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When designing attB1 and attB2 primers two points must first be addressed:
This will determine which ribosome recognition sequence(s) (Shine-Dalgarno for E. coli
and/Kozak for eukaryotic expression) need to be present.
rrs rrs
Figure 1. Structures of Expression Clones for native, N-terminal and C-terminal fusion expression. Note
the position of start codons, rrs, and stop codons with respect to the recombination sites.
Table 1 provides examples of primer pairs (attB1 and attB2) which can be used to amplify
genes for specific protein expression formats. Shine-Dalgarno (E. coli ribosome binding
sequence) and Kozak (eukaryotic ribosome recognition sequence) show are typical sequences.
Other sequences than those depicted may also function well for protein expression. Choose the
primer pair that will allow for the appropriate expression format.
How to design primers
Table 1. Suggested attB1&2 primer pairs to be used for different expression formats.
Choose organism(s) attB1 attB2 attB1 attB2 attB1 attB2 attB1 attB2
for expression
E. coli only A K C K A L A K
Yeast only B K C K B L B K
Baculovirus only B K C K B L B K
Mammalian only B K C K B L B K
Yeast, Baculovirus B K C K B L B K
and Mammalian only
E. coli, Yeast, A K C K A L A K
Baculovirus &
Mammalian
5' GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTC* GAA GGA GAT AGA ACC ATG (18-24 gsp) 3'
A. 5' GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTC GAA GGA GAT AGA ACC ATG (18-24 gsp) 3'
B. 5' GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTC - - - - - - - - - - - - - - - ACC ATG (18-24 gsp) 3'
C. 5' GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTC (ATG) - - - - - - - - - - - - - - - - - - - (18-24 gsp) 3'
GGGG-attB2 Stop
K. 5' GGGG AC CAC TTT GTA CAA GAA AGC TGG GTT TTA** (18-24 gsp)
L. 5' GGGG AC CAC TTT GTA CAA GAA AGC TGG GTG*** (18-24 gsp)
A & B. To leave the possibility open for N-terminal fusion, keep the ATG in-frame with the reading
frame of attB1.
C. For N-terminal fusion only primer, including the ATG of the open reading frame is optional. The
reading frame of the gene must match the reading frame through attB1.
L. The reading frame of the gsp must match the reading frame of the attB2 sequence.
* The following combinations are NOT possible because they form a STOP codon: TTA, TAG, TGA.
** CTA and TCA may also be used here for stop codon.
*** One base must be added here to complete this codon.
B. Adapter PCR Protocol----To save cost and avoid possible primer dimers
5' AA AAA GCA GGC TTC* GAA GGA GAT AGA ACC ATG (18-24 gsp) 3'
A. 5' AA AAA GCA GGC TTC GAA GGA GAT AGA ACC ATG (18-24 gsp) 3'
B. 5' AA AAA GCA GGC TTC - - - - - - - - - - - - - - - ACC ATG (18-24 gsp) 3'
GGGG-attB2 Stop
Adapter attB1: G GGG ACA AGT TTG TAC AAA AAA GCA GGC T
Adapter attB2: GGG GAC CAC TTT GTA CAA GAA AGC TGG GT
If you with to insert a TEV protease cleavage site after the N-terminal tag for removal of the tag the following
forward primer is recommended:
5' GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTC GAA-AAC-CTG-TAT-TTT-CAG-GGC-ATG-forward
gene specific sequence-3'
TEV will cleave between the 6th and 7th amino acid residues in the recognition site. Therefore, your protein will
contain a single glycine residue on the N-terminus of the protein. If this is undesirable, the ATG in your protein
can be replaced by the GGC (encoding a glycine residue) codon. Following cleavage with TEV, your protein will
contain a single Met -> Gly substitution.