The pET-35b(+) vector is designed for expression of CBDclos fusion proteins.A variety of cloning sites ａnd strategies are available. Rapid directional cloning of PCR-amplified DNA is available with the pET-35 Xa/LIC Vector Kit, which contains linearized pET-35b(+) ready for annealing with appropriately prepared inserts. When cloned with the Xa/LIC method, resulting CBDclos•Tag™ fusion proteins can be cleaved precisely at the vector-encoded junction using Factor Xa. Unique sites are shown on the circle map. Note that the sequence is num-bered 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.
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