One of the widely used shRNA expression vectors is pSuper described in 2002. It uses Pol III promoter H1 to transcribe a shRNA with a 21 bp stem and 9 nt loop structure. Furthermore, the first widely used genome-wide shRNA library utilized a lentiviral AbMole (R)-(-)-Modafinic acid vector named pLKO.1-puro and this library has generated much of data that allowed for a better understanding of the diverse cellular processes associated with virology and cancer. We AbMole Corosolic-acid observed that the pLKO.1-puro vector possessed a unique palindromic loop different from other shRNA expression vectors such as pSuper. This observation resulted in the hypothesis that a shRNA structure could be constructed using only a single long or two short oligonucleotides. We further describe a strategy for rapid cloning of multiple shRNAs which permits easier combination of the most efficient promoter-shRNA cassettes for the simultaneous knockdown of multiple genes or different targets of the same gene. Here we gave proofs that our thought was feasible, and a shRNA could be constructed by only 1 long oligonucleotide or 2 short oligonucleotides with half the cost of conventional shRNA clone methods. Various parameters for the design of effective shRNAs based on our strategy were compared such as the palindromic loop sequences. Finally, the loop sequence “TTCTAGAA” was selected for shRNAs construction and then we gave examples that our method could apply to other genes such as the bacterial enzyme b-galactosidase. This method was also used to successfully inhibit hepatitis B virus antigen expression both in vitro and in vivo. This approach is cost effective and more easily applied to many areas of basic or applied research utilizing RNAi technology. In a previous report that used the pSuper vector, shRNA efficiency was also influenced by the position of the antisense and sense strands. We therefore cloned an shRNA named SA1856 with an sense-antisense stem also containing the TTCTAGAA loop and assessed its ability to inhibit HBsAg and HBeAg expression compared to the AS isoform. In 3 independent experiments, we did not identify discernible differences in the inhibition rates between these constructs, but the other two shRNAs with different stem structures targeting another HBV sequence showed a different result. In this study, we have constructed a shRNA vector with several characteristics for its better and easier use. First, we employed the H1 RNA polymerase III promoter instead of U6 whose toxicity has been previously identified. The Pol III H1 promoter has a well-defined transcription start site proven to be more flexible than the U6 promoter with regard to +1 sequence changes. Second, proper isocaudomers were used for easier cascade connected shRNAs construction. Third, the CMVemGFP cassette was used to track shRNA transfection. This cassette could be replaced easily by other therapeutic genes as a means of overexpressing one gene while concomitantly knocking down another gene. The resulting vector was named pshOK-basic. For efficient and lower-cost shRNA construction, several routes was reported previously including the one-oligonucleotide method combined with PCR or the four short oligonucleotides based strategy. The most significant advantage to the method described in this study is that only single long or two short oligonucleotides were required for cloning shRNAs. To accomplish this goal, a unique palindromic shRNA scaffold was screened and optimized.