P5E-CMV/SP6: Difference between revisions

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== Construction Details ==
== Construction Details ==
p5E-''hsp70l'' was made by PCR amplification of the 1.5 kb hsp70l promoter (Genbank AF158020), using primers to add att sites, followed by a BP reaction. Note that hsp70l is the official ZFIN gene name for the gene originally named hsp70 by Halloran et al., 2000. Thanks to Ceri van Slyke of ZFIN for pointing this out.
P5E-CMV/SP6 was made by PCR amplification of the sCMV and SP6 promoters from pCS2+, using primers that added att sites, followed by a BP reaction.


Sequencing shows a single base mutation in the hsp70l promoter (T1068G), but this does not appear to be a critical base as constructs made with this clone show normal induction by heatshock.
This 5' element can be used to build pCS2-like constructs, either to make capped mRNA for injection, or to drive expression from the CMV promoter after DNA injection.
 
The insert has been completely sequenced, revealing two point mutations (T889C and G1508T) in the CMV promoter; however, DNA injection of constructs made with this clone show that the CMV appears to be functional.


== Crude Map ==
== Crude Map ==
Screenshot from Sequencher showing locations of components:
Screenshot from ApE showing locations of components:
P5E-hsp70.png
 
:[[File:382 p5E-CMV-SP6.PNG|thumb|none|350px|382 p5E-CMV-SP6]]


==Download the Sequence ==
==Download the Sequence ==
* '''Annotated Sequence'''(Genbank format) and '''Full-Length Sequence with individual component sequences included'''(FASTA file)
* '''Annotated Sequence'''(Genbank format) and '''Full-Length Sequence with individual component sequences included'''(FASTA file)
: [[Media:382.p5E-CMV-SP6.zip|Download p5E-CMV-SP6]]
: [[Media:382.p5E-CMV-SP6.zip|Download p5E-CMV-SP6]]
== Reference ==
Higashijima S, Okamoto H, Ueno N, Hotta Y, Eguchi G. (1997) Dev Biol.15;192:289-99. "High-frequency generation of transgenic zebrafish which
reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin."

Latest revision as of 19:54, 29 May 2025

Construction Details

P5E-CMV/SP6 was made by PCR amplification of the sCMV and SP6 promoters from pCS2+, using primers that added att sites, followed by a BP reaction.

This 5' element can be used to build pCS2-like constructs, either to make capped mRNA for injection, or to drive expression from the CMV promoter after DNA injection.

The insert has been completely sequenced, revealing two point mutations (T889C and G1508T) in the CMV promoter; however, DNA injection of constructs made with this clone show that the CMV appears to be functional.

Crude Map

Screenshot from ApE showing locations of components:

382 p5E-CMV-SP6

Download the Sequence

  • Annotated Sequence(Genbank format) and Full-Length Sequence with individual component sequences included(FASTA file)
Download p5E-CMV-SP6
Retrieved from "https://tol2kitkwan.genetics.utah.edu/index.php?title=P5E-CMV/SP6&oldid=353"