IMPACT™ Vectors and Applications

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  • Vectors Site of Target
    Protein Fusion
    Intein Tag
    (kDa)
    Recommended
    Cloning Sites
    Preferred Residues
    at Cleavage Sitea
    Method of
    Cleavageb,c
    Applications
    pTXB1
    pTXB3
    C-terminus Mxe GyrA
    intein (28)
    NdeI-SapI (or SpeI) Y, F, Q, N, T, K, A, H, M, LEM
    (Unfavorable residues- S, P, D, G)
    DTT (or MESNA)
    pH 8.0-8.5, 4°C
    Purification; C-terminal
    thioester for ligation and modification
    pTYB21
    pTYB22
    N-terminus Sce VMA1
    intein (56)
    SapI-PstI
    BsmI (or NdeI)-PstI
    A, Q, M, G, L, N, W, F, Y
    (Unfavorable residues- P, S, C, T, R)
    DTTc
    pH 8.0-8.5, 25°C
    Purification
    pTYB1
    pTYB2
    pTYB3
    pTYB4
    C-terminus Sce VMA1
    intein (56)
    NdeI-SapI
    NdeI-SmaI (or XhoI)
    NcoI-SapI
    NcoI-SmaI (or XhoI)
    G, A, M, F, Q, W, Y, K, LEG
    (Unfavorable residues- P, C, N, D, R)
    DTT (or MESNA)
    pH 8.0-8.5, 4°C
    Purification and ligation
    pTWIN1 C-terminus
    (Intein 2)
    Mxe GyrA
    intein (28)
    NdeI-SapI (or SpeI) Y, F, Q, N, T, K, A, H, M, LEM
    (Unfavorable residues- S, P, D, G)
    Purification; C-terminal
    thioester for ligation and modification
    pTWIN2 C-terminus
    (Intein 2)
    Mth RIR1
    intein (22)
    NdeI-SapI (or SpeI) G, A, LEG
    (Unfavorable residues- P, E, D)
    pTWIN1d N-terminus
    (Intein 1)
    Ssp DnaB
    mini-intein
    (27)
    SapI-SapI
    SapI-PstI (or BamHI)
    BsrGI-PstI (or BamHI)
    C, S, A, G, M, T, CRAM
    (Unfavorable residue- P)
    pH 6.0-7.0,
    25°C
    Purification; Defined
    N-terminus (e.g. Cys);
    Ligation
    pTWIN1d N-terminus &
    (Intein 1)

    C-terminus
    (Intein 2)
    Ssp DnaB
    mini-intein (27)

    Mxe GyrA
    intein (28)
    SapI-SapI C, S, A, G, M, T, CRAM
    (Unfavorable residue- P)

    Y, F, Q, N, T, K, A, H, M, LEM
    (Unfavorable residues- S, P, D, G)
    Step 1:
    pH 6.0-7.0, 25°C

    Step 2:
    DTT (or MESNA)
    pH 8.0-8.5, 4°C
    Purification; Defined
    N-terminus (e.g. Cys);
    Ligation and Cyclizatione
    pTWIN2d N-terminus &
    (Intein 1)

    C-terminus
    (Intein 2)
    Ssp DnaB
    mini-intein (27)

    Mth RIR1
    intein (22)
    SapI-SapI C, S, A, G, M, T, CRAM
    (Unfavorable residue- P)

    G, A, LEG
    (Unfavorable residues- P, E, D)

    a Actual cleavage efficiency is dependent on the adjacent residues as well as the folding of the fusion protein.
    b Dithiothreitol (DTT) is used only for protein purification. 2-mercaptoethanesulfonic acid (MESNA) is used for isolation of proteins possessing a C-terminal thioester for ligation, labeling and cyclization.
    c Cysteine can be used in the place of DTT.
    d pTWIN vectors allow for induction of intein cleavage by a pH/temperature shift.
    e When creating circular proteins it is typical that the linear form will be co-purified, requiring a further step to separate the two protein species.