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. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: Yeast. 2012 Jun 6;29(7):275–291. doi: 10.1002/yea.2908

Figure 1.

Figure 1

Plasmids and integration strategies. Tetracycline inducible I-PpoI integration plasmids are shown in (A) and (B), I-PpoI cleavage site integration plasmids in (C), and integration strategies in (D) and (E) (not drawn to scale). (A) The I-PpoI ORF was cloned under the control of the CaMV35S promoter (p) and the nmt1 terminator (t) in the pDUAL-tet-rpsL-neo vector (Erler et al., 2006) to create pSS12. The plasmid also contains a tetracycline repressor (TetR) that is constitutively expressed from an ADH1 promoter and terminator. The TETp-I-PpoI NotI fragment from pSS12 (sites shown) is excised and integrated at leu1-32 as detailed (Matsuyama et al., 2004). (B) A second I-PpoI expression plasmid was created by transferring the entire TETp-I-PpoI control unit from pSS12 into a plasmid such that it was flanked by a clonNAT selectable marker (nat+) and sequences homologous to the regions immediately up (5′) and downstream (ORF) of the arg3+ ATG start codon. The TETp-I-PpoI NotI fragment is excised and integrated as shown in (D). PCR primers (355/512 Table I) used to verify correct integration at arg3+ are illustrated as half arrows. (C) To integrate an I-PpoI cleavage site at ura5+, pSS21 was created such that a single cleavage site was flanked by a hygromycin B selectable marker (hph+) marker and sequences homologous to the regions immediately up (5′) and downstream (ORF) of the ura5+ ATG start codon. A NotI fragment with the cleavage site (black triangle) is then excised and integrated at ura5+ as shown in (E). PCR primers used to verify correct integration across each junction (396/611 and 397/612 Tabel I) are illustrated. The pSS23 plasmid was created in a similar fashion for integration of a single I-PpoI cleavage site immediately upstream of the lys1+ ATG start codon (E). Verification primers for lys1+ integration correspond to 396/844 and 397/356 (Table I). For (B) and (C) plasmids, integrations produce an auxotrophic phenotype for the corresponding amino acid that can be used as a second means to verify correct integration. See Material and methods for further details.