LOCUS       sequence-437725-        8387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mKelly-T2A-mtq2 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8387)
  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 8387)
  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 mKelly-T2A-mtq2
FEATURES             Location/Qualifiers
     source          1..8387
                     /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             1473..1526
                     /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      1542..1551
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1548..1550,1551..1553,1554..2267)
                     /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(1593..1614)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1854..1873)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2201..2222
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2277..2301)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2363..2382)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2364..2382)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2530..2717
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2585..2609)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2729..2746)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2729..2745)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2738..2760)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3181..4527
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4638..5441)
                     /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(5442..5657)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5682..5700)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5768..5872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5873..5941,5942..6733)
                     /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(6091..6110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6904..7492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7393..7412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7646..7663
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7780..7801
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7816..7821,7822..7839,7840..7846)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7854..7870
                     /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     7859..7881
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7878..7894
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7878..7894
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7924..8379)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8061..8082)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8273..8292
                     /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 atgcgtatga
      841 aactgtacat ggaaggtacc gttggcaacc atcacttcaa atgcaccgcg gaaggtgagg
      901 gtaaaccgta cgaaggcacc cagacccagc gtattaaagt tgttgaaggt ggtccgctgc
      961 cgttcgcgtt cgacatcctg gcgacctgct tcatgtatgg ttctaaaact tttatcaact
     1021 accctcaggg tatcccggac ttcttcaaac agtctttccc agaaggtttc acctgggaac
     1081 gtgttaccac ctacgaagac ggcggtgttc tgaccgttac ccaggacacc tctctgcagg
     1141 acggttacct gatctacaac gttaaactgc gtggtgttaa cttcccgtct aacggccctg
     1201 ttatgcagaa aaagactctg ggttgggaac cgaccaccga gtacctggtc ccggttgacg
     1261 gtggcctgga gggtacatgt gatatggcgc tgcgtctggt tggtggcggt cacctgaagt
     1321 gcaatctgaa aaccacgtat cgttccaaaa aaccagcgaa aaacctgaag atgccgggtg
     1381 agcaccaggt tgaccgtaag ctggagcgta tcaaagaggc ggacaacgaa acctacgttg
     1441 aacagcacga agttgcggtt gcgagatccg gagagggcag aggaagtctt ctaacatgcg
     1501 gtgacgtgga ggagaatccc ggccctgggg atccaccggt cgccaccatg gtgagcaagg
     1561 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1621 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1681 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1741 tgtcctgggg cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct
     1801 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1861 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1921 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1981 actactttag cgacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca
     2041 acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac cactaccagc
     2101 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     2161 agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     2221 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc ggccgcaccc
     2281 agctttcttg tacaaagtgg tgtagttaat tcatgatggc cgctgcaggt cgacctcgag
     2341 ggggggcccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgga tccagctttg
     2401 gacttcttcg ccagaggttt ggtcaagtct ccaatcaagg ttgtcggctt gtctaccttg
     2461 ccagaaattt acgaaaagat ggaaaagggt caaatcgttg gtagatacgt tgttgacact
     2521 tctaaataag cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa
     2581 ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg
     2641 agtaactctt tcctgtaggt caggttgctt tctcaggtat agcatgaggt cgctcttatt
     2701 gaccacacct ctaccggcat gccaattcac tggccgtcgt tttacaacgt cgtgactggg
     2761 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     2821 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2881 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcataa
     2941 tcggatcgta cttgttaccc atcattgaat tttgaacatc cgaacctggg agttttccct
     3001 gaaacagata gtatatttga acctgtataa taatatatag tctagcgctt tacggaagac
     3061 aatgtatgta tttcggttcc tggagaaact attgcatcta ttgcataggt aatcttgcac
     3121 gtcgcatccc cggttcattt tctgcgtttc catcttgcac ttcaatagca tatctttgtt
     3181 aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc gctaattttt
     3241 caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgaa agcgctattt
