Table 1.
Namea | 20 nt spacer sequenceb | Hairpin | GC (%) | ΔGc |
---|---|---|---|---|
11-1 | TGCAAATCGGAGTAGAAGCG CGG | N | 50 | −39.6 |
11-2 | GCCGACGCCGCCCTTGATGG TGG | N | 75 | −49.0 |
11-3 | GCGTGAGGAGGAAGCGGCGG TGG | N | 75 | −47.3 |
11-4 | TCCTGTCCGAACCGCCCGCC CGG | N | 75 | −48.4 |
17-4 | GACGAGGCACCTAGAGACAC TGG | N | 60 | −35.8 |
17-5 | CCACTCATCCAGGACGCTAC TGG | N | 60 | −37.8 |
17-6 | CGCGAGGCTGCGGCTGCCAC TGG | Y | 80 | −49.3 |
17-7 | GACGCCCCATCCCCAACCAC TGG | N | 70 | −44.7 |
17-8 | CGAGAGGTCGCTCTCCAAAC TGG | Y | 60 | −39.3 |
17-9 | GGCGATGATGAGGAACCAAC TGG | N | 55 | −39.2 |
17-10 | CCTGCTCCTGCGCCTTCCAC TGG | N | 70 | −44.0 |
Degenerated | NVH-N2-BNH-N2-H-N5-HHAC TGG |
aThe locations are referred to Fig. 4 for 11-1 to 11-4 and Fig. 5 for 17-4 to 17-10
bDisfavored nucleotides are shown in italics
cΔG indicates free energy (kcals/mol). The calculation of free energy and prediction of hairpin in 20 nt spacer were performed by primer premier 5.00
dH: A/T/C, V: G/A/C, B: G/T/C, N: any nucleotide; the C-terminus TGG or CGG is the PAM motif