Question about the sequence of plasmid pSpCas9(BB)-2A-GFP (PX458)

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Xiaoyu Zhao

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Oct 4, 2015, 9:25:18 PM10/4/15

to Genome Engineering using CRISPR/Cas Systems

I have a question about the sequence of 2A-EGFP in pSpCas9(BB)-2A-GFP (PX458). According to the sequence on Addgene, T2A sequence is correct, but the EGFP sequence following T2A has no ATG start codon. I am wondering whether ATG start codon is not required here or just some error on Addgene? Looking forward to any replies. Thanks!

Alex Ling

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Oct 4, 2015, 11:25:07 PM10/4/15

to Genome Engineering using CRISPR/Cas Systems

It doesn't need a start codon as translation continues from the Cas9 coding sequence. The T2A sequence mediates self peptide splicing to yield separate Cas9 and gfp, in 1:1 ratio.

Xiaoyu Zhao

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Oct 5, 2015, 1:57:56 PM10/5/15

to Genome Engineering using CRISPR/Cas Systems

Thanks a lot! I think I got it.

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