Loop: The default is a 10-nucleotide spacer (TTGATATCCG), and other spacers have been reported as well: CCACC , CTCGAG , AAGCTT , CCACACC , TTCAAGAGA.
Transcription Start Site: RNA Polymerase III prefers to initiate transcription with a purine . If the hairpin insert does not start with a "G" or "A", an additional "G" will be added to the 5' end of the hairpin insert sequence.
Vector: Vector specific sequences will be added based on vendor recommendation. Those sequences could include:
Transcription Start Site
Dinucleotide Leader Sequence: Please do not include the dinucleotide leader sequence "AA" in the sense target sequence. The "AA" leader is not required for vector-based siRNA contruct.
Orientation of hairpin insert:. Hairpin insert can be constructed in these orientations: antisense-loop-sense (Default), or sense-loop-antisense (Reverse). Both orientations can function effectively.Use this tool to build small hairpin inserts for use as siRNA expression vectors.