User:Etienne Robillard/Notebook/Agent Ecoli:Plasmid cloning vectors for gene therapy with phosphohistidine based oligos

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(Artificial DNA promoters: phosphoramidate-based oligonucleotide synthesis)
(Artificial DNA promoters: phosphoramidate-based oligonucleotide synthesis)
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[http://openwetware.org/wiki/20.109(S07):Start-up_genome_engineering#Part_3:_Digest_M13K07 M13K07] cloning in ''E. coli'':
[http://openwetware.org/wiki/20.109(S07):Start-up_genome_engineering#Part_3:_Digest_M13K07 M13K07] cloning in ''E. coli'':
* amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL
* amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL
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Orthogonal DNA shell with Benzyl-derived (Bz) [http://en.wikipedia.org/wiki/Protecting_group#Carboxylic_acid_protecting_groups protecting groups]:
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* note! note! Important: Can you elucidate the reasoning of using an carboxilic acid as protecting group for DNA assembly?
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References:
References:

Revision as of 07:27, 12 November 2012

Plasmid cloning vectors for gene therapy with phosphohistidine-based oligos

Contents

Artificial DNA promoters: phosphoramidate-based oligonucleotide synthesis

cDNA:

  • N-6-benzoyl-deoxyadenosine phosphoramidite - Notice the P-N bound indicating the linkage of a phosphonate diester to a benzoyl atom :)
  • N-3-oxo-hexanoyl-L-homoserine lactone - A light-inducible phosphohistidine-bound AHL ? A(denosine)TP/cAMP dependent.
  • Reminder:
    • NHC = (N heterocycle carbene) - Chemical compounds with a N chiral prodrug switch - highly enantioselective molecules
    • N = indicates a Nitrogen atom
    • P = a Phosphonate (inorganic state) - a compound for oligo synthesis using the nucleoside phosphoramidite method.
    • P-N = phosphoramidate (a Nitrogen atom bound to a Phosphonate ester in nucleophilic substitution).

deoxynucleotide synthesis:

  • cDNA synthesis building block = deoxynucleoside + 3'-phosphonate = synthetic nucleotide analog (dATP) 1

oligodeoxyribonucleotide synthesis:

  • short 50-200(?) bp restriction endonucleases enzymes for cDNA catalysis/plasmid vector cloning in E. coli
  • building blocks for oligonucleotides assembly with phosphoramidites method 1
  • see also Oligonucleotide Synthesis
  • 1 mutation, 1 oligo assay (phosphoramidite-based oligo synthesis): [1]

M13K07 cloning in E. coli:

  • amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL

Orthogonal DNA shell with Benzyl-derived (Bz) protecting groups:

  • note! note! Important: Can you elucidate the reasoning of using an carboxilic acid as protecting group for DNA assembly?


References:

References

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