pACYCentry16

This expression vector simplifies identification of recombinant clones using blue/white selection, allows to use phage T7 regulatory elements or E. coli arabinose promoter for expression and allows to connect His-tag to the N-termini of recombinant proteins.

This vector is a small, medium copy number, E.coli plasmid, 3397 bp in length.

The plasmid contains:
1) the p15A replicon (source – plasmid pACYC184);
2) the Cm(R) gene, coding for chloramphenicol acetyl transferase, which confers resistance to chloramphenicol (source – plasmid pACYC184);
3) the region of E.coli lac operon containing a CAP protein binding site, promoter Plac, lac repressor binding site, and the 5’-terminal part of the lacZ gene encoding the 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 encodes positively 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 the sequence encoding alpha-peptide, but in the opposite orientation. In-frame cloning into this reading frame allows generation of polypeptides that will possess His-tag at their N-termini and can be purified by Ni-affinity chromatography. This reading frame is controlled by the 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 any mechanism controlling expression of the T7 RNA polymerase.

The map shows enzymes that cut pACYCentry16 once.

The plasmid is fully compatible with those carrying the pMB1 and ColE1 replicons.