LOCUS       sequence-437580-        8717 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  equimolar expression of ymiRFP680 and mTurquoise2 in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8717)
  AUTHORS   Teusink B
  TITLE     Expression of FPs in budding yeast
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8717)
  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            "Unpublished"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 ymiRFP680-T2A-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..8717
                     /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             1803..1856
                     /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      1872..1881
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1878..1880,1881..1883,1884..2597)
                     /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(1923..1944)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2184..2203)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2531..2552
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2607..2631)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2693..2712)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2694..2712)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2860..3047
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2915..2939)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3059..3076)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3059..3075)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3068..3090)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3511..4857
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4968..5771)
                     /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(5772..5987)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6012..6030)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6098..6202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6203..6271,6272..7063)
                     /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(6421..6440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7234..7822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7723..7742
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7976..7993
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8110..8131
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8146..8151,8152..8169,8170..8176)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8184..8200
                     /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     8189..8211
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8208..8224
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8208..8224
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(8254..8709)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8391..8412)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8603..8622
                     /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 tgtgatgatg
      901 aaccaattca tattccaggt gctattcaac cacatggttt gttattagct ttggctgctg
      961 atatgaccat tgttgctggt tctgataact tgcctgaatt gactggttta gctattggtg
     1021 ccttgatcgg tagatctgct gctgatgttt tcgattctga aacccataat agattgacta
     1081 ttgctttggc tgaaccaggt gctgctgtcg gtgctccaat taccgtcggt ttcaccatgc
     1141 gtaaggatgc cggtttcatt ggttcttggc atagacatga tcaattaatt ttcttggaat
     1201 tggaaccacc acaacgtgac gttgccgaac cacaggcttt cttccgtaga accaactctg
     1261 ctattagaag attacaagct gctgaaactt tggaatctgc ttgtgctgct gctgctcaag
     1321 aagttcgtaa gattactggt ttcgatagag ttatgatcta cagatttgct tctgatttct
     1381 ctggtgaagt tattgctgaa gatagatgtg ctgaagttga atctaagttg ggtttgcatt
     1441 acccagcttc tactgttcca gctcaagcta gaagattgta cactattaat ccagttagaa
     1501 ttattccaga tatcaactac agaccagtcc cagttacccc agatttgaac ccagttactg
     1561 gtagacctat tgatttatct ttcgctattt tgagatctgt ttccccatgt catttggaat
     1621 tcatgagaaa catcggtatg catggtacca tgtctatttc tattttgaga ggtgaaagat
     1681 tgtggggttt gattgtttgt catcatagaa ctccatacta cgttgacttg gatggtagac
     1741 aagcctgtaa gagagttgct gaaagattgg ctacccaaat tggtgttatg gaagaatccg
     1801 gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctgggg
     1861 atccaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1921 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1981 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2041 ccgtgccctg gcccaccctc gtgaccaccc tgtcctgggg cgtgcagtgc ttcgcccgct
     2101 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     2161 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     2221 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     2281 gcaacatcct ggggcacaag ctggagtaca actactttag cgacaacgtc tatatcaccg
     2341 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg
     2401 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2461 tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac cccaacgaga
     2521 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2581 acgagctgta caagtaaagc ggccgcaccc agctttcttg tacaaagtgg tgtagttaat
     2641 tcatgatggc cgctgcaggt cgacctcgag ggggggcccg gtacccaatt cgccctatag
     2701 tgagtcgtat tacgcgcgga tccagctttg gacttcttcg ccagaggttt ggtcaagtct
     2761 ccaatcaagg ttgtcggctt gtctaccttg ccagaaattt acgaaaagat ggaaaagggt
     2821 caaatcgttg gtagatacgt tgttgacact tctaaataag cgaatttctt atgatttatg
     2881 atttttatta ttaaataagt tataaaaaaa ataagtgtat acaaatttta aagtgactct
     2941 taggttttaa aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt
     3001 tctcaggtat agcatgaggt cgctcttatt gaccacacct ctaccggcat gccaattcac
     3061 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     3121 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     3181 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
     3241 cgcatctgtg cggtatttca caccgcataa tcggatcgta cttgttaccc atcattgaat
     3301 tttgaacatc cgaacctggg agttttccct gaaacagata gtatatttga acctgtataa
     3361 taatatatag tctagcgctt tacggaagac aatgtatgta tttcggttcc tggagaaact
