pi-entry8 - Alpha Universe LLC

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This vector is a high copy number, E.coli plasmid, 4751 bp in length.

The plasmid contain:
1 - the pMB1 replicon (ori) responsible for the replication of plasmid (source - plasmid pUC19);
2 - the Km(R) gene, coding for neomycin phosphotransferase, that confers resistance to kanamycin
3 – the region of E.coli lac operon containing a promoter Plac, lac repressor binding site and the 5’-terminal part of the lacZ gene encoding the modified N-terminal fragment of beta-galactosidase (alpha-peptide). This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic (alpha) complementation with a defective form of beta-galactosidase encoded by the host (mutation delta(lacZ)M15). In the presence of IPTG, bacteria synthesize both fragments of the enzyme and form blue colonies on media with X-Gal. Insertion of DNA into the Multiple Cloning Sites (MCS) located within the lacZ' gene inactivates the N-terminal fragment of beta-galactosidase and abolishes alpha-complementation. Bacteria carrying recombinant plasmids therefore give rise to white colonies.
4 – the region of E.coli arabinose operon that incodes pozitively regulated promoter Para and gene araC encoding it's positive regulator.
5 -the  short reading frame encoding  His-tag and MSC. This sequence is located within sequence encoding alpha-peptide but in oposite orientation to the last one. In-frame cloning into this reading frame allows generation of polypeptides that will possess His-tag at their N-terminai and can be purified by Ni-affinity chromatography. This reading frame is controlled by efficient translation initiation site and two strong promoters: Para and promoter of bacteriophage T7. Production of the His-tag-carrying polypeptide can be triggered practically in any chosen strain via addition of arabinose. Also, in special  strains encoding RNA-polymerase of bacteriophage T7, expression of the recombinant protein may be triggered by what ether mechanism controlling expression of the T7 RNA polymerase.

The map shows enzymes that cut the plasmid once and can be used for inesrtion of foreign DNAs into lacZ' sequence.

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

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