Restriction digests of the clone give the following sizes (kb): XbaI/SphI--3.4, 1.15; SalI--4.4; BglII--4.4. The insert contains the following restriction sites (bp from the 5' XbaI site): DraI--321; SalI--602; BglII--791. The primer-derived NsiI site is at nt 1059. Clones pSTMV5 and pSTMV6 (ATCC 45135, 45136) can be distinguished by RNase protection assays which give distinct patterns due to an A-C difference at nt 751 of the STMV sequence. The RNA transcribed from this clone is infectious. Contains a complete genome representing a less abundant genome type in the STMV type strain. pSTMV6 is identical to the reported STMV type strain sequence except for a G insertion at the 5' end and three single-base differences: C to U at nt 682, A to C at nt 751, and U to C at nt 805. Linearize with NsiI and transcribe with T7 RNA polymerase to produce a sense strand. Constructed using primers for cDNA synthesis that added NsiI and SphI sites at the 3' end and an XbaI site at the 5' end. |
