Gateway Cloning Primer Design Guide
| Native only | N- terminal fusion only | C-terminal | Native & N-terminal Fusion | |||||
| attB1 | attB2 | attB1 | attB2 | attB1 | attB2 | attB1 | attB2 | |
| 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 and Mammalian | B | K | C | K | B | L | B | K |
| E. coli, Yeast, Baculovirus, Mammalian | A | K | C | K | A | L | A | K |
1. One step PCR Protocol
attB1 Primers to amplify the N-terminal of the gene
GGGG-attB1 ;Shine-Dalgarno Kozak Start
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′
attB2 Primers to amplify the C-terminal of the gene
GGGG-attB2 Stop
K. 5′ GGGG AC CAC TTT GTA CAA GAA AGC TGG GTT TTA** (18-24gsp)
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.
2, Adapter PCR Protocol—-To save money and avoid possible primer dimers, but you need more PCR cycles
attB1 Primers to amplify the N-terminal of the gene
12 attB1 Shine-Dalgarno Kozak Start
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′
C. 5′ AA AAA GCA GGC TTC (ATG) – - – - – - – - – - – - – - – - – - -(18-24 gsp) 3′
attB2 Primers to amplify the C-terminal of the gene
GGGG-attB2 Stop
K. 5′ A GAA AGC TGG GTG TTA** (18-24 gsp)
L. 5′ A GAA AGC TGG GTG *** (18-24 gsp)
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 wish 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.
Sequencing primers for sequencing your entry vectors are:
Clones derived from pDONR201 (kanR) and pDONR207 (genR)
SeqL-A (proximal to attL1) TCGCGTTAACGCTAGCATGGATCTC (106 nt from cloned ORFs)
SeqL-B (proximal to attL2) GTAACATCAGAGATTTTGAGACAC (123 nt from cloned ORFs)
