pJTB126
(Plasmid #248910)

• Purpose: expresses the yeast analog-sensitive Smk1-Q120A and Ssp2 fused to glutathione-S-transferase in bacteria
• Depositing Lab: Edward Winter
• Publication: Rimal et al Mol Cell Biol. 2020 Jul 29;40(16). pii: MCB.00097-20. doi: 10.1128/MCB.00097-20. Print 2020 Jul 29.

Backbone

• Vector backbone: pET-DUET
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5420
• Total vector size (bp): 8024
• Modifications to backbone: minor deletions in cloning sites
• 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: SMK1 Mitogen-activated Protein Kinase -analog-sensitive form
• Alt name: MAPK
• Alt name: Smk1-as1
• Alt name: Smk1-Q120A
• Species: S. cerevisiae (budding yeast); Sequence is from the SK1 isolate of S. cerevisiae and will differ by 1 non-synonomous/several synonymous changes from S288C form
• Insert Size (bp): 1179
• Mutation: codon 120 has been changed from Q to A (Smk1-Q120A) to generate an analog-sensitive kinase (Smk1-as1) also the internal NcoI and NdeI sites were removed for cloning purposes
• GenBank ID: NM_001184151.1
• Entrez Gene: SMK1 (a.k.a. YPR054W)
• Promoter: T7

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: NdeI (not destroyed)
• 3′ cloning site: KpnI (not destroyed)
• 5′ sequencing primer: standard T7 promoter primer
• 3′ sequencing primer: standard T7 promoter primer

Gene/Insert 2

• Gene/Insert name: Ssp2 Activator of the Smk1 MAPK
• Alt name: SSP2
• Species: S. cerevisiae (budding yeast); Sequence is from the SK1 isolate of S. cerevisiae and may differ by synonymous changes from S288C form
• Insert Size (bp): 1377
• Mutation: deleted codons 1-137
• GenBank ID: NM_001183661.3
• Entrez Gene: SSP2 (a.k.a. YOR242C)
• Promoter: T7
• Tag / Fusion Protein:
  • glutathione-S-transferase (C terminal on insert)

Cloning Information for Gene/Insert 2

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

Terms and Licenses

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