Fig. 1 Concept and engineering of pFila.
(a) In this reaction, the primary process was the production of Fluc gene by Pf (Pf is composed of P1 and P2, which are complementary to upstream of Fluc gene and pRL-TK plasmid, respectively) and Pr (Pr is composed of P3 and P4, which are complementary to downstream of Fluc gene and pRL-TK plasmid, respectively) primers. In the secondary process, amplified Fluc gene bearing a deliberately designed region annealed with its homologous sequence in pRL-TK, thus generating a large linear fragment. In order to exponentially amplify the linear fragment, the outermost Pf and P2R (complementary to P2) primer pair initiated the ternary process to produce adequate fused fragment.
(b) Duplex bridge PCR. The 2.4 kb fragment was Fluc gene, while the 6.4 kb fragment was the desired fusion product. M is 1 kb DNA ladder.
(c) Map of pFila. The engineered pFila is 6486 bp in length. XbaI and ApaI restriction sites are located in the 3’UTR of Rluc to facilitate the cloning of miRNA target for reporter assay.
(d) 5’ and 3’ seaming sites of Fluc into pRL-TK. For detailed information, please refer to Gene sequence 1.