Vectors with segmented multiple cloning sites (SMCS) for easy monitoring of restriction digests and post-cloning orientation reversal of genes.
A novel approach is described, through which the transcriptional orientation of cloned genes is manipulated without further subcloning. This is achieved through a restriction/ligation process (without changing the test tube) leading to approx. 1:1 mixture of both possible orientations. The plasmid containing the reversed orientation is easily distinguished from the wild type by built-in restriction-indicator sites for SpeI. Additionally, a large marker fragment is incorporated into the segmented multiple cloning site (SMCS) regions to facilitate monitoring the progress and/or efficiency of vector restrictions. The marker fragment is released upon appropriate double digestion, thus acting as an indicator for complete restriction-enzyme digestion. We have equipped some existing plasmids, commonly used in molecular biology, with these features and demonstrated their superiority to plasmids having regular MCS sequences.
Georges
F
F
Plant Biotechnology Institute, National Research Council of Canada, Saskatoon.
Hussain
A
A
Papish
B
B
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids
D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific