Restriction digests of the clone give the following sizes (kb): EcoRI--4.6; BamHI--4.6; BglI--2.5, 2.1, 0.25. The plasmid contains the following restriction sites (kb from nt 1): BamHI--4.12; BglI--0.87, 3.19, 3.42; BglII--3.99; EcoRI--4.63; HindIII--4.60; NcoI--4.12; NdeI--4.09; PstI--0.75; PvuI--0.62; PvuII--2.29; SalI--3.71; XbaI--4.05; XhoI--4.11. Digestion with NcoI, BamHI or XhoI and subsequent filling of ends, allows cloning of blunt-ended fragments with a +0, +1 (C) or +2 (GA) nucleotide reading frame, respectively. Fusion proteins can be purified using a nickel-chelating column, even under denaturing conditions. Constructed from pET3a by insertion of a double stranded oligonucleotide linker between the NdeI and BamHI sites, creating three restriction sites and encoding six His residues. |
