Of course, you can, if the vector contains the appropriate restriction sites. As usual, check first that the resulting construct will be aequate for the production of the desired recombinant protein, e.g. making sure that the cloned ORF will be in frame with the translation start codon etc. But then, there is no reason to limit yourself to Gateway vectors.

Pierre Béguin

Thanks for answering my question. I am still not sure about the function of attB1 and attB2 site, is it needed for protein expression? During LR cloning, the destination vector's attR1/attR2 will be converted into attB1/attB2 site. If I removed the sequence between attR1/attR2 in the destination vector by restriction enzyme, the whole attR1/attR2 and attB1 and attB2 sequence will be gone. I was wondering whether there is some sequence in that region is also needed for POI expression, such as Kozak sequence or other purposes.

Normally, the attB sites are there just to provide the opportunity to use the Gateway system without depending on restriction cloning. All sequence loci that determine the expression of the ORF (promoter, ribosome-biding site, terminator, poly-A site etc.) lie outside of the region that gets exchanged upon Gateway cloning. As a consequence, the presence of the att sites adds several vector-encoded amino acids to the recombinant protein, which can be an inconvenient in some cases. But the best thing for you would be to check the sequence of your vector and make sure that all sites required for proper expression will be still there if you perform restriction cloning and that the ORF will be cloned in the correct reading frame relative to the translation start site.

Dear Min

you can use PIPE cloning, which is an enzime free cloning approac, to clone ORF in the pdestination vectors suitable for Gateway. Thanks to the presence of the ccdb gene, those vectors are a perfect templates for V-PCR amplification.

you can find more information about PIPE cloning in the attached paper or at the following link on my blog PRoteoCool

cloning methods overview:

https://www.blogger.com/video.g?token=AD6v5dymTB4mmANTKeTOyzXQq7QSUurTLQFPDsGxi4FqyNDpu3YZutcuN0VfLTfDTPRD8edEIDPD6bJZIe_VWirwCNZHkJ1A34BATcDIzXZEbV1UUtRLR6Nc19tU2_t6-Or5Sag2dwU

primer desing for PIPE cloning:

https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8

PIPE cloning is quite cheap. The main cost is associated to hi fidelity polimerases and competent cells

good luck

Manuele