extension at the C-terminus (TPGRPAAKLN) corresponding to the fragment of the pQE30 plasmid polylinker
PCR products were digested with HindIII and EcoRV and cloned into the polylinker
region of the mammalian cell expression vector pcDNA3 (Invitrogen, San Diego, CA).
Subsequent sequencing of the 3' end of the insert and the flanking genomic DNA indicated that the polylinker
sequence containing a HindIII site was truncated by 10 bp.
8 kb of Pu DNA subcloned into the EcoRI restriction site within the polylinker region of pBluescribe (Stratagene) (McLean et al.
The gel purified insert DNA was ligated overnight into the EcoRI-XbaI restriction sites within the polylinker region of the pJY505 (16[degrees]C; ratio of insert DNA to vector DNA was approximately 10:1).
This 812-bp linked sequence was cloned into the polylinker
region of the pSP64polyA plasmid.
sites permitted directional cloning into single-stranded M13 bacteriophages (Medlin et al.
This includes polylinker
sequence and the first 166 base pairs of P-Ract1 (positions -835 to -670 from the start of transcription as defined by McElroy et al.
Vector pAMV75 contains the AMV NZ1 CP coding region inserted in the pGA643 polylinker
downstream from the CaMV 35S promoter.
NIa fragments were cloned into the appropriate sites of the polylinker of the plasmid pBluescript KS+ (Stratagene, La Jolla, CA) (named pTVNIa).
The sequence encoding PVY-NIa was excised from the pPVNIa clone using XhoI and XbaI and ligated into like sites in the polylinker of pKYLX71:35S2.