Cloning Chimeric 5'UTR-Luciferase Constructs

Chimeric 5′UTR-luciferase constructs were cloned by FastCloning using Phusion HF polymerase according to manufacturer’s protocol (NEB) (Li et al, 2011 (link)). pGEM-luc and pSP64 Poly(A) vectors were purchased from Promega. NotI and BglII sites were first cloned into pSP64 Poly(A) vector, and luciferase was cloned upstream of the poly(A) sequence. 5′UTR from intestine and kidney/liver isoforms were cloned from GeneBlocks (Integrated DNA Technologies) directly 5′ to the luciferase sequence. Plasmid sequences were all verified by Sanger sequencing across inserts.

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Bracey N.A., Platnich J.M., Lau A., Chung H., Hyndman M.E., MacDonald J.A., Chun J., Beck P.L., Girardin S.E., Gordon P.M, & Muruve D.A. (2020). Tissue-selective alternate promoters guide NLRP6 expression. Life Science Alliance, 4(3), e202000897.

Publication 2020

pGEM-luc pSP64 Poly(A) GeneBlocks

5 utr Chimeric Intestine Isoforms Kidney Liver Luciferase Pgem Plasmid Poly a Promega Vector

Corresponding Organization : University of Calgary

Other organizations : University of Alberta, University of Toronto

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Variable analysis

independent variables

5'UTR from intestine and kidney/liver isoforms

dependent variables

Luciferase activity (measured outcome)

control variables

PGEM-luc and pSP64 Poly(A) vectors
NotI and BglII sites in pSP64 Poly(A) vector
Luciferase cloned upstream of the poly(A) sequence

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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