pACYCentry16 - Alpha Universe LLC

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

The plasmid contain:
1 – the p15A replicon (source – plasmid pACYC184);
2 – the Cm(R) gene, coding for chloramphenicol acetyl transferase, that 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 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 pACYCentry16 once.

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

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