Pcmv6 ac gfp survivin

Briefly, plasmid encoding the entire coding region of human BIRC5 (coding for survivin) gene (pCMV6-AC-GFP-Survivin, sequence verified, cat# RG205935) was purchased from OriGene (Rockville, MD, USA). Construction of the antisense survivin gene (AS-Sur) with extra BspHI and EcoRI enzymatic cleavage site located on the 5′ and 3′ end, respectively, was carried out by PCR (95°C for 30 seconds, followed up by 68°C for 30 seconds, then 72°C for 30 seconds, for 30 cycles) using the forward primer 5′-AATCATGAATCCATGGCAGCCAG-3′ and the reverse primer 5′-AAGAATTCATGGGTGCCCCGA-3′. The PCR product (AS-Sur) was ligated to the PCR products cloning vector pJET1.2 (cat# K1231, Thermo Fisher Scientific) and then transformed into DH5-α Escherichia coli cells. DNA sequencing was preformed to validate the sequence of the recombinant AS-Sur gene. AS-Sur was excised by digestion with BspHI and EcoRI, and subcloned into the LacZ gene-removed pDRIVE-hSurvivin, which is a mammalian transfectable vector bearing a human survivin promoter. The final product, pSur/AS-Sur, was transformed into DH5-α E. coli cells for long-term storage.