Answer Happy

In lab, you amplified the GFP coding sequence by PCR as an XhoI
fragment, in order to clone it into the XhoI site of the vector
pSK.
The GFP insert was amplified in such a way that, after ligation,
the GFP coding sequence was in frame with LacZ present on the
plasmid, thereby producing a construct expressing a LacZ-GFP
fusion protein in E. coli.
Your task is to design the 5’ oligonucleotide used to amplify
GFP in order to obtain the correct PCR product to clone into pSK
(assume you have the 3’ oligonucleotide available). Your 5’
oligonucleotide needs an XhoI site, which, after restriction, will
produce an overhang that can be ligated in frame into the pSK
vector.
You will need to write down the sequence of your 5’
oligonucleotide as follows:
1) the restriction site (6 nucleotides)
2) Any nucleotide you may find necessary to include to
preserve the frame (0,1 or 2 nucleotides)
3) ) 5 codons (15 nucleotides) corresponding to the
relevant GFP coding sequence, starting from the second codon of
GFP.