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. 2003 Sep 15;31(18):5221–5228. doi: 10.1093/nar/gkg743

Figure 2.

Figure 2

Plasmid substrates. The plasmid pAT153 has a single recognition site for BspMI denoted by the arrowhead (the tip points to the site of cleavage) and further restriction sites at the positions shown. To make pNAG1, pAT153 was cleaved with HindIII and BamHI and ligated to a duplex (thick line) made from two 32 base oligonucleotides: the duplex contained a double-stranded segment of 28 bp that has a recognition site for BspMI (arrowhead, orientation as indicated) and 5′ single-strand extensions of 4 bases at both ends, which match, respectively, HindIII and BamHI termini. The plasmid pNAG2 was constructed in an identical manner except that the sequence of the double-stranded segment of the duplex was reversed relative to the duplex in pNAG1. The plasmids pNAG4 and pNAG5 were constructed from pNAG1 and pNAG2, respectively, by inserting at their PshAI sites a 905 bp fragment from pBR322 (zigzag line) that lacks BspMI sites.