Part:BBa_K1128001
Targetted Methylation Assay
MaGellin is a platform that streamlines the process of testing the specificity of DNA binding domains and the activity of methyltransferase-DNA binding domain fusion proteins. This technology combines the use of methylation-sensitive restriction enzymes with a powerful software package to identify and quantify the location and amount of CpG DNA methylation. The one-plasmid design ensures simple customization of the DNA-binding domain and targeting sequence, easy readout by agarose gel electrophoresis and analysis by included built-in software, and pre-designed bisulfite sequencing primers.
The plasmid itself is a modified version of Novagen's pET-26(b)+ vector with additional sites cloned in, as well as the standard BioBrick prefix and suffix for cloning in standard parts from the registry. The plasmid backbone contains two AvaI restriction sites, which are composed of CYCGRG and are cut by the AvaI restriction enzyme. These sites can be CpG methylated, which blocks the activity of the AvaI enzyme. The backbone also contains a 9 base pair region that can be removed and replaced. This site is upstream of one of the AvaI sites and can act as a transcription factor "target site", used to determine where certain DNA transcription factors bind. This biobrick has the BBa_J04450 insert, which is an RFP expression cassette with a pLac promoter. This backbone has a lac repressor gene, and will NOT express the RFP in vivo. The backbone has a gene for Kanamycin resistance.
For a detailed, graphical explanation of the MaGellin work flow and case study experimental data, please download the MaGellin Workflow Specifications Sheet , which includes all of the steps in the MaGellin workflow.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found at 5068
Illegal suffix found at 6159 - 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 5068
Illegal NheI site found at 6269
Illegal NheI site found at 6292
Illegal SpeI site found at 6160
Illegal PstI site found at 6174
Illegal NotI site found at 5074
Illegal NotI site found at 6167 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 5068
Illegal BglII site found at 4964
Illegal BamHI site found at 6452
Illegal XhoI site found at 6488 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 5068
Illegal suffix found at 6160 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 5068
Plasmid lacks a suffix.
Illegal XbaI site found at 5083
Illegal SpeI site found at 6160
Illegal PstI site found at 6174
Illegal NgoMIV site found at 137
Illegal NgoMIV site found at 3184
Illegal NgoMIV site found at 3344
Illegal NgoMIV site found at 4932
Illegal NgoMIV site found at 6203
Illegal AgeI site found at 5870
Illegal AgeI site found at 5982 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal SapI.rc site found at 2263
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