The insert contains internal SacI, AccI, HincII and PstI sites. Restriction digests of the clone give the following sizes (kb): BamHI/XbaI--4.65; BamHI--4.65; XbaI--uncut; PstI--4.1, 0.65; EcoRI--4.75; EcoRI/HindIII--3.05, 1.6; BamHI/HindIII--3.05, 1.6. Plasmid must be transformed into a dam- Escherichia coli strain - otherwise XbaI will not cut. Linearize the plasmid with XbaI for in vitro transcription. This is a full-length clone of virus segment S8. This construct gives a transcript with termini identical to the authentic S8 plus-strand sequence (5'-GGUAUU---UGAU-3') when linearized with XbaI for transcription and subsequent mung bean nuclease digestion. The cDNA was tailored to a 5'-terminal BamHI site followed by the T7 promoter fused to the S8 5'-end. The 3'-end was tailored to an XbaI site. This construct was cloned into BamHI-XbaI digested pSP64. |
