Department of Molecular and Cell Biology
Primer Design
Primer design for pLEICS vectors is very simple:
- Decide which vector family you want to use (see below)
- Copy the appropriate 5' vector homology region for forward and reverse primers
- Design the insert homology region according to your desired insert 15-25 bp (longer if AT rich, shorter if GC rich) ensure 8bp at 3' end are unique if possible, and end (3') in a A or T.
An example of Vector family A primer design:
Key:
Your protein sequence
Homology tag
1. Find which Family your vector is from the website.
2. Family A homology sequence from PROTEX website:
N-ter: GTATTTTCAGGGCGCC
C-ter: GACGGAGCTCGAATTTCA
3. Primer design:
Met Glu Gly Arg Gly Asn Thr
5’ GTATTTTCAGGGCGCCATGGAGGGCCGTGGAAATACG------------
3’ TACCTCCCGGCACCTTTATGC------------
Ala Ser Lys Leu Lys Cys Gly
-------GCTAGCAAGTTAAAATGTGGC 3’
-------CGATCGTTCAATTTTACTCCGACTTTAAGCTCGAGGCAG 5’
Forward primer:
5’ GTATTTTCAGGGCGCCATGGAGGGCCGTGGAAATACG 3'
Reverse primer:
5’ GACGGAGCTCGAATTTCAGCCTCATTTTAACTTGCTAGC 3‘
*Remember to design 5’ to 3’ by reversing your DNA sequence, (in green.)
An example of Vector Family A Mutagenesis primer design:
Key: Homology tag
Your protein sequence
Region of mutation
Forward 1→ Met Asn Val Ser Lys Gly Asp5’ GTATTTTCAGGGCGCCATGAACGTGAGCAAGGGCGAT---------
TACTTGCACTCGTTCCCGCTA---------
Forward 2→ Mutate to AAC
----CCACCTACGGCAAGCTGAcccTGAAGTTCATCTGCACCA----
----GGTGGATGCCGTTCGTCTgggACTTCAAGTAGACGTGGT----
Mutate to TTG ← Reverse1
Tyr Val Thr Lys Arg Ala Thr
------TACGTCACGAAGCGGGCGACA 3’
------ATGCAGTGCTTCGCCCGCTGTACTTTAAGCTCGAGGCAG 5’ ←Reverse2
Primers:
PCR 1(a)
Forward 1: 5’ GTATTTTCAGGGCGCCATGAACGTGAGCAAGGGCGAT 3’
Reverse 1: 5’ TGGTGCAGATGAACTTCAGTTTCAGCTTGCCGTAGGTGG 3’
PCR 1(b)
Forward 2: 5’ CCACCTACGGCAAGCTGAAACTCAGCTTGCCGTAGGTGG 3’Reverse 2: 5’ GACGGAGCTCGAATTTCATGTCGCCCGCTTCGTGACGTA 3’
*Remember to design 5’ to 3’ by reversing your DNA sequence.
PCR 2: F1+R2 (using products from PCR 1 (a) + 1 (b) as a template to give whole DNA.
Family A homology sequence from PROTEX website
N-ter: GTATTTTCAGGGCGCC
C-ter: GACGGAGCTCGAATTTCA
Primer F1: 5' GTATTTTCAGGGCGCCATGAACGTGAGCAAGGGCGA
Primer R1: 5' GACGGAGCTCGAATTTCAGCGGGCGAAGCACTGCA3'
18nt..................Mutation..................18nt
Mutation F2: 5' CCACCTACGGCAAGCTGAAACTGAAGTTCATCTGCACCA 3'
Mutation R2: 5' TGGTGCAGATGAACTTCAGTTTCAGCTTGCCGTAGGTGG 3'
We will provide advice for customers to design vector based primers for the cloning using their vectors
Family A vectors homology region:
Family A1 pLEICS-12 vector homology region without Tag (Cut with KpnI and EcoRI has a different N-ter primer):
N-ter TATAGGGAGACCCAAGCTTGGTACCFamily B vectors homology region:
Family C vectors homology region:
Family D vectors homology region (with TEV site):
Family E vectors homology region (without TEV site):
Family F vectors homology region:
Family G vectors homology region:
Family H vectors homology region:
Family I vectors homology region:
Family J vectors homology region (with TEV site):
Family K vectors homology region (without TEV site):
pLEICS-12 vector homology region without His10-3xFLAG Tag (Cut with KpnI and EcoRI):
Family 29T vector homology region (with TEV site):
Family 29 vector homology region (without TEV site):
Family 35 vector homology region:
Family 36 vector homology region:
Family 37 vector homology region:
Family 39C-Tag vector homology region:
Family 39NC-Tag vector homology region:
Family 40C-Tag vector homology region:
Family 40NC-Tag vector homology region:
Family 49 vector homology region:
Family 69 vector homology region:
Family 72 vector homology region:
Family 73 vector homology region:
Family 74 vectors homology region:
Family 76 vector homology region:
Family 77 vector homology region:
Family 79 vector homology region:
Family 80 vector homology region:
Family 81 vector homology region:
Family 82 vector homology region:
Family 85 vector homology region:
Family 89 vector homology region:
Family 90 vector homology region:
Family 94 vector homology region:
Family 95 vector homology region:
Family 98 vector homology region:
Family 118 vector homology region:
Family 131-A homology region (Tag: N-His10-3xFlag, C-EGFP/mCherry-APEX2/miniSOG):
Family 131-B homology region (Tag: N-His10-3xFlag, C-APEX2/miniSOG Tag):
Family 131-C homology region (Tag: C-EGFP/mCherry-APEX2/miniSOG Tag):
N-ter: TATAGGGAGACCCAAGCTTGGTACCATG ...
C-ter: GCCCTTGCTCACCATGAATTC ...
Family 131-D homology region (Tag: C-APEX2/miniSOG Tag):
Family 135 vector homology region:
Family 136 vector homology region:
Family 136 vector homology region without His10-3xFLAG Tag (Cut with KpnI and EcoRI):
Family 144 vector homology region:
N: 20-nt guild sequence; n: Reverse complementary of N
Family 151 vector homology region: