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Prices are in US dollars and valid only for US orders.
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Description:
pKYB1 is an E. coli expression vector for use with the IMPACT™ Protein Purification System (1,2). It is designed for in-frame insertion of a target gene into the polylinker, upstream of the Sce VMA intein/chitin binding domain (55 kDa)(1,3). pKYB1 carries the kanamycin resistance gene (Kn) from Tn 903. This double stranded vector is 8,393 bp in length.
Source:
pKYB1 is isolated from an E. coli strain (r- m-) by a standard plasmid purification procedure.
Advantages:- A pBR322 derivative.
- The NdeI site in the polylinker contains an ATG sequence for translation initiation.
- Use of the SapI site allows for cloning of the target protein adjacent to the intein, resulting in purification of the target protein without any additional amino acids at its C-terminus.
- Unique sites are in bold. NheI and NruI are not unique.
- Expression of the fusion gene is under the stringent control of the T7 promoter (4) and is regulated by IPTG due to the presence of a lac I gene.
- Origin of DNA replication from bacteriophage M13, which allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid (M13K07 Helper Phage, NEB #N0315S).
- Kanamycin resistant.
- T7 Universal Primer (NEB #S1248S) and Intein Reverse Primer (NEB #S1261S) are available for sequencing the insert.
- Compatible restriction sites for subcloning a fusion gene from other IMPACT vectors.
- A wide range of E. coli host strains: ER2566 (NEB) or BL21 (DE3) and derivatives.
Concentration:
200 μg/ml
Storage Conditions
Storage Conditions:
10 mM Tris-HCl
1 mM EDTA
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- E. coli strain ER2566 is only available to purchasers of the IMPACT™ T7 System or replacement vectors.
References
- Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H. and Xu, M.-Q. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene, 192, 271-281.
- Chong, S., Shao, Y., Paulus, H., Benner, J., Perler F.B. and Xu, M.-Q. (1996) Protein splicing involving the Saccharomyces, cerevisiae VMA intein: the steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J. Biol. Chem., 271, 22159-22168.
- Watanabe, T., Ito, Y., Yamada, T., Hashimoto, M., Sekine, S. and Tanaka, H. (1994) The role of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J. Bacteriol., 176, 4465-4472.
- Dubendorff, J.W. and Studier, F.W. (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol., 219, 45-59.
Companion Products
pTYB1 Vector
pTYB11 Vector
pTYB12 Vector
pTYB2 Vector
pTYB3 Vector
pTYB4 Vector
Legal
Research Use Assurance:
The buyer and user have a nonexclusive sublicense to use this system for any component thereof for RESEARCH PURPOSES ONLY, based upon agreement to the following assurances.
Transfer of this vector in host cells that contain the cloned copy of the T7 gene 1 to third parties is explicitly prohibited.
Licenses/Patents/Disclaimers:
Notice to Buyer/User: the buyer/user has a non-exclusive license to use this system or any components thereof for RESEARCH PURPOSES ONLY. See Research Use Assurance Statement for details on terms of the license granted hereunder.
A license to use this system or any components thereof for commercial purposes may be obtained from New England Biolabs, Inc.:
Commercial Laboratory Buyer/User: Use of the vector in host cells that contain the cloned copy of T7 gene 1, the gene for T7 RNA polymerase for any purpose other than in combination with the IMPACT™ System is explicitly prohibited.
Use of the host cells that contain the copy of the T7 gene 1, the gene for T7 RNA polymerase with any other vector(s) containing a T7 promoter to direct the production of RNA or protein requires a license from Brookhaven National Laboratory. Information about research-use or commercial-use license agreements may be obtained from the Office of Technology Transfer, Brookhaven National Laboratory, Building 475D, P.O. Box 5000, Upton, New York 11973-5000; Tel. 631-344-7134, Fax: 631-344-3729.
You may refuse this non-exclusive research license agreement by returning the enclosed materials unused. By keeping or using the enclosed materials, you agree to be bound by the terms of this sub-license.
Note: E. coli strain ER2566 is only available to purchasers of the IMPACT™ System or replacement vectors.
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