Research Article

Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters

Figure 1

Physical map of the E. coli-P. pastoris shuttle plasmid pPPE17 and its derivatives. The restriction enzyme sites shown are unique. Abbreviations: : P. pastoris AOX1 promoter; : Zeocin resistance gene; AOX1 TT: P. pastoris AOX1 transcription termination region; : yeast promoter TEF1; : bacterial promoter EM7; : kanamycin resistance gene (confers resistance against kanamycin in bacteria and G418 in yeast); PARS1: P. pastoris autonomous replication sequence; pUC origin: bacterial replication sequence; : ampicillin resistance gene (bla). DNA sequence of the 3′-part of the wild-type promoter (base pairs 670 to 950) is displayed above the plasmid map. The promoter core region is shown in bold (see also text). The transcriptional start site (A) and the putative TATA-box (TATATAAA) are highlighted in grey. The BspHI (TCATGA) restriction enzyme site is written in bold and underlined and is deleted by the introduction of a point mutation (TAATGA) in plasmids pPPE20 and pPPE50. GGATGA is mutated to the KpnI (GGTACC) restriction enzyme site in plasmids pPPE50. TAACCC is mutated to the EcoRI (GAATTC) restriction enzyme site in plasmids pPPE20 and pPPE50. CCAATT is mutated to the NheI (GCTAGC) restriction enzyme site in plasmid pPPE20 and pPPE50.
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