LOCUS       sequence-407633-        8483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mfRFP-T2A-mtq2 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8483)
  AUTHORS   Botman D, de Groot DH, Wehrens M, van Rossum K, Yska S, van der 
            Kieft B, Albert V, Minghetti N, Goedhart J, Teusink B
  TITLE     Side-by-side systematic characterization of novel FPs in budding 
            yeast
  JOURNAL   bioRxiv 2025.06.27.661975; doi: 
            https://doi.org/10.1101/2025.06.27.661975
REFERENCE   2  (bases 1 to 8483)
  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: "bioRxiv 
            2025.06.27.661975; doi: https://doi.org/10.1101/2025.06.27.661975"
COMMENT     Sequence Label: pDRF1-GW mfRFP-T2A-mtq2
FEATURES             Location/Qualifiers
     source          1..8483
                     /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             1569..1622
                     /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      1638..1647
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1644..1646,1647..1649,1650..2363)
                     /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(1689..1710)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1950..1969)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2297..2318
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2373..2397)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2459..2478)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2460..2478)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2626..2813
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2681..2705)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2825..2842)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2825..2841)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2834..2856)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3277..4623
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4734..5537)
                     /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(5538..5753)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5778..5796)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5864..5968
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5969..6037,6038..6829)
                     /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(6187..6206)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7000..7588
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7489..7508
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7742..7759
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7876..7897
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7912..7917,7918..7935,7936..7942)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7950..7966
                     /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     7955..7977
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7974..7990
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7974..7990
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(8020..8475)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8157..8178)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8369..8388
                     /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 atggtgagca
      841 agggcgagga gctgattaag gagaacatgc cactgaagct gtacatggag ggcaccgtta
      901 acaatcacca cttcaagtgc acagctgagg gcgaaggcaa gccctacgag ggcacccaga
      961 cccagagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta
     1021 ccagtttcat gtacggcagt cacaccttca tcgaccacac cccgggcatc cccgacttct
     1081 ggaagcagtc cttccccgag ggcttcacgt gggagagagt caccacatac gaagacgggg
     1141 gcgtcctgat cgcgacccag gacaccagcc tccatgacgg ctgcctcatc tacaacgtca
     1201 agttgagagg ggtgaacttc ccatccaacg gccctgtgat gcaaaagaaa acactcggct
     1261 gggaggcaca caccgagctt ttgtacccca ctgacggcgg tctggaaggc agaaccgcgc
     1321 tggccctgaa gcttgtgggc gggggccacc tattgtgtaa cttcaagact atatacagat
     1381 ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac tacagactgg
     1441 aaagaatcga ggaggccgac aatgagacct acgtcgagca gcacgaggtg gctgtggcca
     1501 gatactgcga cctccctacc aaactggggc acaagcttaa gggcatggac gagctgtaca
     1561 agtccggaga gggcagagga agtcttctaa catgcggtga cgtggaggag aatcccggcc
     1621 ctggggatcc accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg
     1681 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1741 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1801 agctgcccgt gccctggccc accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg
     1861 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1921 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1981 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2041 aggacggcaa catcctgggg cacaagctgg agtacaacta ctttagcgac aacgtctata
     2101 tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg
     2161 aggacggcgg cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2221 ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc aaagacccca
     2281 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2341 gcatggacga gctgtacaag taaagcggcc gcacccagct ttcttgtaca aagtggtgta
     2401 gttaattcat gatggccgct gcaggtcgac ctcgaggggg ggcccggtac ccaattcgcc
     2461 ctatagtgag tcgtattacg cgcggatcca gctttggact tcttcgccag aggtttggtc
     2521 aagtctccaa tcaaggttgt cggcttgtct accttgccag aaatttacga aaagatggaa
     2581 aagggtcaaa tcgttggtag atacgttgtt gacacttcta aataagcgaa tttcttatga
     2641 tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt
     2701 gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg
     2761 ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgcca
     2821 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     2881 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     2941 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     3001 tccttacgca tctgtgcggt atttcacacc gcataatcgg atcgtacttg ttacccatca
     3061 ttgaattttg aacatccgaa cctgggagtt ttccctgaaa cagatagtat atttgaacct
     3121 gtataataat atatagtcta gcgctttacg gaagacaatg tatgtatttc ggttcctgga
