chloramphenicol acetyltransferase reporter gene plasmids pcat3-enhancer vector contains sv40 enhancer  (Promega)

Journal:

Article Title: Molecular Characterization and Placental Expression of HERV-W, a New Human Endogenous Retrovirus Family

doi:

Figure Lengend Snippet: Definition of a putative LTR. (A) The 2,364- to 2,720-nt region of the cl.PH74 clone, identified as a putative promoter region with Signalscan 4.05, was PCR amplified and subcloned into pCAT3 reporter vector. The resulting pCAT3-3′LTR plasmid and pCAT3 control plasmid were used to transfect HeLa cells prior to CAT activity determination. Cells, HeLa cell extract; pCAT3 and pCAT3-3′LTR, extract of HeLa cells transfected with control plasmid and promoter plasmid, respectively. (B) Alignment of 5′ and 3′ UTRs of placental experimental clones with the 5′ (5-RG-28000-28872) and 3′ (3-RG-37500-38314) repeated sequences of the 28,000- to 38,314-nt fragment of human DNA sequence RG083M05. The end of the 3′-most ORF is indicated (env orf). The tandemly repeated CAAC flanking sequences are doubly underlined on DNA sequences. The 783-bp LTR consensus sequence is positioned at the bottom. The polypurine tract (PPT) upstream from the 5′ end of the LTR and the tRNATrp PBS downstream from the 3′ end of LTR are indicated. U3, R, and U5 subparts are indicated, except for the U3-R junction. Transcription factor sites determined with Signalscan 4.05 are underlined and labelled from I to VI. The sites V and VI may correspond to CCAAT box and TATA box, respectively. The 3′ region of clone cl.PH74 used in the CAT assay is underlined on the cl.PH74 sequence. ∗ and ▴, potential cap sites obtained in 5′ RACE experiments with human placental poly(A)+ RNA and total RNA extracted from pCAT-3′LTR-transfected HeLa cells, respectively. [polyA], polyadenylation signal.

Article Snippet: The putative promoter region was cloned into pCAT3 Enhancer reporter vector purchased from Promega Biotec (Madison, Wis.).

Techniques: Amplification, Plasmid Preparation, Activity Assay, Transfection, Clone Assay, Sequencing