Available with the CloneJET™ PCR Cloning Kit

The positive selection vector pJET1.2 is a small, high copy number E.coli plasmid, 2974 bp in length. It was derived from pUC19 by replacing the 5’-terminal part of the lacZ gene encoding the N‑terminal fragment of beta-galactosidase with the gene coding for the Eco47I restriction endonuclease. In the absence of cognate methylation, the endonuclease is lethal to E.coli host cells. Insertional inactivation of the eco47IR gene is the mechanism to obtain a positive selection using the plasmid pJET1.2. For convenience, the multiple cloning site (MCS) used for positive selection, as well as a T7 polymerase promoter, were incorporated into eco47IR by silent mutagenesis. The pJET1.2 plasmid contains: (1) the pMB1 replicon rep responsible for the replication of plasmid (source – plasmid pUC19). The high copy number of pJET1.2 is a result of the lack of the rop gene and a single point mutation in the replicon rep of pMB1; (2) the bla gene, coding for beta-lactamase, that confers resistance to ampicillin (source – plasmid pUC19); (3) the region of E.coli lac operon containing a CAP protein binding site, promoter P_lacUV5 which differs from the wild-type promoter P_lac by two point mutations in -10 region, and lac repressor binding site which contains two mutations resulting in reduced binding of the repressor (source – plasmid pUC19; mutations were introduced by site-specific mutagenesis); (4) the modified eco47IR gene, coding for Eco47I restriction endonuclease. The gene is toxic for all E.coli strains that are not protected by cognate methylation. The background transcription from P_lacUV5 ensures the efficient killing of recipient cells even in case if they encode the lacI^q mutation which ensures elevated intracellular concentration of LacI repressor. Therefore, the induction of Eco47I synthesis by IPTG is not required for positive selection purposes. After the cloning of DNA into eco47IR the integrity of this gene is disrupted. Therefore, only those cells survive after the transformation and are able to form a colony in presence of ampicillin which have the recombinant plasmid. This feature ensures positive selection of recombinant clones.
The map shows enzymes which have unique targets within pJET1.2. The coordinates refer to the position of the first nucleotide in each recognition sequence.
The exact positions of the genetic elements are shown on the map (termination codons included). The bla gene nucleotides 2782-2714 (complementary strand) code for a signal peptide. The indicated rep region is sufficient to promote replication. DNA replication initiates at position 1162 (+/-1) and proceeds in the direction indicated. Plasmids carrying the pMB1 and ColE1 replicons are incompatible, but they are fully compatible with those carrying the p15A replicon (pACYC177, pACYC184). pMB1-derived plasmids can be amplified using chloramphenicol.

GenBank/EMBL Accession Number
EF694056

Additional Information
CAP protein binding site – 884-847 (compl. strand).

Sequence here

Multiple Cloning Site

REviewer™ - DNA sequence analysis and map creation