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At a Glance
|Origin of Replication||
|Link to Sequence|
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"The pBADX53 expression plasmid was constructed by making the following modifications to the pBAD30 plasmid obtained from American Type Culture Collection (ATCC): (i) the origin of replication was replaced with the low-copy SC101 origin of replication; (ii) the araC gene was removed, leaving the araC pro- moter intact; (iii) the ribosome binding site from the Pbad promoter in the pBAD18s (ATCC) plasmid was inserted for use with the luciferase gene in control cells; and (iv) an n-myc DNA fragment was inserted upstream of the rrn T1/T2 transcription terminators to provide an alter- native unique priming site for real-time PCR. Plasmids were constructed using basic molecular cloning techniques described in standard cloning manuals (, ). Copies of all transcripts in the SOS test network were obtained by PCR amplification of cDNA using PfuTurbo. cDNA was prepared from total RNA as described below. PCR primers included overhanging ends containing the appropriate restriction sites for cloning into the pBADX53 plasmid. Endogenous ribosome binding sites were included in the cDNA fragments for all SOS test network genes that were cloned into the pBADX53 plasmid. Sequences of the cloned SOS test network genes and their ribosome binding sites were verified using an Applied Biosystems Prism 377 Sequencer. All cloning was performed by TSS transformation ()."
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See Help:References for how to manage references in EcoliWiki.
- Ausubel, F et al. eds. (2007) Current Protocols in Molecular Biology Wiley.
- Sambrook, J and Russell DW (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)
- Gardner, TS et al. (2003) Inferring genetic networks and identifying compound mode of action via expression profiling. Science 301 102-5 PubMed
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