Addgene - pET His6 GFP TEV LIC cloning vector (1GFP) Plasmid Data

Plasmid 29663: pET His6 GFP TEV LIC cloning vector (1GFP)

Gene/insert name:
None
Fusion protein or tag:
His6-GFP-TEV
Terminal:
N terminal on backbone
Vector backbone:
pET
(Search Vector Database)
Vector type:
Bacterial Expression
Backbone size (bp):
6075
Modifications to Backbone:
The GFP is the version described in Pedelacq et. al. (Jan 2006, Nature Biotechnology). The following mutations are included: F100S, M154T, V164A, S30R, Y39N, N105T, Y145F, I171V and A206K. These mutations have been shown to enhance brightness and solubility, and to inhibit dimerization.
Cloning site 5':
LIC tag
Site destroyed during cloning:
Yes
Cloning site 3':
LIC tag
Site destroyed during cloning:
Yes
5' sequencing primer:
GFP forward (5'cagctcgccgaccacta) List of Sequencing Primers
3' sequencing primer:
T7 reverse
Bacterial resistance(s)
Kanamycin
Growth strain(s)
DH5alpha
Growth temperature (℃):
37
High or low copy:
Low Copy
Sequence: View sequences (2)
Map:
View map
Principal Investigator:
Scott Gradia
Terms and Licenses:
MTA
EMD Millipore Limited Use Label License/Brookhaven pET Assurance Letter

Comments:

This plasmid is an empty vector to be used with a LIC cloning protocol.

It has a TEV-cleavable His6 fusion tag on its N-terminus. GFP can enhance your protein's expression and solubility. It can also be used as a reporter gene.

To clone into this vector, add LIC fusion tags to the 5' end of your PCR primers.

Forward - 5'TACTTCCAATCCAATGCA3'

Reverse - 5'TTATCCACTTCCAATGTTATTA3'

Linearize the plasmid with SspI and gel purify.

When digesting the DNA with T4 polymerase, use dCTP for insert and dGTP for vector.

More information on this vector can be found through http://qb3.berkeley.edu/qb3/macrolab/

Addgene has sequenced a portion of this plasmid for verification. Click here for the sequencing result.

Please acknowledge the principal investigator and cite this article if you use this plasmid in a publication. Also, please include the text "Addgene plasmid 29663" in your Materials and Methods section.