KG#62
(Plasmid #110874)

• Purpose: Expresses the C. elegans acy-1 cDNA pan-neuronally
• 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): 2617
• 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 cDNA
• Species: C. elegans (nematode)
• Insert Size (bp): 3762

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 intron just upstream and 1 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 cDNA coding region from KG#61 and cloned into Age I/ Xho I cut KG#59 (rab-3:: expression vector). Transform 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 made glycerol stock.

Features of the expression construct: This expression construct has a promoter (rab-3) that will drive strong and specific expression of acy-1 cDNA in the entire nervous system of juvenile and adult animals. Other features of the expression vector include a "decoy" 5' to the promoter to reduce non-specific expression in the gut (see Fire Lab 1995 Kit documentation for description), 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. The vector provides the unc-54 3' UTR and control region. 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