pKJ1267 (aka pAC-alphaGal4)
(Plasmid #21869)

• Purpose: (Empty Backbone)
• Depositing Lab: Keith Joung
• Publication: Maeder et al Nat Protoc. 2009. 4(10):1471-501.

Backbone

• Vector backbone: pKJ1267
• Backbone size (bp): 3428
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Growth instructions: Grow in LB supplemented with 30 µg/ml chloramphenicol. Due to toxicity associated with high expression from the dual lpp/lacUV5 promoter, this plasmid must be propagated in a lacIq strain (e.g.--XL-1 Blue).
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: None

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: lac promoter

Resource Information

• Addgene Notes:
  • Addgene diagnostic digest

Terms and Licenses

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

Depositor Comments

Bacterial expression vector for the alpha-Gal4 hybrid protein (required for the B2H system). The tandem lpp/lacUV5 promoter which drives expression of the Gal11P fusion protein contains a lac operator and therefore these dual promoters can be repressed by lac repressor.

This plasmid is a very low copy number plasmid and therefore we recommend that maxipreps be performed from a 200 ml culture. A miniprep will not be sufficient.

Annotations for pKJ1267:

2988- 219: Chloramphenicol-resistance gene (complementary strand)/ 581-1493: p15A origin of replication/ 1552-1557: lpp promoter -35 box / 1576-1581: lpp promoter -10 box / 1624-1629 : lacUV5 promoter -35 box / 1648-1653 : lacUV5 promoter -10 box / 1748-2623 : alpha-Gal4 hybrid protein gene (E. coli RNA polymerase alpha subunit aa 1-248 fused by an Ala-Ala-Ala linker to aa 58-97 of the yeast Gal4 protein)