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Many expression vectors contain an Nco I site (CCATGG) at the 5'-end of the multiple cloning site. The ATG within this site can be used directly to create the ATG start codon and/or the ATG codon for the N-terminal methionine residue. Target genes or PCR products that contain either Nco I sites or sites that generate compatible overhangs at their 5'-ends can be cloned into these Nco I sites (see table below).

Enzyme restriction site
Afl III A C Pu Py G T
BspH I T C A T G A
Btg I C C Pu Py G G
Nco I C C A T G G
Pci I A C A T G T
Sty I C C A/T A/T G G

Usually Nco I will be used but the other listed enzymes are useful alternatives in case the target gene contains internal Nco I sites. However, each of these restriction sites dictates what the first nucleotide of the next triplet codon must be, which may prevent the generation of native target protein. An alternative strategy is to create the right 5'-overhangs by using restriction enzymes that cleave downstream of their recognition site (see figure below).

These restriction sites can be engineered into PCR primers such that Nco I compatible overhangs can be generated. Here we show an example of a PCR primer that encodes a Bsa I site (red) for the generation of an Nco I compatible overhang (blue):

5'-XXXXGGTCTCNCATG + 18-21 specific bases-3'

Since the Bsa I enzyme cleaves downstream of its recognition site it will generate an Nco I compatible overhang without dictating the sequence of the 3'-flanking base, i.e. the second codon triplet is completely unrestricted.

From: inNovations 10, 13 (1999).