LOCUS       sequence-421715-        6481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the mRuby2 fluorescent protein (Part3b') with the Aga2 
            signal peptide (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 6481)
  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 6481)
  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: pYSD141
FEATURES             Location/Qualifiers
     source          1..6481
                     /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             962..1669
                     /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             1676..1747
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             1751..1768
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        2423..2830
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2831..3925
                     /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    4104..4607
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(join(4718..5509,5510..5578))
                     /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     5341..5360
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5579..5683)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5786..6374
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6275..6294
                     /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 atctatgatg cagttacttc gctgtttttc aatattttct gttattgctt cagttcttgc
      961 tgtgtccaaa ggagaggagt taatcaagga aaacatgaga atgaaagttg tcatggaggg
     1021 ctccgttaat ggtcaccaat tcaagtgtac aggggaaggt gaaggtaatc cttacatggg
     1081 tacacaaact atgagaatta aagtaattga aggcggacca ctaccatttg catttgacat
     1141 tctggcaacg tcattcatgt acggatcacg aactttcatc aagtacccta aaggtatacc
     1201 agactttttc aagcaatctt ttccagaggg ttttacatgg gaaagggtta caagatacga
     1261 agatgggggt gtcgtcacag ttatgcaaga tacttcatta gaagatggct gccttgtcta
     1321 tcatgtgcaa gtaagagggg tgaattttcc ttctaacgga cctgtgatgc agaaaaagac
     1381 caaaggttgg gaaccaaata ctgaaatgat gtacccagct gatggaggtt tgagaggcta
     1441 cacacacatg gcgcttaaag ttgatggtgg aggtcatttg tcttgtagtt ttgttaccac
     1501 ttatcgttct aaaaagactg ttggcaatat caaaatgcca ggaatacatg ctgtagacca
     1561 cagactagaa agactcgaag agagcgataa cgaaatgttc gttgtacaga gagagcatgc
     1621 cgtagccaaa tttgctggct taggcggtgg tatggatgaa ttgtataagg gatccgatta
     1681 caaggacgat gatgataagg actacaaaga tgatgatgac aaagattata aggacgatga
     1741 tgataaaggt catcatcatc atcatcacta actcgagtgg cataaagcaa tcttgatgag
     1801 gataatgatt tttttttgaa tatacataaa tactaccgtt tttctgctag attttgtgat
     1861 gacgtaaata agtacatatt actttttaag ccaagacaag attaagcatt aactttaccc
     1921 ttttctttct aagtttcaat attagttatc actgtttaaa agttatggcg agaacgtcgg
     1981 cggttaaaat atattaccct gaacggctgg atgtgagacg ataaatcgca gccaagtgag
     2041 tgaatagatg acgcaccacg gtcagacacg gccacatcgt atctcacagg agcaagcgcg
     2101 ataggagcac tcacacatag tacggtgatc cgctgactcc tttgcccaaa taagacgtga
     2161 gccactagtg cactgcagta catcgaggag aacttctagt atatctacat acctaatatt
     2221 attgccttat taaaaatgga atcccaacaa ttacatcaaa atccacattc tcttcaaaat
     2281 caattgtcct gtacttcctt gttcatgtgt gttcaaaaac gttatattta taggataatt
     2341 atactctatt tctcaacaag taattggttg tttggccgag cggtctaagg cgcctgattc
     2401 aagaaatatc ttgaccgcag ttaactgtgg gaatactcag gtatcgtaag atgcaagagt
     2461 tcgaatctct tagcaaccat tatttttttc ctcaacataa cgagaacaca caggggcgct
     2521 atcgcacaga atcaaattcg atgactggaa attttttgtt aatttcagag gtcgcctgac
     2581 gcatatacct ttttcaactg aaaaattggg agaaaaagga aaggtgagag cgccggaacc
     2641 ggcttttcat atagaataga gaagcgttca tgactaaatg cttgcatcac aatacttgaa
     2701 gttgacaata ttatttaagg acctattgtt ttttccaata ggtggttagc aatcgtctta
     2761 ctttctaact tttcttacct tttacatttc agcaatatat atatatatat ttcaaggata
     2821 taccattcta atgtctgccc ctaagaagat cgtcgttttg ccaggtgacc acgttggtca
     2881 agaaatcaca gccgaagcca ttaaggttct taaagctatt tctgatgttc gttccaatgt
