LOCUS       sequence-407635-        8468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of hfYFP-T2A-mtq2  in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8468)
  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 8468)
  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 hfYFP-T2A-mtq2
FEATURES             Location/Qualifiers
     source          1..8468
                     /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             1554..1607
                     /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      1623..1632
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1629..1631,1632..1634,1635..2348)
                     /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(1935..1954)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2282..2303
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2358..2382)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2444..2463)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2445..2463)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2611..2798
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2666..2690)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2810..2827)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2810..2826)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2819..2841)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3262..4608
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4719..5522)
                     /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(5523..5738)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5763..5781)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5849..5953
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5954..6022,6023..6814)
                     /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(6172..6191)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6985..7573
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7474..7493
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7727..7744
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7861..7882
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7897..7902,7903..7920,7921..7927)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7935..7951
                     /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     7940..7962
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7959..7975
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7959..7975
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(8005..8460)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8142..8163)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8354..8373
                     /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 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      901 acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac ggcaagctga
      961 ccctgaagct catctccacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1021 ccttaggcta cggcctgatg gtgttcgccc gctaccccga ccacatgaag cagcacgact
     1081 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct ttcgaggacg
     1141 acggttacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1201 tcgtgctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1261 acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg
     1321 ctaacttcaa gatccgccac aacgttgagg acggcggcgt gcagctcgcc gaccactacc
     1381 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct
     1441 accagtccgt cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgaaggaga
     1501 gggtgaccgc cgccgggatt acacatgaca tgaacgagct gtacaagtcc ggagagggca
     1561 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctggg gatccaccgg
     1621 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1681 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1741 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1801 ggcccaccct cgtgaccacc ctgtcctggg gcgtgcagtg cttcgcccgc taccccgacc
     1861 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1921 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1981 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2041 tggggcacaa gctggagtac aactacttta gcgacaacgt ctatatcacc gccgacaagc
     2101 agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcggcgtgc
     2161 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2221 acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     2281 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2341 acaagtaaag cggccgcacc cagctttctt gtacaaagtg gtgtagttaa ttcatgatgg
     2401 ccgctgcagg tcgacctcga gggggggccc ggtacccaat tcgccctata gtgagtcgta
     2461 ttacgcgcgg atccagcttt ggacttcttc gccagaggtt tggtcaagtc tccaatcaag
     2521 gttgtcggct tgtctacctt gccagaaatt tacgaaaaga tggaaaaggg tcaaatcgtt
     2581 ggtagatacg ttgttgacac ttctaaataa gcgaatttct tatgatttat gatttttatt
     2641 attaaataag ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta
     2701 aaacgaaaat tcttattctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta
     2761 tagcatgagg tcgctcttat tgaccacacc tctaccggca tgccaattca ctggccgtcg
     2821 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2881 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2941 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     3001 gcggtatttc acaccgcata atcggatcgt acttgttacc catcattgaa ttttgaacat
     3061 ccgaacctgg gagttttccc tgaaacagat agtatatttg aacctgtata ataatatata
     3121 gtctagcgct ttacggaaga caatgtatgt atttcggttc ctggagaaac tattgcatct
     3181 attgcatagg taatcttgca cgtcgcatcc ccggttcatt ttctgcgttt ccatcttgca
     3241 cttcaatagc atatctttgt taacgaagca tctgtgcttc attttgtaga acaaaaatgc
     3301 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
     3361 tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt gtaaaacaaa
     3421 aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac
     3481 agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct
     3541 acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc
     3601 tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag
     3661 gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact
     3721 tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc
     3781 ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt
     3841 tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac
     3901 tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt
     3961 actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg
     4021 agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca
     4081 cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaata
     4141 ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc
     4201 gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagctagag aataggaact
     4261 tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc
     4321 tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata
     4381 tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct
     4441 atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat tccatgcggg
     4501 gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc actcctcaat
     4561 tggattagtc tcatccttca atgctatcat ttcctttgat attggatcga tccgatgata
     4621 agctgtcaaa catgagaatt gggtaataac tgatataatt aaattgaagc tctaatttgt
     4681 gagtttagta tacatgcatt tacttataat acagtttttt agttttgctg gccgcatctt
     4741 ctcaaatatg cttcccagcc tgcttttctg taacgttcac cctctacctt agcatccctt
     4801 ccctttgcaa atagtcctct tccaacaata ataatgtcag atcctgtaga gaccacatca
     4861 tccacggttc tatactgttg acccaatgcg tctcccttgt catctaaacc cacaccgggt
     4921 gtcataatca accaatcgta accttcatct cttccaccca tgtctctttg agcaataaag
     4981 ccgataacaa aatctttgtc gctcttcgca atgtcaacag tacccttagt atattctcca
     5041 gtagataggg agcccttgca tgacaattct gctaacatca aaaggcctct aggttccttt
     5101 gttacttctt ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt
     5161 gcattcgtaa tgtctgccca ttctgctatt ctgtatacac ccgcagagta ctgcaatttg
     5221 actgtattac caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg
     5281 gataatgcct ttagcggctt aactgtgccc tccatggaaa aatcagtcaa gatatccaca
     5341 tgtgttttta gtaaacaaat tttgggacct aatgcttcaa ctaactccag taattccttg
     5401 gtggtacgaa catccaatga agcacacaag tttgtttgct tttcgtgcat gatattaaat
     5461 agcttggcag caacaggact aggatgagta gcagcacgtt ccttatatgt agctttcgac
     5521 atgatttatc ttcgtttcct gcatgttttt gttctgtgca gttgggttaa gaatactggg
     5581 caatttcatg tttcttcaac actacatatg cgtatatata ccaatctaag tctgtgctcc
     5641 ttccttcgtt cttccttctg ttcggagatt accgaatcaa aaaaatttca aggaaaccga
     5701 aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa gctaattctt gaagacgaaa
     5761 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     5821 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5881 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5941 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     6001 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     6061 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     6121 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     6181 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6241 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6301 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6361 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6421 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6481 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6541 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6601 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6661 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6721 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6781 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6841 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6901 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6961 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     7021 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7081 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     7141 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7201 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7261 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7321 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7381 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     7441 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     7501 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7561 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7621 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     7681 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     7741 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     7801 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     7861 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     7921 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     7981 ttacgccaag cttaccgcat caggaaattg taagcgttaa tattttgtta aaattcgcgt
     8041 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     8101 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     8161 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     8221 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     8281 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     8341 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     8401 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     8461 ccattcgc
//
