tRNA–gRNA Primer Generator

Design Golden Gate assembly primers for Polycistronic tRNA-gRNA (PTG) gene construction

1 Spacer Input

20 bp Spacer Sequence 5′ → 3′, upstream of NGG PAM

0 / 20

gRNA Identifier optional

BsaI Overhang Position any 4 consecutive nt — default nt 9–12

Enter spacer above to preview

Position nt 9–12

4 bp overhang

F-primer spacer region

R-primer spacer region

2 Primer Sequences

Forward —

Reverse —

Random bases

BsaI site (GGTCTC)

4 bp overhang

Spacer

gRNA scaffold

tRNA anchor

3 Validation Checks

4 Export — Plain Text

gRNA	Spacer (5′→3′)	Primer	Sequence (5′→3′)	Length

↺ Session History

Design Rules — Xie et al. 2015 (SI Methods, Table S3)

F primer: ta·GGTCTCC·[overhang 4 bp][spacer nt(oh+4)–20]·gttttagagctagaa

R primer: cg·GGTCTCA·[RC overhang][RC spacer nt1–(oh-1)]·tgcaccagccgggaa

⚠ Forbidden overhangs: GGCA · AAAC — reserved for terminal adaptors in pRGE32 / pRGEB32

Ramesh R

Genome Editing Lab · Division of Plant Physiology
ICAR–Indian Agricultural Research Institute, New Delhi

Developer

Reference

Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. PNAS 112(11): 3570–3575.

doi: 10.1073/pnas.1420294112