     2941 caagttcgat ttcgaaaatc atttaattgg tggtgctgct atcgatgcta caggtgttcc
     3001 acttccagat gaggcgctgg aagcctccaa gaaggctgat gccgttttgt taggtgctgt
     3061 gggtggtcct aaatggggaa ccggtagtgt tagacctgaa caaggtttac taaaaatccg
     3121 taaagaactt caattgtacg ccaacttaag accatgtaac tttgcatccg actctctttt
     3181 agacttatct ccaatcaagc cacaatttgc taaaggtact gacttcgttg ttgtcagaga
     3241 attagtggga ggtatttact ttggtaagag aaaggaggac gatggtgatg gtgtcgcttg
     3301 ggatagtgaa caatacaccg ttccagaagt gcaaagaatc acaagaatgg ccgctttcat
     3361 ggccctacaa catgagccac cattgcctat ttggtccttg gataaagcta atgttttggc
     3421 ctcttcaaga ttatggagaa aaactgtgga ggaaaccatc aagaacgaat ttcctacatt
     3481 gaaggttcaa catcaattga ttgattctgc cgccatgatc ctagttaaga acccaaccca
     3541 cctaaatggt attataatca ccagcaacat gtttggtgat atcatctccg atgaagcctc
     3601 cgttatccca ggttccttgg gtttgttgcc atctgcgtcc ttggcctctt tgccagacaa
     3661 gaacaccgca tttggtttgt acgaaccatg ccacggttct gctccagatt tgccaaagaa
     3721 taaggtcaac cctatcgcca ctatcttgtc tgctgcaatg atgttgaaat tgtcattgaa
     3781 cttgcctgaa gaaggtaagg ccattgaaga tgcagttaaa aaggttttgg atgcaggtat
     3841 cagaactggt gatttaggtg gttccaacag taccaccgaa gtcggtgatg ctgtcgccga
     3901 agaagttaag aaaatccttg cttaacctgg aggacccttc tctttagact attctactct
     3961 tatgcacgta aaaaattcta ggaaatatgt attaactagg agtaaaataa ccggctagtg
     4021 gcattcatat agccgtctgt ttacatctac atcacacatt tcgagtgtat atctcgcaac
     4081 gttggcgtta aataggcagg agtatcacgt gctataaaaa taattataat ttaaattttt
     4141 taatataaat atataaatta aaaatagaaa gtaaaaaaag aaattaaaga aaaaatagtt
     4201 tttgttttcc gaagatgtaa aagactctag ggggatcgcc aacaaatact accttttatc
     4261 ttgctcttcc tgctctcagg tattaatgcc gaattgtttc atcttgtctg tgtagaaaac
     4321 cacacacgaa aatcctgtga ttttacattt tacttatcgt taatcgaatg tatatctatt
     4381 taatctgctt ttcttgtcta ataaatatat atgtaaagta cgctttttgt tgaaattttt
     4441 taaacctttg tttatttttt tttcttcatt ccgtaactct tctaccttct ttatttactt
     4501 tctaaaatcc aaatacaaaa cataaaaata aataaacaca gagtaaattc ccaaattatt
     4561 ccatcattaa aagatacgag gcgcgtgtaa gttacaggca agcgatccgt cccgagcggc
     4621 cgcgattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4681 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4741 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4801 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcggg
     4861 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4921 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4981 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5041 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5101 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5161 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5221 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5281 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5341 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5401 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5461 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5521 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5581 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5641 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5701 tgccacctgt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5761 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5821 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5881 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5941 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6001 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6061 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6121 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6181 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6241 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6301 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6361 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6421 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtgcggccg
     6481 c
//
