pJK500
(Plasmid #72245)

• Purpose: Produces E. coli N5-carboxyaminoimidazole ribonucleotide mutase, Trp20>Phe mutant (EcPurE1-W20F), and N5-carboxyaminoimidazole ribonucleotide synthetase (EcPurK)
• Depositing Lab: T. J. Kappock
• Publication: Single Trp variants of Acetobacter aceti PurE (unpublished)

Backbone

• Vector backbone: pET23a
• Backbone size w/o insert (bp): 3666
• Total vector size (bp): 5191
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: N5-carboxyaminoimidazole ribonucleotide mutase
• Alt name: purE
• Alt name: JW0512
• Alt name: b0523
• Species: Escherichia coli str. K-12 substr. MG1655
• Mutation: changes tryptophan-20 to phenylalanine.
• GenBank ID: NP_415056.1
• Promoter: T7

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: NdeI (not destroyed)
• 3′ cloning site: n/a (unknown if destroyed)
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: n/a

Gene/Insert 2

• Gene/Insert name: N5-carboxyaminoimidazole ribonucleotide synthetase
• Alt name: purK
• Alt name: JW0511
• Alt name: b0522
• Species: Escherichia coli str. K-12 substr. MG1655
• Mutation: Pro64 to Arg; Asp65 to His; Gln206 to Arg
• GenBank ID: NP_415055.1
• Promoter: endogenous

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: n/a (unknown if destroyed)
• 3′ cloning site: HindIII (not destroyed)
• 5′ sequencing primer: n/a
• 3′ sequencing primer: T7 terminator

Terms and Licenses

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

Depositor Comments

This construct is based on the pNC2 (Plasmid #72438) plasmid. It encodes sequence variants in the purK region described there.

This plasmid encodes arginine-64 and histidine-65, an apparently natural variant in some E. coli accessions, as well as a glutamine-205 to arginine mutant introduced during the cloning of parental plasmid pNC2 (Addgene Plasmid #72438)