Avoid these codons if you want to express your protein in BL21:

AGG and AGA for arginine
AUA for isoleucine
CUA for leucine
GGA for glycine
CCC for proline

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'

Oligos for making shRNA:
F: 5’ CCGG CCAGACCACTACTGAATATAA CTCGAG TTATATTCAGTAGTGGTCTGG TTTTT 3’
R: 5’AATT AAAAACCAGACCACTACTGAATATAACTCGAGTTATATTCAGTAGTGGTCTGG 3’

50 µl reaction mix for LCR:

*5 µl of 10x T4 ligase buffer (NEB)
*5 µl of 10x Ampligase buffer (Epicentre Biotechnology)
*0.67 µM of TmPrime-optimized oligonucleotides for target gene (phosphorylated using 20 U of T4 polynucleotide kinase, NEB), and 20 U of Ampligase (Epicentre Biotechnology)
* H2O to 5 µl.

LCR:
37°C for 4 hrs, denatured at 95°C for 3 min, ramped to 60°C at 0.1°C/s for annealing, and incubated at 60°C for 2–8 hrs.

PCR mix:
*2 µl of LCR product
*0.4 µM of outer primers
*2 mM of MgSO4
*500 µM of each dNTP (Stratagene)
*500 µg/ml of bovine serum albumin
*1 U of KOD Hot Start (Novagen)
*1x PCR buffer (Novagen)
*H2O to 50 µl.

PCR:
–2 min of initial denaturation at 95°C
–30 cycles of:
*95°C for 5 sec
*55°C for 30 sec
*72°C for 30 sec
–Last Extension at 72°C for 10 min.

1 TmPrime: http://prime.ibn.a-star.edu.sg/
Accepted annealing temperature: 50–70 °C
Design conditions: Oligos with Tm of 55 or 65 +/- 2 °C

2 DNAWorks: http://helixweb.nih.gov/dnaworks/
Accepted annealing temperature: 58–70 °C
Design conditions: Oligo size: 50 nt Random; Annealing temp: 65 +/- 1 °C; No gaps are allowed in assembled gene

3 GeneDesign: http://baderlab.bme.jhu.edu/gd/
Accepted annealing temperature: no information
Users can not specify the oligonucleotide concentration and PCR buffer conditions.
Software does not provide the information of melting temperature derivation of oligonucleotide set.
Design conditions: Target oligo length: 60 bp; Chunk overlap 20 bp
Oligonucleotide design may fail when the sequence of consecutive oligonucleotides collides.

4 Gene2oligo: http://berry.engin.umich.edu/gene2oligo/
Accepted annealing temperature: no information
Design condition: Oligo Tm priority with of Tm of 55 or 65 +/- 4 °C
Oligonucleotide design may fail to converge.

5 Assembly PCR Oligo Maker: http://startrek.ccs.yorku.ca/~pjohnson/AssemblyPCRoligomaker.html
Accepted assembly (annealing) temperature: 50–60 °C
Accepted oligonucleotide length: 40–70 nt
Design condition: Maximum oligonucleotide length: 70 nt
Software does not provide the information of melting temperature derivation of oligonucleotide set.