Pcdna3.1 v5 his expression vector

A putative S. mansoni Akt sequence was identified in Genbank (XM_002578520.1) by tBLASTp analyzes of the S. mansoni genome data bases (Berriman et al., 2009 (link)). 5′ and 3′ UTR were determined with the SmAkt encoding ESTs AM042871.1, CF497410.2, AA169931.1 and CF498818.1. The cDNA sequence of SmAkt was obtained by PCR amplification of total cDNA obtained from adult S. mansoni RNA (prepared using TRIzol® reagent, Invitrogen) by reverse transcription using the Superscript III (Invitrogen). The SmAkt sequence was amplified using SmAktFLf (5′-CGGCACGAGGCCAAGTCTTAAATGCTAGT-3′) and SmAktFLr (5′-GTTAAATCATGTTGGTGGCAGTCAATTGAACT-3′) primers, cloned into a pCR2.1 TOPO cloning vector (Invitrogen) and sequenced (EurofinsDNA). A second PCR was performed using as primers SmAktFLRE-f (5′-CCggatccGTTATCGAGATTGCAGATTTTCTGGG-3′) and SmAktFLRE-r (5′-GCctcgagAAAATGTGTCACCAAAACTATAACCAC-3′) containing, respectively, BamHI and XhoI restriction sites and the fragment was inserted in frame in the pcDNA3.1-V5/His expression vector (Invitrogen). Site-directed mutagenesis was performed on the wild-type (WT) SmAkt construct to produce SmAkt E₁₁₇K and SmAkt L₁₅₀R active mutants using, respectively, the 5′-GGCTTATGAAACGCGGCAAACATATTAAAAATTGGCGACG-3′ and 5′-TAAAGATGATATGGCGCAACCTCGAAATAATTTTACTGTTCGCG-3′ mutated sequences and their reverse complement as primers.