KG#80
(Plasmid #110875)

• Purpose: Expresses the C. elegans acy-1 cDNA in body wall muscle
• 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
• 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 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 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

Note: 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 cDNA from KG#62 and cloned into the like-digested myo-3 expression vector pPD96.52 (6100 bp). Transform into XL1-Blue electrocompetent cells. Miniprep 4 clones to find one with correct size insert and vector and make glycerol stock and save the DNA.

Features of the construct: This expression construct has a promoter (myo-3) that will drive expression of the acy-1 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]. The vector 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. An unc-54 3' end containing a poly A addition signal is downstream of the MCS to stop transcription and provide a 3' UTR for the transcript. Three introns in the UTR's are included 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). This construct includes only the 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 for a consensus stop site. 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