pVGI-0020-dC
(Plasmid #261735)

• Purpose: Expresses murine CD45.1 with a deletion of the cytosolic domain such that only the 12 post-transmembrane amino acids of the cytosolic domain remain. Correct isoform expression remains intact.
• Depositing Lab: William Raschke
• Depositing Organization: Virogenics, Inc.
  Located in the United States of America
• Publication: Virts et al Blood. 2003 Feb 1;101(3):849-55. doi: 10.1182/blood-2002-07-1969. Epub 2002 Sep 19.

Backbone

• Vector backbone: pcDNA3.1
• Backbone size w/o insert (bp): 5341
• Total vector size (bp): 21101
• Modifications to backbone: SfiI and NotI restriction sites were introduced into the multiple cloning site of pcDNA3.1 which were used to insert the CD45.1 minigene.
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Murine CD45.1 Minigene with deleted cytosolic domain (dC)
• Alt name: PTPRC-a Minigene dC
• Species: M. musculus (mouse)
• Insert Size (bp): 15760
• Mutation: Changed T to G at nt 15,759, which converts a TTA leucine codon (the 12th codon of the cytosolic domain) to a TGA stop codon, and deleted the following 2864 bp.
• GenBank ID: NC_000067.7
• Entrez Gene: Ptprc (a.k.a. B220, CD45R, Cd45, L-CA, Ly-5, Lyt-4, T200, loc)
• Promoter: human LFA

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SfiI1 introduced into the multiple cloning site. (not destroyed)
• 3′ cloning site: Not1 introduced into the multiple cloning site (not destroyed)
• 5′ sequencing primer: ccgacaattgcatgaag
• 3′ sequencing primer: ggcacagtcgaggctg

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

pVGI-0020-dC differs from pVGI-0020 (Addgene #261733) by the following:
(1) a change of a T to G at position 15,759, which converts a TTA leucine codon to a TGA stop codon and
(2) the next 2,864 nucleotides following this TGA are deleted. This deletion removes the rest of the cytosolic domain and the natural stop codon, but leaves intact the natural CD45 poly A signal sequence.

CD45.1 protein expressed from pVGI-0020dC is truncated at the C' end but with intact extracellular and transmembrane sequences. The truncated CD45.1 protein ais expressed on the cell surface and regulates isoform expression normally and in the correct cell lineage-specific manner. The truncated protein expressed from this minigene has no protein tyrosine phosphatase activity as a result of the deleted sequence.