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Room temperature, protected from moisture
ARP-3622 (pR7-GFP) expresses a modified green fluorescent protein (GFP), with the GFP coding sequences substituted for HIV-1 nef coding sequences. Alanine was substituted for serine at amino acid 65 in the modified jellyfish (Aequorea victoria) GFP, resulting in markedly increased fluorescence as compared to wild type GFP. This modification is similar to the traditional S65T modification used for EGFP. Cloning vector SP65 was modified to include a xanthine guanine phosphoribosyltransferase (gpt) gene under the control of simian virus 40 (SV40) early genes' regulating sequences (SV-gpt) within the vector and an SV40 origin of replication for amplification in T antigen-containing cells. SP65 also has an ampicillin-resistance gene. Propagate in Escherichia coli (E. coli) STBL2 grown at 30°C or E. coli HB101. For additional vector details please refer to the provided sequence file.
Since there is a gpt gene in the plasmid but not in the provirus, infected cells cannot be selected with any drug. Transfected cells can be selected with mycophenolic acid/hypoxanthine/xanthine. The amount to use depends on the cell type. Selection would ensure isolation of cells that have integrated the part of the construct that includes the SV-gpt gene (in the vector) but does not ensure that an intact provirus is integrated. One can easily screen selected clones for those that secrete virus by doing p24 assays on the culture supernatant.
Each vial of ARP-3422 contains approximately 5 µg of dried purified DNA stabilized in DNAstable®PLUS. Please refer to the appropriate data sheet for lot-specific information.
Page, K. A., T. Liegler and M. A. Feinberg. “Use of a Green Fluorescent Protein as a Marker for Human Immunodeficiency Virus Type I Infection”. AIDS Res. Hum. Retroviruses 13 (1997): 1077-1081. PubMed: 9282811.
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