KG#45
(Plasmid #110926)

• Purpose: Vector for expressing proteins in most or all tissues, including nervous system, under control of the inducible heat shock promoter in C. elegans
• Depositing Lab: Kenneth Miller
• Publication: Reynolds et al Genetics. 2005 Feb;169(2):651-70. doi: 10.1534/genetics.104.031286. Epub 2004 Oct 16.

Backbone

• Vector backbone: pUC
• Backbone size w/o insert (bp): 2632
• Total vector size (bp): 4123
• 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: hsp-16-2 promoter
• Species: C. elegans (nematode)
• Insert Size (bp): 425

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: Unknown

Gene/Insert 2

• Gene/Insert name: unc-54 3' control region
• Species: C. elegans (nematode)
• Insert Size (bp): 940

Cloning Information for Gene/Insert 2

• Cloning method: Unknown
• 5′ sequencing primer: Unknown

Resource Information

• Supplemental Documents:
  • Annotated ApE and Strider formatted file--download free ApE plasmid editor to view

Terms and Licenses

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

Depositor Comments

Unique sites in multi-cloning site: Nhe I/ Kpn I/ Age I/ Eco RV/ Xho I/ Sac I/ Bgl II

We used Hind III/ Bam HI to cut out the 2410 bp myo-3 promoter from pPD96.52 (Fire Lab plasmid), leaving the 3684 bp vector fragment. To this vector fragment, we ligated the ~430 bp hsp16-2 promoter Hind III/ Bam HI fragment cut from pPD49.78 (Fire Lab plasmid).

Note: this vector can be converted to a vector that fuses GFP, YFP, or CFP onto the C-terminus of the protein as follows:

Remove the ~1000 bp (Kpn I or Age I) + (Spe I or BsiW I [expensive, 55 C cutter with 50% activity at 37 C; heat inactivate 80 C] or Apa I) fragment containing the unc-54 3’ control region from this plasmid and replace it with the ~1800 bp like-digested fragment containing 3-intron S65C GFP (see pPD94.81 in Fire Lab plasmids), YFP (see pPD136.64 in Fire Lab plasmids) or CFP (see pPD136.61 in Fire Lab plasmids) + the unc-54 3’ UTR and control region. Note if Spe I is used to cut these Fire Lab vectors, you get 3 bands: 1100, 1800, and 4000 (total size 6.9 Kb). The 1800 bp band is the XFP-containing band.

For C-terminal fusions, remember to leave the stop codon off of the upstream protein. For the above-mentioned Fire Lab vectors, the reading frame for the downstream XFP relative to the Kpn I and Age I sites is as follows (triplets are the reading frame codons):

Kpn I
G GTA CCG GT
Age I

Alternatively, use Gibson Assembly/ NEBuilder to insert any genetically encoded fluorescent protein at the N- or C-terminus of your protein.