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URL: http://www.addgene.org/110926

Proper Citation: RRID:Addgene_110926

Insert Name: hsp-16-2 promoter

Organism: Caenorhabditis elegans

Bacterial Resistance: Ampicillin

Defining Citation: PMID:15489511

Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin

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.

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