LOCUS       sequence-421795-        6427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the mRuby2 fluorescent protein (Part3) without any signal 
            peptide assembled with S. cerevisiae pTEF1 promoter, tTDH1 
            terminator, LEU2 selectable marker and CEN/ARS yeast origin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6427)
  AUTHORS   O'Riordan NM, Juric V, O'Neill SK, Roche AP, Young PW
  TITLE     A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization 
            of Heterologous Protein Secretion and Surface Display in 
            Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2024 Mar 14. doi: 10.1021/acssynbio.3c00743.
  PUBMED    38483353
REFERENCE   2  (bases 1 to 6427)
  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: "ACS Synth 
            Biol. 2024 Mar 14. doi: 10.1021/acssynbio.3c00743."
COMMENT     Sequence Label: pYSD140
FEATURES             Location/Qualifiers
     source          1..6427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(729..746)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(750..821)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             complement(828..1538)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     promoter        complement(1558..1971)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     rep_origin      complement(2550..3138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2630..2649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3241..3345
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3346..3414,3415..4206)
                     /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(3564..3583)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_feature    4317..4820
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(4999..6093)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(join(6094..6427,1..74))
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga
       61 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg
      121 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt
      181 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg
      241 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt agatatacta
      301 gaagttctcc tcgatgtact gcagtgcact agtggctcac gtcttatttg ggcaaaggag
      361 tcagcggatc accgtactat gtgtgagtgc tcctatcgcg cttgctcctg tgagatacga
      421 tgtggccgtg tctgaccgtg gtgcgtcatc tattcactca cttggctgcg atttatcgtc
      481 tcacatccag ccgttcaggg taatatattt taaccgccga cgttctcgcc ataactttta
      541 aacagtgata actaatattg aaacttagaa agaaaagggt aaagttaatg cttaatcttg
      601 tcttggctta aaaagtaata tgtacttatt tacgtcatca caaaatctag cagaaaaacg
      661 gtagtattta tgtatattca aaaaaaaatc attatcctca tcaagattgc tttatgccac
      721 tcgagttagt gatgatgatg atgatgacct ttatcatcat cgtccttata atctttgtca
      781 tcatcatctt tgtagtcctt atcatcatcg tccttgtaat cggatccctt atacaattca
      841 tccataccac cgcctaagcc agcaaatttg gctacggcat gctctctctg tacaacgaac
      901 atttcgttat cgctctcttc gagtctttct agtctgtggt ctacagcatg tattcctggc
      961 attttgatat tgccaacagt ctttttagaa cgataagtgg taacaaaact acaagacaaa
     1021 tgacctccac catcaacttt aagcgccatg tgtgtgtagc ctctcaaacc tccatcagct
     1081 gggtacatca tttcagtatt tggttcccaa cctttggtct ttttctgcat cacaggtccg
     1141 ttagaaggaa aattcacccc tcttacttgc acatgataga caaggcagcc atcttctaat
     1201 gaagtatctt gcataactgt gacgacaccc ccatcttcgt atcttgtaac cctttcccat
     1261 gtaaaaccct ctggaaaaga ttgcttgaaa aagtctggta tacctttagg gtacttgatg
     1321 aaagttcgtg atccgtacat gaatgacgtt gccagaatgt caaatgcaaa tggtagtggt
     1381 ccgccttcaa ttactttaat tctcatagtt tgtgtaccca tgtaaggatt accttcacct
     1441 tcccctgtac acttgaattg gtgaccatta acggagccct ccatgacaac tttcattctc
     1501 atgttttcct tgattaactc ctctcctttg gacaccatag atcttttgta attaaaactt
     1561 agattagatt gctatgcttt ctttctaatg agcaagaagt aaaaaaagtt gtaatagaac
     1621 aagaaaaatg aaactgaaac ttgagaaatt gatgaccgtt tattaactta aatatcaatg
     1681 ggaggtcatc gaaagagaaa aaaatcaaaa aaaaaaattt tcaagaaaaa gaaacgtgat
     1741 aaaaattttt attgcctttt tcgacgaaga aaaagaaacg aggcggtgtc ttttttcttt
     1801 tccaaacctt tagtacgggt aattaacgac accctagagg aagaaagagg ggaaatttag
     1861 tatgctgtgc ttgggtgttt tgaagtggta cggcgatgcg cggagtccga gaaaatctgg
     1921 aagagtaaaa aaggagtaga aacattttga agctatggtg tgtgggggat cacttgtggg
     1981 ggattgggtg tgatgtaagg attcgcggtc ctcgaaaatt aaaagtccaa cgcgcctgtt
     2041 gcttcctatg tgatatgtat tatatgtaat atgcataaat atatctactg cattgtattt
     2101 tgaacgtaca aagtatgcat tgtttatacg ctattatcag ccaaagttgg gtggtcgctt
     2161 tctgttgtat gactattgat gtctaggctg tcaataattt cgttttgagc ctccatgtct
     2221 ctgaagaact ccctgttggc aaggcgttgt agctgtgttg acatctggtt tggtgagacg
     2281 taactgcctt gatctgtcgg gtgtctgctt ctctcacagg cttaggtgga tctgctgtcc
