KG#205
(Plasmid #110881)

• Purpose: Expresses the C. elegans pde-4 (isoform d) D448N cDNA pan-neuronally
• Depositing Lab: Kenneth Miller
• Publication: Charlie et al Genetics. 2006 May;173(1):111-30. doi: 10.1534/genetics.105.054007. Epub 2006 Apr 19.

Backbone

• Vector backbone: pUC
• Backbone size w/o insert (bp): 2617
• Total vector size (bp): 6936
• 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: rab-3 promoter
• Species: C. elegans (nematode)
• Insert Size (bp): 1208

Cloning Information for Gene/Insert 1

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

Gene/Insert 2

• Gene/Insert name: pde-4d (isoform d) D448N cDNA
• Species: C. elegans (nematode)
• Insert Size (bp): 2025
• Mutation: changed Asp 448 to Asn

Cloning Information for Gene/Insert 2

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

Gene/Insert 3

• Gene/Insert name: unc-54 3' control region with 1 artificial intron just upstream and 1 artificial intron in control region
• Species: C. elegans (nematode)
• Insert Size (bp): 993

Cloning Information for Gene/Insert 3

• 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

Started with KG#203 (contains pde-4d(+) coding region cDNA in rab-3:: expression vector) and use the QuikChange site directed mutagenesis method to introduce the D448N mutation. The mutation changes codon 448 of pde-4d from GAT(Asp) to AAT (Asn) (HDVDH to HNVDH). Miniprepped 4 clones and chose 2 with the correct size insert to sequence. Sequenced the entire insert of both clones, and kept one clone that has the correct mutation, but no other mutations. After sequencing, this insert was "re-cloned" into a fresh rab-3:: expression vector to ensure that there are no other vector mutations that could affect its performance or expression in transgenic worms.

Features of the expression construct: This expression construct has a promoter (rab-3) that will drive expression of the cDNA throughout the nervous system. This construct includes only the 2025 bp coding region of pde-4d (and contains the D448N mutation), and the sequence is taken from the Wormbase WS134 release, gene designation R153.1d (the d isoform appears to be the most common based on analysis of EST cDNAs). The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG. An AT immediately follows the TAA stop codon for a consensus stop site. Three introns (all located in untranslated regions of the vector) are present to give stable and uniform expression (1 in the 5' UTR, 1 just upstream of the unc-54 3' end, and 1 in the unc-54 3' end sequence). The vector includes the unc-54 3' untranslated end containing a poly A addition signal to stop transcription and provide a 3' UTR for the transcript. The vector also contains a "decoy" (see 1995 vector kit documentation) upstream of the promoter to reduce background expression in the gut and pharynx from read-through transcription.