We describe the construction of a novel herpes simplex virus (HSV) vector containing a unique I restriction enzyme cloning site in an intergenic position in the short unique genome region. Sequences can be inserted at this site with high efficiency by ligation with I-digested vector DNA. A series of plasmids has been designed for use with this vector. These allow protein coding sequences to be placed under the control of various transcriptional regulation signals and then isolated as I fragments ready for insertion into the vector. The I fragments also contain the β-galactosidase gene thereby facilitating selection of recombinant virus by screening for blue plaques. A variant of the vector has been made based on the temperature-sensitive (ts) mutant tsK, which expresses only immediate early (IE) genes at non-permissive tempratures. Chloramphenicol acetyltransferase was used as a reporter gene to assess the fidelity of expression of sequences cloned into this position. Under these circumstances IE and early HSV promoters were shown to behave as expected in both wild-type and ts vectors.


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