     3301 taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg agagcgctaa
     3361 tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgagagcgc
     3421 tattttacca acaaagaatc tatacttctt ttttgttcta caaaaatgca tcccgagagc
     3481 gctatttttc taacaaagca tcttagatta ctttttttct cctttgtgcg ctctataatg
     3541 cagtctcttg ataacttttt gcactgtagg tccgttaagg ttagaagaag gctactttgg
     3601 tgtctatttt ctcttccata aaaaaagcct gactccactt cccgcgttta ctgattacta
     3661 gcgaagctgc gggtgcattt tttcaagata aaggcatccc cgattatatt ctataccgat
     3721 gtggattgcg catactttgt gaacagaaag tgatagcgtt gatgattctt cattggtcag
     3781 aaaattatga acggtttctt ctattttgtc tctatatact acgtatagga aatgtttaca
     3841 ttttcgtatt gttttcgatt cactctatga atagttctta ctacaatttt tttgtctaaa
     3901 gagtaatact agagataaac ataaaaaatg tagaggtcga gtttagatgc aagttcaagg
     3961 agcgaaaggt ggatgggtag gttatatagg gatatagcac agagatatat agcaaagaga
     4021 tacttttgag caatgtttgt ggaagcggta ttcgcaatat tttagtagct cgttacagtc
     4081 cggtgcgttt ttggtttttt gaaagtgcgt cttcagagcg cttttggttt tcaaaagcgc
     4141 tctgaagttc ctatactttc tagctagaga ataggaactt cggaatagga acttcaaagc
     4201 gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt
     4261 tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat
     4321 agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg atgaaaggta
     4381 gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc
     4441 actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct catccttcaa
     4501 tgctatcatt tcctttgata ttggatcgat ccgatgataa gctgtcaaac atgagaattg
     4561 ggtaataact gatataatta aattgaagct ctaatttgtg agtttagtat acatgcattt
     4621 acttataata cagtttttta gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct
     4681 gcttttctgt aacgttcacc ctctacctta gcatcccttc cctttgcaaa tagtcctctt
     4741 ccaacaataa taatgtcaga tcctgtagag accacatcat ccacggttct atactgttga
     4801 cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa
     4861 ccttcatctc ttccacccat gtctctttga gcaataaagc cgataacaaa atctttgtcg
     4921 ctcttcgcaa tgtcaacagt acccttagta tattctccag tagataggga gcccttgcat
     4981 gacaattctg ctaacatcaa aaggcctcta ggttcctttg ttacttcttc tgccgcctgc
     5041 ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg cattcgtaat gtctgcccat
     5101 tctgctattc tgtatacacc cgcagagtac tgcaatttga ctgtattacc aatgtcagca
     5161 aattttctgt cttcgaagag taaaaaattg tacttggcgg ataatgcctt tagcggctta
     5221 actgtgccct ccatggaaaa atcagtcaag atatccacat gtgtttttag taaacaaatt
     5281 ttgggaccta atgcttcaac taactccagt aattccttgg tggtacgaac atccaatgaa
     5341 gcacacaagt ttgtttgctt ttcgtgcatg atattaaata gcttggcagc aacaggacta
     5401 ggatgagtag cagcacgttc cttatatgta gctttcgaca tgatttatct tcgtttcctg
     5461 catgtttttg ttctgtgcag ttgggttaag aatactgggc aatttcatgt ttcttcaaca
     5521 ctacatatgc gtatatatac caatctaagt ctgtgctcct tccttcgttc ttccttctgt
     5581 tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa
     5641 aaaaatgatg aattgaaaag ctaattcttg aagacgaaag ggcctcgtga tacgcctatt
     5701 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5761 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5821 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5881 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5941 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     6001 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     6061 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6121 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6181 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6241 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6301 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6361 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6421 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6481 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6541 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6601 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6661 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6721 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6781 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6841 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6901 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6961 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     7021 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     7081 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7141 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7201 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7261 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7321 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7381 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7441 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7501 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     7561 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     7621 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     7681 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     7741 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     7801 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     7861 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc ttaccgcatc
     7921 aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     7981 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     8041 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     8101 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     8161 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     8221 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     8281 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     8341 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgc
//