     2341 ccagtcagtg cggtcatcat gcagtcatcc gagcgtgtat tgcacacatt tgtttggcgc
     2401 atgatgagcc gtgatgaccc cgttctagat gcgaattcag gggcggccgc acggttatcc
     2461 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2521 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2581 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2641 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2701 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2761 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2821 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2881 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2941 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3001 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3061 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3121 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3181 aacgaaaact cacgttaagg gattttggtc atgacaggtg gcacttttcg gggaaatgtg
     3241 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3301 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3361 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3421 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3481 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3541 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3601 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3661 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3721 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3781 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3841 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3901 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3961 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4021 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtcccgcgg tatcattgca
     4081 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4141 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4201 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4261 taatttaaaa ggatctaggt gaagatcctt tttgataatc gcggccgctc gggacggatc
     4321 gcttgcctgt aacttacacg cgcctcgtat cttttaatga tggaataatt tgggaattta
     4381 ctctgtgttt atttattttt atgttttgta tttggatttt agaaagtaaa taaagaaggt
     4441 agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt ttaaaaaatt tcaacaaaaa
     4501 gcgtacttta catatatatt tattagacaa gaaaagcaga ttaaatagat atacattcga
     4561 ttaacgataa gtaaaatgta aaatcacagg attttcgtgt gtggttttct acacagacaa
     4621 gatgaaacaa ttcggcatta atacctgaga gcaggaagag caagataaaa ggtagtattt
     4681 gttggcgatc cccctagagt cttttacatc ttcggaaaac aaaaactatt ttttctttaa
     4741 tttctttttt tactttctat ttttaattta tatatttata ttaaaaaatt taaattataa
     4801 ttatttttat agcacgtgat actcctgcct atttaacgcc aacgttgcga gatatacact
     4861 cgaaatgtgt gatgtagatg taaacagacg gctatatgaa tgccactagc cggttatttt
     4921 actcctagtt aatacatatt tcctagaatt ttttacgtgc ataagagtag aatagtctaa
     4981 agagaagggt cctccaggtt aagcaaggat tttcttaact tcttcggcga cagcatcacc
     5041 gacttcggtg gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac
     5101 ctttttaact gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa
     5161 catcattgca gcagacaaga tagtggcgat agggttgacc ttattctttg gcaaatctgg
     5221 agcagaaccg tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc
     5281 caaggacgca gatggcaaca aacccaagga acctgggata acggaggctt catcggagat
     5341 gatatcacca aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac
     5401 taggatcatg gcggcagaat caatcaattg atgttgaacc ttcaatgtag gaaattcgtt
     5461 cttgatggtt tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt
     5521 atccaaggac caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct
     5581 tgtgattctt tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc
     5641 atcgtcctcc tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa
     5701 gtcagtacct ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc
     5761 aaagttacat ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc
     5821 ttgttcaggt ctaacactac cggttcccca tttaggacca cccacagcac ctaacaaaac
     5881 ggcatcagcc ttcttggagg cttccagcgc ctcatctgga agtggaacac ctgtagcatc
     5941 gatagcagca ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc
     6001 agaaatagct ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc
     6061 tggcaaaacg acgatcttct taggggcaga cattagaatg gtatatcctt gaaatatata
     6121 tatatatatt gctgaaatgt aaaaggtaag aaaagttaga aagtaagacg attgctaacc
     6181 acctattgga aaaaacaata ggtccttaaa taatattgtc aacttcaagt attgtgatgc
     6241 aagcatttag tcatgaacgc ttctctattc tatatgaaaa gccggttccg gcgctctcac
     6301 ctttcctttt tctcccaatt tttcagttga aaaaggtata tgcgtcaggc gacctctgaa
     6361 attaacaaaa aatttccagt catcgaattt gattctgtgc gatagcgccc ctgtgtgttc
     6421 tcgttat
//