     3421 attgcatcta ttgcataggt aatcttgcac gtcgcatccc cggttcattt tctgcgtttc
     3481 catcttgcac ttcaatagca tatctttgtt aacgaagcat ctgtgcttca ttttgtagaa
     3541 caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca
     3601 gaacagaaat gcaacgcgaa agcgctattt taccaacgaa gaatctgtgc ttcatttttg
     3661 taaaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt
     3721 ttacagaaca gaaatgcaac gcgagagcgc tattttacca acaaagaatc tatacttctt
     3781 ttttgttcta caaaaatgca tcccgagagc gctatttttc taacaaagca tcttagatta
     3841 ctttttttct cctttgtgcg ctctataatg cagtctcttg ataacttttt gcactgtagg
     3901 tccgttaagg ttagaagaag gctactttgg tgtctatttt ctcttccata aaaaaagcct
     3961 gactccactt cccgcgttta ctgattacta gcgaagctgc gggtgcattt tttcaagata
     4021 aaggcatccc cgattatatt ctataccgat gtggattgcg catactttgt gaacagaaag
     4081 tgatagcgtt gatgattctt cattggtcag aaaattatga acggtttctt ctattttgtc
     4141 tctatatact acgtatagga aatgtttaca ttttcgtatt gttttcgatt cactctatga
     4201 atagttctta ctacaatttt tttgtctaaa gagtaatact agagataaac ataaaaaatg
     4261 tagaggtcga gtttagatgc aagttcaagg agcgaaaggt ggatgggtag gttatatagg
     4321 gatatagcac agagatatat agcaaagaga tacttttgag caatgtttgt ggaagcggta
     4381 ttcgcaatat tttagtagct cgttacagtc cggtgcgttt ttggtttttt gaaagtgcgt
     4441 cttcagagcg cttttggttt tcaaaagcgc tctgaagttc ctatactttc tagctagaga
     4501 ataggaactt cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc
     4561 aacgcgagct gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct
     4621 gtatatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta
     4681 tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt
     4741 ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca
     4801 ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcgat
     4861 ccgatgataa gctgtcaaac atgagaattg ggtaataact gatataatta aattgaagct
     4921 ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta gttttgctgg
     4981 ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc ctctacctta
     5041 gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga tcctgtagag
     5101 accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc atctaaaccc
     5161 acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat gtctctttga
     5221 gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt acccttagta
     5281 tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa aaggcctcta
     5341 ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc
     5401 acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc cgcagagtac
     5461 tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag taaaaaattg
     5521 tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa atcagtcaag
     5581 atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac taactccagt
     5641 aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg
     5701 atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc cttatatgta
     5761 gctttcgaca tgatttatct tcgtttcctg catgtttttg ttctgtgcag ttgggttaag
     5821 aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac caatctaagt
     5881 ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa
     5941 ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag ctaattcttg
     6001 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     6061 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     6121 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     6181 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     6241 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     6301 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     6361 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     6421 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     6481 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     6541 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6601 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6661 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6721 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6781 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6841 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6901 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6961 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     7021 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     7081 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     7141 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     7201 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     7261 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     7321 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     7381 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     7441 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     7501 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     7561 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7621 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7681 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7741 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7801 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7861 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7921 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     7981 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     8041 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     8101 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     8161 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     8221 tgaccatgat tacgccaagc ttaccgcatc aggaaattgt aagcgttaat attttgttaa
     8281 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     8341 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     8401 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     8461 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     8521 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     8581 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     8641 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     8701 agggcgcgtc cattcgc
//
