pTYB21 Vector

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
N6709S     -20       pTYB21 Vector N6709SVIAL -20 1 x 0.05 ml 200 µg/ml

Features

• The multiple cloning site (MCS) is compatible with the multiple cloning sites of vectors in the pMAL Protein Fusion and Purification System (NEB #E8200) and the K. lactis Protein Expression Kit (NEB #E1000).
• When the SapI (or BspQI) site in the MCS is used for cloning the 5´ end of the target gene, the N-terminus of the target protein is immediately adjacent to the intein cleavage site. This results in the purification of a target protein without any extra vector-derived residues at its N-terminus. After cloning the target gene in the MCS using SapI, the recognition sequence of SapI is lost; therefore, the vector cannot be recut with SapI. For details, see the IMPACT Manual.
• When NdeI is used for cloning the 5’ end of the target gene, extra amino acids (Gly-Arg-Ala-His) will be added to the N-terminus of the target protein.
• A stop codon should be included in the reverse primer.
• A pBR322 derivative with a ColE1 replication origin.
• Expression of the fusion gene is under the control of the T7/lac promoter and can be induced by IPTG due to the presence of a lacI gene (5).
• Expression requires an E. coli host that carries the T7 RNA Polymerase gene [e.g., T7 Express Competent E. coli, (NEB #C2566) or BL21(DE3) Competent E.coli (NEB #2527) and derivatives].
• Ampicillin resistance.
• When pTYB21 or pTYB22 is used, a small peptide (15 amino acids, 1.6 kDa) is also cleaved from the intein tag and co-eluted with the target protein. It cannot be detected on a regular SDS-PAGE and can be dialyzed out.
• Origin of DNA replication from the bacteriophage M13 allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid. M13K07 Helper Phage (NEB #N0315) is available.
• Other IMPACT vectors are available which allow for fusion of a target gene to N- or C- terminus of an intein and a cleavage reaction which can be induced by thiol reagent or temperature/pH shift.
• Intein Forward Primer (NEB #S1263) and T7 Terminator Reverse Primer (NEB #S1271) are available for sequencing the target gene.

Companion Products

• T7 Express Competent E. coli (High Efficiency)
• BL21(DE3) Competent E. coli

1. Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.-Q., Benner, J. (1998). Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res. 26, 5109-5115.
2. 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-cleavableaffinity tag derived from a protein splicing element. Gene. 192, 277-281.
3. Chong, S., Williams, K.S., Wotkowicz, C., and Xu, M.Q. (1998). Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J. Biol. Chem. 273, 10567-77.
4. 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.
5. 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, 45-49.

• Intein-Mediated Protein Ligation IPL and Labeling with the IMPACT™ Kit

• IMPACT™ Vectors and Applications

• DNA Sequences and Maps Tool

1. What is the DNA sequence of this product?

• Over 40 years in protein expression and purification – a historical perspective

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

• N6709S_v1

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

• N6709S_v1_10008699
• N6709S_v1_10047010
• N6709S_v1_10056433
• N6709S_v1_10063182
• N6709S_v1_10077148
• N6709S_v1_10078210
• N6709S_v1_10136119
• N6709S_v1_10156478
• N6709S_v1_10174201
• N6709S_v1_10195926
• N6709S_v1_10243986

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

• pTYB21 Vector

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