The pET-44 Ek/LIC Vector is prepared for rapid, directional cloning of PCR-amplified DNA for high-level expression of polypeptides fused with N-terminal His?Tag®,Nus?Tag? and S?Tag? sequences. Using specifically designed primers for amplification and the pET-44 Ek/LIC Vector Kit (Cat. No. 71144-3), inserts can be efficiently cloned without the need for restriction digestion or ligation. 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 COLIDOWN Primer (Cat. No. 70845-3). Vector encoded sequences can be completely removed when cloning into the Ek/LIC site (as shown below left) by cleaving the fusion protein with enterokinase.