Cloning system and Primer D'Signer

Cloning procedure

The polylinkers of the expression vectors carry the restriction sites BsaI (isoschizomer Eco31I) and BsmFI (New England Biolabs, MBI Fermentas) which allow the precise fusion of the structural gene with the vector-encoded functional elements (including Strep-tag^® II, 6xHistidine-tag and, depending on the vector, OmpA-signal sequence, start codon, protease cleavage site or stop codon. This is easily achieved by adapting both ends of the coding region of the structural gene via PCR.
The cloning strategy is described for pASK-IBA3 (see cloning scheme). If a different vector is to be used, the cloning strategy has to be adapted accordingly. The essential primer sequences for each vector are described here or may be deduced by using the Primer D'Signer software (see below).
In cases where other restriction sites are intended to be used for cloning, care must be taken to ensure the in-frame fusion of the structural gene and the vector encoded functional elements.
In the vectors pASK-IBA4 to pASK-IBA7, pASK-IBA35, pASK-IBA37, pASK-IBA44 and pASK-IBA45 with N-terminal affinity tags (see vector overview) the tag is followed by the linker sequence 5'-GGCGCC-3', which is recognized by four different restriction enzymes (KasI, NarI, EheI and BbeI). These four enzymes cut the linker sequence in four different ways. Thus, cleavage with the suitable enzyme and a subsequent filling reaction enable the production of blunt ends in all reading frames in case the target gene insert requires a particular reading frame.
To avoid the incorporation of base substitutions, PCR should be performed with a proof-reading DNA polymerase such as Pfu (Stratagene). 3' phosphorothioate-protected primers should be used in order to avoid 3'5' degradation by the proof-reading activity.

Eco31I and BsaI belong to the Type IIS restriction enzymes which cleave the DNA double strand outside their recognition site. Thereby, the digestion with one single enzyme can generate two different independent sticky ends with 4-base 5'-overhangs allowing directional cloning. In addition, the digestion reaction removes the recognition sequence not affecting the encoded amino acid sequence and expressing authentic protein.

Cloning scheme (demonstrated for pASK-IBA3)

Precise fusion using Eco31I or BsaI

Free Primer D'Signer 1.1 Software for pASK-IBA, pEXPR-IBA and pPR-IBA Vectors

To facilitate the design of primers for PCR cloning of ORFs (open reading frames) into pASK-IBA, pEXPR-IBA and pPR-IBA expression vectors we are offering the software Primer D'Signer (1.1). The Microsoft Windows software is available free of charge for download (758 KB). This version is suitable for all pASK-IBA, pEXPR-IBA and pPR-IBA vectors. The easy-to-handle software reduces time consuming primer design work to several mouse clicks only. It checks the reading frame, looks for gene internal restriction sites and provides the optimal cloning strategy. Therefore, Primer D'Signer 1.1 is extremely convenient for all Strep-tag/6xHistidine-tag expression system users interested in e.g. the expression of authentic proteins. Now also suitable for all One-STrEP vectors!