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. 2005 Jan;187(2):747–751. doi: 10.1128/JB.187.2.747-751.2005

FIG. 5.

FIG. 5.

Creation of pKV258 and pKV264. 1. The 7.5-kb SphI-BamHI fragment from pKV208 was subcloned into the corresponding sites of pALTER-1 (Promega). 2. Into this was cloned the 1.5-kb BamHI fragment spanning the 5′ end of MPN141. 3. A 7.7-kb BsiWI fragment was removed to reduce the size of the plasmid temporarily for efficient silent mutagenesis. 4. The second and third EcoRI sites in MPN142 were silently changed to GGATTC using an AlteredSites II mutagenesis kit (Promega), and the SphI site was replaced with an MfeI site using complementary synthetic linkers (5′-CAATTGTCATG and 5′-ACAATTGCATG). 5. The resulting 1.4-kb BamHI-MfeI fragment was cloned into the BamHI and EcoRI sites of the Tn4001mod-bearing plasmid pKV74 (5). 6. The 7.7-kb BsiWI fragment was then restored to yield pKV258. To create pKV264, the 4.6-kb BsaBI-StuI fragment in MPN141 was removed to create a massive internal in-frame deletion, as described in the text. B, BamHI; Bi, BsiWI; Bs, BsaBI; E, EcoRI; e, destroyed EcoRI site; S, StuI; Sm/P, SmaI/PmeI junction; M/E, MfeI/EcoRI junction at the former SphI site. Open arrow from Tn4001mod in pKV74 shows the location and direction of the Pout promoter. Note that the inserts are inverted when cloned into Tn4001mod. The figure is not created to scale.