The pET-32 Ek/LIC vector is prepared for rapid, directional cloning of PCR-amplified DNA for high-level expression of polypeptides fused with the 109aa Trx?Tag? thioredoxin protein. Using specifically designed primers for amplification and the pET-32 Ek/LIC Cloning Kit (Cat. No. 69076-3), inserts can be efficiently cloned without the need for restriction digestion or ligation. Fusion proteins also contain cleavable His?Tag® and S?Tag? sequences for detection and purification. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circle map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single stranded DNA that corresponds to the coding strand. Therefore, single stranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).
