pBGP3-NtEG

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	The resulting linear DNA fragment was self-ligated, giving rise to pBGP3 (Fig. 1A and B). The DNA sequences of all the plasmids were checked.

Protocol tips
The resulting linear DNA fragment was self-ligated, giving rise to pBGP3 (Fig. 1A and B). The DNA sequences of all the plasmids were checked.

Publication protocol

Briefly, a sequence encodingmyc-epitope and six consecutive histidines (6
His) of pBGP1 was amplified by PCR using Pyrobest DNA polymerase (Takara, Japan) and a set of forward (5 0 -CAATTGGAACAAAAACTCATC TCAGAAGAGGATCG-3 0 ) and reverse (5 0 -GAATTC- ATGATGATGATGATGATGGTCGACGG-3 0 ) primers containingMunI andEcoR I restriction sites respec- tively (underlined) at the 5 0 -ends. The amplified DNA fragment was phosphorylated and inserted into the EcoR V site of pBluescript II SK(þ). This plasmid, pBS-BGP2, was digested withMunI andEcoR I and inserted into theEcoR I site of pBGP1, generating pBGP2. Since only the use of N-terminal tags was intended, the presence of C-terminal tags in pBGP2 was undesired and useless. To remove the repetitive C-terminal tag sequence from pBGP2, a pair of back-to- back, oppositely directed primers (5 0 -GTTTTAGCCTT- AGACATGACTGTTCCTCA-3 0 and 5 0 -TTCTAGAAA- GCTGGCGGCCG-3 0 ) was used to amplify the entire sequence of pBGP2, except for the region coding for the redundant C-terminal tags. The resulting linear DNA fragment was self-ligated, giving rise to pBGP3 (Fig. 1A and B). The DNA sequences of all the plasmids were checked.

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