Part:BBa_K2429145
HBG Intron Guide Family Member
Parts K2429145 through K2429152 and parts K2429153 through K2429167 are a family of guide sequences used by either a dCas13a variant or Ms2 to target the second human betaglobin intron of our various reporters (BBa_K2429034, BBa_K2429035, BBa_K2429036, BBa_K2429050, BBa_K2429051, BBa_K2429052). These guides lie downstream of a U6 promoter, which is a constitutive promoter typically used for the production of guide RNA.
Our team used multiple guide sequences to "tile" along the reporter’s intron to determine what part of the intron is best to target when trying to control its splicing efficiency. Based on the level of fluorescence associated with each guide, we can compare the results to see whether a specific targeted sequence is integral in the process of splicing, and whether the binding of the RNA binding protein to said sequence changes the splicing efficiency.
The guides shift by a few base pairs so we can make more precise conclusions about what intronic sequences are important for splicing.
Parts K-145 through K-152 lead to guide RNA sequences that serve as a guide for the dCas13a protein.
Parts K-153 through K-159 lead to the production of antisense oligonucleotides (ASOs). Parts K-160 through K-167 consist of the same ASO sequences corresponding to the same order (i.e. K-153 sequence is used in K-160); however, they have an additional portion of nucleotides that will form a hairpin loop which Ms2 can bind. The addition of oligonucleotides without hairpins loops serves as a control to test whether it’s the guide sequence or the protein that is actually affecting splicing.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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