Restriction digests of the clone give the following sizes (kb): SalI--3.1, 0.5; HindIII--3.6; BamHI/PvuII--2.5, 0.8, 0.25. The insert contains the following restriction sites (approximate kb from the 5' end): PstI--0.15, 0.61; SacII--0.59; SalI--0.21. The insert gene was cloned by PCR amplification of a provirus isolated from a naturally infected cow. The insert contains artificially introduced start and stop codons flanking the coding region. The insert also contains a BamHI site, immediately upstream from the start codon and a PvuII site, immediately downstream from the ochre stop codon introduced by PCR. The Pfu polymerase-generated blunt end PCR product was cloned into the EcoRV site of pBluescript SK+ and the EcoRV site was destroyed after cloning. |
