KG#81
(Plasmid #110876)

• Purpose: Expresses the C. elegans acy-1 P260S gain-of-function cDNA in body wall muscle
• 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): 9853
• 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: myo-3 promoter
• Species: C. elegans (nematode)
• Insert Size (bp): 2387

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 Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 3.8 Kb acy-1(P260S) cDNA coding region from KG#63 and cloned into Age I/ Xho I cut pPD96.52 (myo-3:: expression vector). Transformed into XL1-Blue electrocompetent cells. Miniprepped 8 clones to find those with correct size insert and vector, then submitted 2 clones for sequence. Chose one clone that has correct sequence and make glycerol stock and save the DNA.

Features of the expression construct: This expression construct has a promoter (myo-3) that will drive strong expression of acy-1 (P260S) cDNA in the body wall muscle cells. The myo-3 promoter is generally active in body muscles (body wall, vulval, and intestine associated muscles including the anal depressor [Okkema et al., 1993 Genetics; Ardizzi and Epstein 1987 JCB]. Other features of the expression vector include the unc-54 3' end including the poly A addition signal, and 3 introns (1 in 5' UTR, 1 just upstream of the unc-54 3' end, and 1 inserted in the unc-54 3' end). These introns help provide more uniform and robust expression, especially if you are expressing a cDNA with no introns (as we are) as opposed to a gene with introns. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG to provide a consensus translation start site. The sequence AT follows the stop codon and immediately precedes the 3' restriction site to provide a consensus post - stop codon sequence. 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.