Lentiviral vectors are an important tool for gene delivery in vivo and in vitro. The success of gene transfer approaches relies on high and stable levels of gene expression. To this end, several molecular strategies have been employed to manipulate these vectors towards improving gene expression in the targeted animal cells. Low gene expression can be accepted due to the weak transcription from the majority of available mammalian promoters; however, this obstacle can be in part overcome by the insertion of cis-acting elements that enhance gene expression in various expression contexts. In this work, we created different lentiviral vectors in which several posttranscriptional regulatory elements, namely the Woodchuck hepatitis posttranscriptional regulatory element (WPRE) and different specialized poly(A) termination sequences (BGH and SV40) were used to develop vectors leading to improved transgene expression. These vectors combine the advantages of restriction enzyme/ligation-independent cloning eliminating the instability and recombinogenic problems occurring from traditional cloning methods in lentiviral expression vectors and were tested by expressing GFP and the firefly Luciferase reporter gene from different cellular promoters in different cell lines. We show that the promoter activity varies between cell lines and is affected by the lentiviral genomic context. Moreover, we show that the combination of the WPRE element with the BGH poly(A) signal significantly enhances transgene expression. The vectors herein created can be easily modified and adapted without the need for extensive recloning making them a valuable tool for viral vector development.