KG#83
(Plasmid #110877)

• Purpose: Expresses the C. elegans acy--1 P260S gain-of-function cDNA pan-neuronally
• Depositing Lab: Kenneth Miller
• Publication: Schade et al Genetics. 2005 Feb;169(2):631-49. doi: 10.1534/genetics.104.032334. Epub 2004 Oct 16.

Backbone

• Vector backbone: pUC
• Backbone size w/o insert (bp): 2617
• Total vector size (bp): 8673
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Always streak out this culture for single colonies and do minipreps on >= 4 clones (~1/2 of colonies will be right). Choose small colonies after 18 - 20h growth. DNA yeild is low (~170 ng/ ul x 30 ul for a Qiagen 3 ml miniprep).
• 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: acy-1 P260S gain-of-function cDNA
• Species: C. elegans (nematode)
• Insert Size (bp): 3762
• Mutation: Changed Proline to Serine at amino acid 260

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): 978

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

Always streak out this culture for single colonies and do minipreps on >= 4 clones (~1/2 of colonies will be right). Choose small colonies after 18 - 20h growth. DNA yeild is low (~170 ng/ ul x 30 ul for a Qiagen 3 ml miniprep).

Used Age I/ Xho I to cut out the 3800 bp acy-1(P260S) cDNA from KG#81 and cloned into the like-digested rab-3 expression vector KG#59 (4900 bp). Transformed into XL1-Blue electrocompetent cells. Miniprepped 4 clones to find one with correct size insert and vector and made glycerol stock.

Features of the construct: This expression construct has a promoter (rab-3) that will drive strong expression of the acy-1(P260S) cDNA in the nervous system. An unc-54 3' end containing a poly A addition signal is downstream of the MCS to provide a 3' UTR for the transcript. Three introns in the UTR's are included to help expression (1 in the 5' UTR, 1 just upstream of the unc-54 3' end, and 1 in the unc-54 3' end sequence). This construct includes only the acy-1 coding region. 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. Note: the acy-1 cDNA from which this clone was produced corresponds to the single acy-1 cDNA predicted by wormbase, which is F17C8.1 except for the gf mutation in this clone.