatatgt
     1561 ctggttgttt cttaagatct atgtctcctt gtcatttgca attcttgaag gatatgggtg
     1621 ttagagctac tttggccgtt tctttagttg ttggtggtaa gttgtggggt ttggttgttt
     1681 gtcaccatta cttgccaaga ttcattagat tcgaattaag agctatctgt aagagattgg
     1741 ctgaaagaat tgctactaga attaccgctt tagaatcttc cggagagggc agaggaagtc
     1801 ttctaacatg cggtgacgtg gaggagaatc ccggccctgg ggatccaccg gtcgccacca
     1861 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1921 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1981 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     2041 tcgtgaccac cctgtcctgg ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc
     2101 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2161 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2221 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2281 agctggagta caactacttt agcgacaacg tctatatcac cgccgacaag cagaagaacg
     2341 gcatcaaggc caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg
     2401 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2461 acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2521 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     2581 gcggccgcac ccagctttct tgtacaaagt ggtgtagtta attcatgatg gccgctgcag
     2641 gtcgacctcg agggggggcc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg
     2701 gatccagctt tggacttctt cgccagaggt ttggtcaagt ctccaatcaa ggttgtcggc
     2761 ttgtctacct tgccagaaat ttacgaaaag atggaaaagg gtcaaatcgt tggtagatac
     2821 gttgttgaca cttctaaata agcgaatttc ttatgattta tgatttttat tattaaataa
     2881 gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa
     2941 ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagcatgag
     3001 gtcgctctta ttgaccacac ctctaccggc atgccaattc actggccgtc gttttacaac
     3061 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3121 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3181 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3241 cacaccgcat aatcggatcg tacttgttac ccatcattga attttgaaca tccgaacctg
     3301 ggagttttcc ctgaaacaga tagtatattt gaacctgtat aataatatat agtctagcgc
     3361 tttacggaag acaatgtatg tatttcggtt cctggagaaa ctattgcatc tattgcatag
     3421 gtaatcttgc acgtcgcatc cccggttcat tttctgcgtt tccatcttgc acttcaatag
     3481 catatctttg ttaacgaagc atctgtgctt cattttgtag aacaaaaatg caacgcgaga
     3541 gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg
     3601 aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa aaatgcaacg
     3661 cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa cagaaatgca
     3721 acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc tacaaaaatg
     3781 catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt ctcctttgtg
     3841 cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa ggttagaaga
     3901 aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac ttcccgcgtt
     3961 tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc cccgattata
     4021 ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg ttgatgattc
     4081 ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata ctacgtatag
     4141 gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct tactacaatt
     4201 tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc gagtttagat
     4261 gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc acagagatat
     4321 atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat attttagtag
     4381 ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag cgcttttggt
     4441 tttcaaaagc gctctgaagt tcctatactt tctagctaga gaataggaac ttcggaatag
     4501 gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat
     4561 acagctcact gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga
     4621 gaagaacggc atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta
     4681 ggatgaaagg tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat
     4741 gcttccttca gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt
     4801 ctcatccttc aatgctatca tttcctttga tattggatcg atccgatgat aagctgtcaa
     4861 acatgagaat tgggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt
     4921 atacatgcat ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat
     4981 gcttcccagc ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca
     5041 aatagtcctc ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt
     5101 ctatactgtt gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc
     5161 aaccaatcgt aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca
     5221 aaatctttgt cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg
     5281 gagcccttgc atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct
     5341 tctgccgcct gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta
     5401 atgtctgccc attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta
     5461 ccaatgtcag caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc
     5521 tttagcggct taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt
     5581 agtaaacaaa ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga
     5641 acatccaatg aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca
     5701 gcaacaggac taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat
     5761 cttcgtttcc tgcatgtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat
     5821 gtttcttcaa cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt
     5881 tcttccttct gttcggagat taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa
     5941 aaagaataaa aaaaaaatga tgaattgaaa agctaattct tgaagacgaa agggcctcgt
     6001 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     6061 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6121 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     6181 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6241 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6301 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6361 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6421 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6481 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6541 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6601 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6661 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6721 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6781 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6841 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6901 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6961 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     7021 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     7081 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     7141 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     7201 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     7261 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     7321 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     7381 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     7441 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     7501 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     7561 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7621 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7681 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7741 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7801 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7861 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7921 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     7981 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     8041 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     8101 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     8161 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     8221 gcttaccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     8281 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     8341 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     8401 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     8461 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     8521 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     8581 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     8641 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgc
//
