pShuttle2腺病毒穿梭表達載體

BioVector NTCC質粒載體菌種細胞蛋白抗體基因保藏中心

Description

pShuttle2 is a cloning vector designed for use with the Adeno-X? Expression System

(Cat. No. 631513; 631022). In the Adeno-X System, standard ligation techniques are used to

transfer a gene-specific expression cassette from pShuttle2 to a replication-deficient, Ad5 genome

(2). The pShuttle2 expression cassette consists of the human cytomegalovirus immediate early

promoter (P-CMV IE), a multiple cloning site (MCS), and the SV40 polyadenylation signal (SV40 poly

A). The entire cassette is flanked by unique I-Ceu I and PI-Sce I restriction sites so that it can be

excised and directly ligated to Adeno-X Viral DNA—the adenoviral genome. The vector backbone

also contains the pUC origin (pUC ori) and a kanamycin resistance gene (Kanr) for propagation and

selection in E. coli.

Use

Insert your full-length cDNA into the MCS of pShuttle2 using any of the unique restriction sites shown.

Your cDNA insert must contain a start codon for proper promoter driven expression in mammalian

cells. After the gene of interest has been cloned into pShuttle2, excise the expression cassette by

digesting the plasmid with I-Ceu I and PI-Sce I according to the one-step double digestion procedure

in the Adeno-X Expression System User Manual (PT3414-1). Because the termini of the excised

expression cassette fragment are compatible with the cloning site in Adeno-X Viral DNA, the cassette

can be directly ligated to Adeno-X DNA to form a recombinant adenoviral vector.

Location of features

? Human immediate early cytomegalovirus promoter (PCMV IE)

Enhancer region: 336–832

TATA Box: 827–832

Transcription start point: 853

? Multiple Cloning Site (MCS): 918–995

? SV40 polyadenylation signal: 1038–1043

? pUC origin of replication: 1393–2036

? Kanamycin resistance (aminoglycoside phosphotransferase) gene: 2710–3525

? I-Ceu I Recognition sequence: 3–28

Propagation in E. coli

? Suitable host strains: DH5α and other general purpose strains.

? Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.

? E. coli replication origin: pUC

? Copy number: high

References

1. Adeno-X Expression System (January 2000) Clontechniques XV (1):8–10.

2. Mizuguchi, H. & Kay, M. A. (1999) Hum. Gene Ther. 10:2013–2017.

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