pα1 cloning fragment

Catalog: AUV34

This vector is a ready-to-use DNA fragment that allows unidirectional insertion of fragments and positive selection of recombinant constructs.

This fragment is 4,578 bp in length.

The vector contains:
1) the ColE1 replicon responsible for plasmid replication (source - plasmid pU119);
2) the Ap(R) gene, coding for beta-lactamase, which confers resistance to ampicillin;
3) the selection/expression module that contains:
a) a sequence containing multiple unique restriction sites that offer additional opportunities for cloning;
b) the reading frame that contains a Ni-specific tag in the N-terminal region and a cysteine residue in the C-terminal region;
c) arabinose (Para) and bacteriophage T7 (T7) promoters and bacteriophage T7 transcription terminator (T7 terminator) that control expression of the above-listed reading frame;
d) the reading frame that encodes a chimera selection protein (selector) whose expression is toxic for αRD1 cells;
e) Plac promoter that controls expression of the selector protein;
4) a sequence (terminator) that prevents extension of the uncontrolled transcription from the rest of the vector onto the reading frame that contains the Ni-specific tag;
5) non-complementary cohesive ends that prevent self-ligation of the fragment and allow unidirectional insertion of DNA possessing corresponding cohesive ends.

The appropriate junction of reading frames of the vector and inserts allows generation of proteins that can be affinity purified using Ni-containing resins and/or specifically conjugated to other molecules via the cysteine residue.

Interruption of the reading frame of the selector sequence as a result of insertion of foreign DNA will lead to predominant survival of recombinant constructs when ligation mixtures are transformed into αRD1 cells.

The plasmid is fully compatible with those carrying the p15A replicon.