How to design shRNA for pLKO.1-puro and TRC2-pLKO-puro
General guidelines for shRNA design
1. siRNA targeted sequence is usually 21 nt in length.
2. Avoid regions within 50-100 bp of the start codon and the termination codon.
3. Avoid intron regions.
4. Avoid stretches of 4 or more bases such as AAAA, CCCC.
5. Avoid regions with GC content <30% or > 60%.
6. Avoid repeats and low complex sequence.
7. Avoid single nucleotide polymorphism (SNP) sites.
8. Perform BLAST homology search to avoid off-target effects on other genes or sequences.
9, Design Sense & Antisense oligos
Forward oligo:
5′ CCGG—21bp sense—CTCGAG—21bp antisense—TTTTTG 3′
Reverse oligo:
5′ AATTCAAAAA—21bp sense—CTCGAG—21bp antisense 3′
For example, if the target sequence is (AA)TGCCTACGTTAAGCTATAC, the oligos would be:
Forward oligo:
5′ CCGGAATGCCTACGTTAAGCTATACCTCGAGGTATAGCTTAACGTAGGCATTTTTTTG 3′
Reverse oligo:
5′ AATTCAAAAAAATGCCTACGTTAAGCTATACCTCGAGGTATAGCTTAACGTAGGCATT 3′
For eGFP:
Age1 Xho1 (loop) EcoR1
F: 5' CCGG TACAACAGCCACAACGTCTAT CTCGAG ATAGACGTTGTGGCTGTTGTA TTTTT G 3'
R: 3' C ATGTTGTCGGTGTTGCAGATA GAGCTC TATCTGCAACACCGACAACAT AAAAA CTTAA 5'
R: 5' AATTCAAAAATACAACAGCCACAACGTCTATCTCGAGATAGACGTTGTGGCTGTTGTAC 3'
Another example:
AgeI Sense Loop (XhoI) Antisense Termination
F: 5’ CCGG CCAGACCACTACTGAATATAA CTCGAG TTATATTCAGTAGTGGTCTGG TTTTT 3’
compl 3' GGCC GGTCTGGTGATGACTTATATT GAGCTC AATATAAGTCATCACCAGACC AAAAA 5'
rev-compl: 5' AAAAACCAGACCACTACTGAATATAACTCGAGTTATATTCAGTAGTGGTCTGGCCGG 3'
add AATT to 5' for EcoR1
remove CCGG from 3' for Age1
R: 5’AATT AAAAACCAGACCACTACTGAATATAACTCGAGTTATATTCAGTAGTGGTCTGG 3’
Oligos for making shRNA:
F: 5’ CCGG CCAGACCACTACTGAATATAA CTCGAG TTATATTCAGTAGTGGTCTGG TTTTT 3’
R: 5’AATT AAAAACCAGACCACTACTGAATATAACTCGAGTTATATTCAGTAGTGGTCTGG 3’