     3181 gaaactattg catctattgc ataggtaatc ttgcacgtcg catccccggt tcattttctg
     3241 cgtttccatc ttgcacttca atagcatatc tttgttaacg aagcatctgt gcttcatttt
     3301 gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt
     3361 tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca
     3421 tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     3481 gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa agaatctata
     3541 cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac aaagcatctt
     3601 agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa ctttttgcac
     3661 tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc tattttctct tccataaaaa
     3721 aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt gcattttttc
     3781 aagataaagg catccccgat tatattctat accgatgtgg attgcgcata ctttgtgaac
     3841 agaaagtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg tttcttctat
     3901 tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt tcgattcact
     3961 ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag ataaacataa
     4021 aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat gggtaggtta
     4081 tatagggata tagcacagag atatatagca aagagatact tttgagcaat gtttgtggaa
     4141 gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa
     4201 gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat actttctagc
     4261 tagagaatag gaacttcgga ataggaactt caaagcgttt ccgaaaacga gcgcttccga
     4321 aaatgcaacg cgagctgcgc acatacagct cactgttcac gtcgcaccta tatctgcgtg
     4381 ttgcctgtat atatatatac atgagaagaa cggcatagtg cgtgtttatg cttaaatgcg
     4441 tacttatatg cgtctattta tgtaggatga aaggtagtct agtacctcct gtgatattat
     4501 cccattccat gcggggtatc gtatgcttcc ttcagcacta ccctttagct gttctatatg
     4561 ctgccactcc tcaattggat tagtctcatc cttcaatgct atcatttcct ttgatattgg
     4621 atcgatccga tgataagctg tcaaacatga gaattgggta ataactgata taattaaatt
     4681 gaagctctaa tttgtgagtt tagtatacat gcatttactt ataatacagt tttttagttt
     4741 tgctggccgc atcttctcaa atatgcttcc cagcctgctt ttctgtaacg ttcaccctct
     4801 accttagcat cccttccctt tgcaaatagt cctcttccaa caataataat gtcagatcct
     4861 gtagagacca catcatccac ggttctatac tgttgaccca atgcgtctcc cttgtcatct
     4921 aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc acccatgtct
     4981 ctttgagcaa taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc aacagtaccc
     5041 ttagtatatt ctccagtaga tagggagccc ttgcatgaca attctgctaa catcaaaagg
     5101 cctctaggtt cctttgttac ttcttctgcc gcctgcttca aaccgctaac aatacctggg
     5161 cccaccacac cgtgtgcatt cgtaatgtct gcccattctg ctattctgta tacacccgca
     5221 gagtactgca atttgactgt attaccaatg tcagcaaatt ttctgtcttc gaagagtaaa
     5281 aaattgtact tggcggataa tgcctttagc ggcttaactg tgccctccat ggaaaaatca
     5341 gtcaagatat ccacatgtgt ttttagtaaa caaattttgg gacctaatgc ttcaactaac
     5401 tccagtaatt ccttggtggt acgaacatcc aatgaagcac acaagtttgt ttgcttttcg
     5461 tgcatgatat taaatagctt ggcagcaaca ggactaggat gagtagcagc acgttcctta
     5521 tatgtagctt tcgacatgat ttatcttcgt ttcctgcatg tttttgttct gtgcagttgg
     5581 gttaagaata ctgggcaatt tcatgtttct tcaacactac atatgcgtat atataccaat
     5641 ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg agattaccga atcaaaaaaa
     5701 tttcaaggaa accgaaatca aaaaaaagaa taaaaaaaaa atgatgaatt gaaaagctaa
     5761 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5821 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5881 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5941 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     6001 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6061 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6121 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6181 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6241 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6301 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6361 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6421 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6481 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6541 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6601 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6661 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6721 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6781 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6841 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6901 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6961 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     7021 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7081 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7141 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7201 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7261 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7321 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7381 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7441 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7501 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7561 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7621 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7681 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     7741 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     7801 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     7861 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     7921 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     7981 cagctatgac catgattacg ccaagcttac cgcatcagga aattgtaagc gttaatattt
     8041 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     8101 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     8161 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     8221 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     8281 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     8341 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     8401 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     8461 cgctacaggg cgcgtccatt cgc
//
