pKSJ376
(Plasmid #103065)

• Purpose: Expresses colicin E1 with R domain deletion and ImmE1
• Depositing Labs: Alan Finkelstein, Karen Jakes
• Publication: Jakes et al J Bacteriol. 2016 Dec 13;199(1). pii: e00412-16. Print 2017 Jan 1.

Backbone

• Vector backbone: pET52-b
• Backbone manufacturer: Novagen EMD/Millipore
• Backbone size w/o insert (bp): 5627
• Total vector size (bp): 6749
• Vector type: Bacterial Expression
• Selectable markers: Immunity to colicin E1

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): BL21(DE3)
• Growth instructions: use BL21(DE3) for protein expression. Expression was done using Millipore Overnight Express Instant TB Medium, which self-induces
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: colicin E1 immunity protein
• Alt name: cei
• Species: E. coli
• Insert Size (bp): 440
• GenBank ID: J01566.1
• Entrez Gene: pColE1_26 (a.k.a. pColE1_26)
• Entrez Gene: imm (a.k.a. pColE1H22_p2)
• Promoter: T7 lac (from pET)

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: AvrII (not destroyed)
• 3′ cloning site: AvrII (not destroyed)
• 5′ sequencing primer: T7-term

Gene/Insert 2

• Gene/Insert name: cea/colicin E1
• Species: E. coli
• Insert Size (bp): 1200
• Mutation: R (receptor-binding domain) deletion in colicin E1 from amino acid residue 191 to 337
• GenBank ID: J01566.1
• Promoter: T7 lac (from pET52-b)

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: NcoI (not destroyed)
• 3′ cloning site: SacI (not destroyed)
• 5′ sequencing primer: T7
• 3′ sequencing primer: not needed

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The genes for colicin E1 with the R domain deletion and E1 immunity protein are transcribed from opposite strands of the vector. Colicin synthesis is under control of the vector inducible promoter; immunity protein synthesis is controlled by its own promoter.