LOCUS       sequence-421717-        6772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the mRuby2 fluorescent protein (Part3b') with the 111AA SCW4
            TFP (Part3a') 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 6772)
  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 6772)
  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: pYSD153
FEATURES             Location/Qualifiers
     source          1..6772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        472..885
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             1253..1960
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     CDS             1967..2038
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             2042..2059
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        2714..3121
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3122..4216
                     /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"
     misc_feature    4395..4898
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(join(5009..5800,5801..5869))
                     /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     5632..5651
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5870..5974)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6077..6665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6566..6585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctgaattc gcatctagaa cggggtcatc acggctcatc atgcgccaaa caaatgtgtg
       61 caatacacgc tcggatgact gcatgatgac cgcactgact ggggacagca gatccaccta
      121 agcctgtgag agaagcagac acccgacaga tcaaggcagt tacgtctcac caaaccagat
      181 gtcaacacag ctacaacgcc ttgccaacag ggagttcttc agagacatgg aggctcaaaa
      241 cgaaattatt gacagcctag acatcaatag tcatacaaca gaaagcgacc acccaacttt
      301 ggctgataat agcgtataaa caatgcatac tttgtacgtt caaaatacaa tgcagtagat
      361 atatttatgc atattacata taatacatat cacataggaa gcaacaggcg cgttggactt
      421 ttaattttcg aggaccgcga atccttacat cacacccaat cccccacaag tgatccccca
      481 cacaccatag cttcaaaatg tttctactcc ttttttactc ttccagattt tctcggactc
      541 cgcgcatcgc cgtaccactt caaaacaccc aagcacagca tactaaattt cccctctttc
      601 ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagacaccgc
      661 ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt tctttttctt
      721 gaaaattttt ttttttgatt tttttctctt tcgatgacct cccattgata tttaagttaa
      781 taaacggtca tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt
      841 ttacttcttg ctcattagaa agaaagcata gcaatctaat ctaagtttta attacaaaag
      901 atctatgcgt ctatctaacc taattgcttc tgcctctctt ttatctgctg ctactcttgc
      961 tgctcccgct aaccacgaac acaaggacaa gcgtgctgtg gtcactacca ctgttcaaaa
     1021 acaaaccact atcattgtta atggtgccgc ttcaactcca gttgctgctt tggaagaaaa
     1081 tgctgttgtc aactccgctc cagctgccgc taccagtaca acatcgtctg ctgcttctgt
     1141 agctaccgct gctgcttcct cttctgagaa caactcacaa gtttctgctg ccgcatctcc
     1201 agcctccagc tctgctgcta catctactca atcttcccta gacaaacgtg ctgtgtccaa
     1261 aggagaggag ttaatcaagg aaaacatgag aatgaaagtt gtcatggagg gctccgttaa
     1321 tggtcaccaa ttcaagtgta caggggaagg tgaaggtaat ccttacatgg gtacacaaac
     1381 tatgagaatt aaagtaattg aaggcggacc actaccattt gcatttgaca ttctggcaac
     1441 gtcattcatg tacggatcac gaactttcat caagtaccct aaaggtatac cagacttttt
     1501 caagcaatct tttccagagg gttttacatg ggaaagggtt acaagatacg aagatggggg
     1561 tgtcgtcaca gttatgcaag atacttcatt agaagatggc tgccttgtct atcatgtgca
     1621 agtaagaggg gtgaattttc cttctaacgg acctgtgatg cagaaaaaga ccaaaggttg
     1681 ggaaccaaat actgaaatga tgtacccagc tgatggaggt ttgagaggct acacacacat
     1741 ggcgcttaaa gttgatggtg gaggtcattt gtcttgtagt tttgttacca cttatcgttc
     1801 taaaaagact gttggcaata tcaaaatgcc aggaatacat gctgtagacc acagactaga
     1861 aagactcgaa gagagcgata acgaaatgtt cgttgtacag agagagcatg ccgtagccaa
     1921 atttgctggc ttaggcggtg gtatggatga attgtataag ggatccgatt acaaggacga
     1981 tgatgataag gactacaaag atgatgatga caaagattat aaggacgatg atgataaagg
     2041 tcatcatcat catcatcact aactcgagtg gcataaagca atcttgatga ggataatgat
     2101 ttttttttga atatacataa atactaccgt ttttctgcta gattttgtga tgacgtaaat
     2161 aagtacatat tactttttaa gccaagacaa gattaagcat taactttacc cttttctttc
     2221 taagtttcaa tattagttat cactgtttaa aagttatggc gagaacgtcg gcggttaaaa
     2281 tatattaccc tgaacggctg gatgtgagac gataaatcgc agccaagtga gtgaatagat
     2341 gacgcaccac ggtcagacac ggccacatcg tatctcacag gagcaagcgc gataggagca
