LOCUS       sequence-421722-        5700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A MoClo Yeast Toolkit LEU2, CEN/ARS empty vector (GFP dropout). 
            Contains ConL1, part234 GFP dropout, ConR2, LEU2, CEN6/ARS4, 
            AmpR-ColE1. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5700)
  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 5700)
  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: pYSD099
FEATURES             Location/Qualifiers
     source          1..5700
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             354..365
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             372..1085
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1097..1168
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1184..1211
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        1642..2049
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2050..3144
                     /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    3323..3826
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(join(3937..4728,4729..4797))
                     /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     4560..4579
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4798..4902)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5005..5593
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5494..5513
                     /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 ctacaacgtg agaccgaaag tgaaacgtga tttcatgcgt cattttgaac
      241 attttgtaaa tcttatttaa taatgtgtgc ggcaattcac atttaattta tgaatgtttt
      301 cttaacatcg cggcaactca agaaacggca ggttcggatc ttagctacta gagaaagagg
      361 agaaatacta gatgcgtaaa ggcgaagagc tgttcactgg tgtcgtccct attctggtgg
      421 aactggatgg tgatgtcaac ggtcataagt tttccgtgcg tggcgagggt gaaggtgacg
      481 caactaatgg taaactgacg ctgaagttca tctgtactac tggtaaactg ccggttcctt
      541 ggccgactct ggtaacgacg ctgacttatg gtgttcagtg ctttgctcgt tatccggacc
      601 atatgaagca gcatgacttc ttcaagtccg ccatgccgga aggctatgtg caggaacgca
      661 cgatttcctt taaggatgac ggcacgtaca aaacgcgtgc ggaagtgaaa tttgaaggcg
      721 ataccctggt aaaccgcatt gagctgaaag gcattgactt taaagaggac ggcaatatcc
      781 tgggccataa gctggaatac aattttaaca gccacaatgt ttacatcacc gccgataaac
      841 aaaaaaatgg cattaaagcg aattttaaaa ttcgccacaa cgtggaggat ggcagcgtgc
      901 agctggctga tcactaccag caaaacactc caatcggtga tggtcctgtt ctgctgccag
      961 acaatcacta tctgagcacg caaagcgttc tgtctaaaga tccgaacgag aaacgcgatc
     1021 atatggttct gctggagttc gtaaccgcag cgggcatcac gcatggtatg gatgaactgt
     1081 acaaatgacc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1141 tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg
     1201 gcctttctgc gtttataggt ctcagctgga tgtgagacga taaatcgcag ccaagtgagt
     1261 gaatagatga cgcaccacgg tcagacacgg ccacatcgta tctcacagga gcaagcgcga
     1321 taggagcact cacacatagt acggtgatcc gctgactcct ttgcccaaat aagacgtgag
     1381 ccactagtgc actgcagtac atcgaggaga acttctagta tatctacata cctaatatta
     1441 ttgccttatt aaaaatggaa tcccaacaat tacatcaaaa tccacattct cttcaaaatc
     1501 aattgtcctg tacttccttg ttcatgtgtg ttcaaaaacg ttatatttat aggataatta
     1561 tactctattt ctcaacaagt aattggttgt ttggccgagc ggtctaaggc gcctgattca
     1621 agaaatatct tgaccgcagt taactgtggg aatactcagg tatcgtaaga tgcaagagtt
     1681 cgaatctctt agcaaccatt atttttttcc tcaacataac gagaacacac aggggcgcta
     1741 tcgcacagaa tcaaattcga tgactggaaa ttttttgtta atttcagagg tcgcctgacg
     1801 catatacctt tttcaactga aaaattggga gaaaaaggaa aggtgagagc gccggaaccg
     1861 gcttttcata tagaatagag aagcgttcat gactaaatgc ttgcatcaca atacttgaag
     1921 ttgacaatat tatttaagga cctattgttt tttccaatag gtggttagca atcgtcttac
     1981 tttctaactt ttcttacctt ttacatttca gcaatatata tatatatatt tcaaggatat
     2041 accattctaa tgtctgcccc taagaagatc gtcgttttgc caggtgacca cgttggtcaa
     2101 gaaatcacag ccgaagccat taaggttctt aaagctattt ctgatgttcg ttccaatgtc
     2161 aagttcgatt tcgaaaatca tttaattggt ggtgctgcta tcgatgctac aggtgttcca
     2221 cttccagatg aggcgctgga agcctccaag aaggctgatg ccgttttgtt aggtgctgtg
     2281 ggtggtccta aatggggaac cggtagtgtt agacctgaac aaggtttact aaaaatccgt
     2341 aaagaacttc aattgtacgc caacttaaga ccatgtaact ttgcatccga ctctctttta
     2401 gacttatctc caatcaagcc acaatttgct aaaggtactg acttcgttgt tgtcagagaa
     2461 ttagtgggag gtatttactt tggtaagaga aaggaggacg atggtgatgg tgtcgcttgg
     2521 gatagtgaac aatacaccgt tccagaagtg caaagaatca caagaatggc cgctttcatg
     2581 gccctacaac atgagccacc attgcctatt tggtccttgg ataaagctaa tgttttggcc
     2641 tcttcaagat tatggagaaa aactgtggag gaaaccatca agaacgaatt tcctacattg
     2701 aaggttcaac atcaattgat tgattctgcc gccatgatcc tagttaagaa cccaacccac
     2761 ctaaatggta ttataatcac cagcaacatg tttggtgata tcatctccga tgaagcctcc
     2821 gttatcccag gttccttggg tttgttgcca tctgcgtcct tggcctcttt gccagacaag
     2881 aacaccgcat ttggtttgta cgaaccatgc cacggttctg ctccagattt gccaaagaat
     2941 aaggtcaacc ctatcgccac tatcttgtct gctgcaatga tgttgaaatt gtcattgaac
     3001 ttgcctgaag aaggtaaggc cattgaagat gcagttaaaa aggttttgga tgcaggtatc
     3061 agaactggtg atttaggtgg ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa
     3121 gaagttaaga aaatccttgc ttaacctgga ggacccttct ctttagacta ttctactctt
     3181 atgcacgtaa aaaattctag gaaatatgta ttaactagga gtaaaataac cggctagtgg
     3241 cattcatata gccgtctgtt tacatctaca tcacacattt cgagtgtata tctcgcaacg
     3301 ttggcgttaa ataggcagga gtatcacgtg ctataaaaat aattataatt taaatttttt
     3361 aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt
     3421 ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct
     3481 tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaaaacc
     3541 acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt
     3601 aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt
     3661 aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt
     3721 ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc
     3781 catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc ccgagcggcc
     3841 gcgattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3901 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3961 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4021 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggga
     4081 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4141 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4201 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4261 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4321 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4381 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4441 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4501 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4561 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4621 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4681 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4741 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4801 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4861 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4921 gccacctgtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4981 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5041 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5101 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     5161 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5221 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5281 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5341 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5401 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5461 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5521 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5581 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5641 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtgcggccgc
//
