Fig. 1
From: HisAK70: progress towards a vaccine against different forms of leishmaniosis
Schematic diagram of the construction of pVAX::HISAK70-asd and pVAX-asd. The kanamycin resistance gene in the eukaryotic expression vectors was replaced by the asd gene. a To generate pVAX::HISAK70 without kanamycin resistance cassette (Kanr), inverse PCR with primers noKanPacI-1R 5’-CTTGTTTAATTAA GCGAAACGATCCTCATCCTGTC-3’ (PacI site underlined) and noKanKpnI-1D 5’-GACGAGGGTACCATTATTAACGCTTACAATTTC-3’ (KpnI site underlined) were employed for outward amplification. The asd gene starting at bp 170 and ending at bp 1345 was PCR amplified from bacterial chromosomal with primers asdKpnI-1D 5’ CTGCAAGGTACCCTACGCCAACTGGCGCAGCAT-3’ (KpnI site underlined) and the asdPacI-1R 5’-TTGGCTGTTAATTAAATGGTGAAGGATGCGCCACAG-3’ (PacI site underlined) creating an amplicon with PacI and KpnI sites on its ends. b To generate pVAX1 without Kanr, inverse PCR with primers noKanPacI-1R and noKanKpnI-1D were employed for outward amplification. c–d The PCR products of asd, ∆kanrpVAX1::HISAK70 and ∆kanrpVAX1 were each double digested with PacI and KpnI, gel isolated, ligated, and transformed. Thus, pVAX1::HisAK70-asd and pVAX1-asd without resistance gene were obtained