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. 2016 Aug 12;90(17):7902–7919. doi: 10.1128/JVI.00283-16

FIG 2.

FIG 2

Cloning strategy for generation of a shuttle vector encoding GP85 under Tet-Off (TRE-tight) promoter control. (Steps 1 and 2) Parts of the GP85 (5′) and GP86 (3′) coding sequences were separately cloned via PCR into pUC19 to generate pGP85FLK and pGP86FLK. (Step 3) The GP86 coding sequence was then cloned into pGP85FLK as a BamHI/HindIII fragment to generate pGP85GP86, which lacked the intergenic sequence between GP85 and GP86. (Steps 4 to 6) A BglII Km cassette was introduced to generate pGP8586Km+ (steps 4 and 5), which was digested with SalI to enable cloning of a SV40 poly(A) cassette to generate pGP8586SV40AKm (step 6). (Step 7) The TREtight promoter from pTREtight (Clontech) was PCR cloned as a BamHI fragment immediately upstream of the GP85 coding sequence to generate pGP8586TRESV40AKm. Shuttle vectors (steps 5, 6, and 7) linearized with PmeI were used to modify the GP85/GP86 intergenic locus in the GPCMV BAC via homologous recombination in separate reactions to introduce the modified sequence, as described in Materials and Methods, to generate the DISC GPCMV BAC and other BAC mutants.