Modified transcriptionally active SP6 plasmid vector

Details Modified transcriptionally active SP6 plasmid vector Modified transcriptionally active SP6 plasmid vector

1986-08-05

1990-08-14

Teskin, Robin L.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435320, 4351723, 435 691, 43525233, 4352536, 935 24, 935 41, 935 56, 935 73, 935 27, 536 27, C12N 100, C12N 1500, C12N 122, C12N 120, C12P 1934, C12P 1206, C07H 1512

Patent

active

049487310

ABSTRACT:
A recombinant plasmid which may be used for propagation of cloned cDNAs and also for the in vitro synthesis of RNA which is an exact copy of the natural sequence, wherein the transcript is devoid of vector-derived sequence. The novel vector was generated de novo by genetic engineering procedures using a synthetic double strand oligodeoxyribonucleotide fragment and a larger DNA fragment derived from plasmid pSP64. The vector, plasmid pHST-O, carried by Escherichia coli HB101, deposited with the ATCC on Dec. 30, 1985 and designated ATCC 53381, is distinguished from other vectors containing the SP6 promoter by the following characteristics: it contains a unique site for the restriction endonuclease BglII; the engineered GBlII site overlaps the downstream border of the SP6 promoter sequence; and the presence and positioning of the BglII restriction site permit insertion of cDNA molecules in such a way that transcription by the SP6 RNA polymerase begins exactly at the 5' terminus of the RNA, providing that the 5' terminal nucleotide of the mRNA transcript is a guanosine (G) residue, thus excluding the transcription of nucleotides derived from the vector. The novel vector permits synthesis of RNA molecules which have a defined 5' terminus and which are devoid of vector-derived sequence. The vector has potential use in research in molecular biology and in the in vitro production of RNA and proteins.

REFERENCES:
patent: 4766072 (1988-08-01), Jendrisak et al.
Itakura et al, Science, vol. 209, pp. 1401-1405 (1980).
Jobling, S. A., Nucl. Acids Res., vol. 18, pp. 4483-4498 (1988).
Janda, M. et al, Virology, vol. 158, pp. 259-262 (1987).
Ahlquist, P. et al, Mol. Cell. Bio., vol. 4, pp. 2876-2882 (1984).
"Synthesis of Infectious Poliovirus RNA by Purified T7 RNA Polymerase", by S. Van Der Werf et al, Proc. Natl, Acad. Sci., U.S.A., 83, 2330-2334 (1986).
"Bacteriophage SP6-14 Specific RNA Polymerase", by E. T. Butler et al, J. Biol. Chem., 257, 5772-5778 and 5779-5788 (1982).
"Efficient in Vitro Synthesis of Biologically Active RNA and RNA Hybridization Probes from Plasmids Containing a Bacteriophage SP6 Promoter", by D. A. Melton et al, Nucl. Acids Res., 12, 7035-7056 and 7057-7070 (1984).

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