Addgene - pET MBP-FlAsH LIC cloning vector (MBP-FlAsH) Plasmid Data

Plasmid 30185: pET MBP-FlAsH LIC cloning vector (MBP-FlAsH)

Gene/insert name:
None
Fusion protein or tag:
FlAsH
Terminal:
C terminal on backbone
Fusion protein or tag:
Biotin-H6-MBP-TEV recognition
Terminal:
N terminal on backbone
Vector backbone:
pET
(Search Vector Database)
Vector type:
Bacterial Expression
Backbone size (bp):
6568
Cloning site 5':
LIC site vGFP
Site destroyed during cloning:
Yes
Cloning site 3':
LIC site vGFP
Site destroyed during cloning:
Yes
5' sequencing primer:
MBP forward (5'ggtcgtcagactgtcgatgaagcc) 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 (3)
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. Your gene can be inserted with a LIC cloning protocol.

This vector adds a short FlAsH tag to the C-terminus of your protein. When your protein is mixed with a biarsenical reagent (see Invitrogen's website), fluorescence will develop. This technique may be useful when a larger fusion interferes with the function of your protein.

Additionally, the vector adds an MBP fusion to the N-terminus of your protein, which may help expression and solubility.

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

Forward - 5' TACTTCCAATCCAATGCA3'

Reverse - 5' CTCCCACTACCAATGCC 3'

Do NOT include a stop codon with your reverse primer.

Linearize the plasmid with SspI, then gel purify.

When digesting the DNA with T4 polymerase, use dCTP for the insert and dGTP for your linearized 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 30185" in your Materials and Methods section.