pBR322 Plasmid DNA

Thermo Scientific pBR322 is one of the most commonly used E.coli cloning vectors.

Highlights

• Isolated from E. coli (dam+, dcm+)
• More than 90% in the supercoiled form
• Purified by chromatography using proprietary patented technology

Applications

• Cloning
• Preparation of DNA molecular weight standards

pBR322 is 4361 bp in length and contains:
• The replicon rep responsible for the replication of plasmid (source – plasmid pMB1)
• The rop gene, coding for the Rop protein, which promotes conversion of the unstable RNA I – RNA II complex to a stable complex and serves to decrease copy number (source – plasmid pMB1)
• The bla gene, coding for beta-lactamase, that confers resistance to ampicillin (source – transposon Tn3)
• The tet gene, encoding tetracycline resistance protein (source – plasmid pSC101)
• The circular sequence is numbered such that 1 is the first T of the unique EcoRI site GAATTC and numbering increases through the tet gene and the pMB1 material and finally through the Tn3 region.

The map shows enzymes that cut pBR322 DNA once. Enzymes produced by Thermo Scientific are shown in orange. 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 4153-4085 (complementary strand) code for a signal peptide. The indicated rep region is sufficient to promote replication. DNA replication initiates at position 2533 (± 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.