Restriction digests of the clone give the following sizes (kb): HindIII, BamHI--3.5, 2.7; EcoRI, BglII--5.6, 0.6; BamHI--6.2; MluI--6.2. Linearizing with MluI before transfection is essential for efficient integration. The MluI site is in the center of the tubulin intergenic sequence. Shuttle vector for integration by homologous recombination targeted to the beta- alpha-tubulin intergenic region of Trypanosoma brucei. Constructed from the PARP promoter and 3' splice site from BNsp-CAT, neo (BglII/BamHI fragment) from pSV2neo, and a tubulin intergenic region (639 bp, blunted EcoRI/BglII) inserted into the SmaI site 3' to the termination codon of neo. |
