I am having trouble finding the exact attR1 and attR2 sequences required for the Gateway LR reaction. Does anyone know the exact, required sequence?

19 May 2017 1 9K Report

I am trying to design my own destination vector. I have found a number of attR1 and attR2 sequences through addgene and through other sources. There is a lot of agreement between the sequences. However, there is also some disagreement. Please let me know what sequences are needed for attR1 and attR2, so that I can design primers. Thanks

Kamal Kumar

The attR1 and attR2 sequences differ at 2 or 3 nucleotides only (at site of recombination) so it is preffered that either you clone ccdB/CmR cassette as a whole or you split the whole ccdB cassette into two halves and clone these two halves of ccdB cassette at desired sites by Gibson assembly/ In-Fusion, etc. A point that needs special attention is the frame of ccdB cassette with regard to downstream ORFs like that of HA, myc, GFP, etc tags.

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