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--4.16; BglI--0.87, 3.19, 3.42; BglII--3.99; EcoRI--4.67; HindIII--4.64; NcoI--4.16; NdeI--4.09; PstI--0.75; PvuI--0.62; PvuII--2.29; SalI--3.71; XbaI--4.05; XhoI--4.15. 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. Expression vector for production of His-tagged fusion proteins, which contain two tandem phosphorylation sites, useful for radiolabeling. Fusion proteins can be purified using a nickel-chelating column, even under denaturing conditions. Constructed from pET-His (ATCC 87036) by insertion of two tandem consensus phosphorylation sites for heart muscle kinase (HMK) between the His coding region and the XhoI site of the polylinker. |