     2401 ctcacacata gtacggtgat ccgctgactc ctttgcccaa ataagacgtg agccactagt
     2461 gcactgcagt acatcgagga gaacttctag tatatctaca tacctaatat tattgcctta
     2521 ttaaaaatgg aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc
     2581 tgtacttcct tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat
     2641 ttctcaacaa gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat
     2701 cttgaccgca gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc
     2761 ttagcaacca ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag
     2821 aatcaaattc gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc
     2881 tttttcaact gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca
     2941 tatagaatag agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat
     3001 attatttaag gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac
     3061 ttttcttacc ttttacattt cagcaatata tatatatata tttcaaggat ataccattct
     3121 aatgtctgcc cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc aagaaatcac
     3181 agccgaagcc attaaggttc ttaaagctat ttctgatgtt cgttccaatg tcaagttcga
     3241 tttcgaaaat catttaattg gtggtgctgc tatcgatgct acaggtgttc cacttccaga
     3301 tgaggcgctg gaagcctcca agaaggctga tgccgttttg ttaggtgctg tgggtggtcc
     3361 taaatgggga accggtagtg ttagacctga acaaggttta ctaaaaatcc gtaaagaact
     3421 tcaattgtac gccaacttaa gaccatgtaa ctttgcatcc gactctcttt tagacttatc
     3481 tccaatcaag ccacaatttg ctaaaggtac tgacttcgtt gttgtcagag aattagtggg
     3541 aggtatttac tttggtaaga gaaaggagga cgatggtgat ggtgtcgctt gggatagtga
     3601 acaatacacc gttccagaag tgcaaagaat cacaagaatg gccgctttca tggccctaca
     3661 acatgagcca ccattgccta tttggtcctt ggataaagct aatgttttgg cctcttcaag
     3721 attatggaga aaaactgtgg aggaaaccat caagaacgaa tttcctacat tgaaggttca
     3781 acatcaattg attgattctg ccgccatgat cctagttaag aacccaaccc acctaaatgg
     3841 tattataatc accagcaaca tgtttggtga tatcatctcc gatgaagcct ccgttatccc
     3901 aggttccttg ggtttgttgc catctgcgtc cttggcctct ttgccagaca agaacaccgc
     3961 atttggtttg tacgaaccat gccacggttc tgctccagat ttgccaaaga ataaggtcaa
     4021 ccctatcgcc actatcttgt ctgctgcaat gatgttgaaa ttgtcattga acttgcctga
     4081 agaaggtaag gccattgaag atgcagttaa aaaggttttg gatgcaggta tcagaactgg
     4141 tgatttaggt ggttccaaca gtaccaccga agtcggtgat gctgtcgccg aagaagttaa
     4201 gaaaatcctt gcttaacctg gaggaccctt ctctttagac tattctactc ttatgcacgt
     4261 aaaaaattct aggaaatatg tattaactag gagtaaaata accggctagt ggcattcata
     4321 tagccgtctg tttacatcta catcacacat ttcgagtgta tatctcgcaa cgttggcgtt
     4381 aaataggcag gagtatcacg tgctataaaa ataattataa tttaaatttt ttaatataaa
     4441 tatataaatt aaaaatagaa agtaaaaaaa gaaattaaag aaaaaatagt ttttgttttc
     4501 cgaagatgta aaagactcta gggggatcgc caacaaatac taccttttat cttgctcttc
     4561 ctgctctcag gtattaatgc cgaattgttt catcttgtct gtgtagaaaa ccacacacga
     4621 aaatcctgtg attttacatt ttacttatcg ttaatcgaat gtatatctat ttaatctgct
     4681 tttcttgtct aataaatata tatgtaaagt acgctttttg ttgaaatttt ttaaaccttt
     4741 gtttattttt ttttcttcat tccgtaactc ttctaccttc tttatttact ttctaaaatc
     4801 caaatacaaa acataaaaat aaataaacac agagtaaatt cccaaattat tccatcatta
     4861 aaagatacga ggcgcgtgta agttacaggc aagcgatccg tcccgagcgg ccgcgattat
     4921 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4981 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5041 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5101 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcgg gacccacgct
     5161 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5221 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5281 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5341 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5401 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5461 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5521 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5581 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5641 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5701 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5761 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5821 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5881 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5941 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6001 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6061 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtgcggcc gc
//
