Purpose
Expression of human codon-optimized dCas9-DNMT3A fusion with T2A-PuroR for targeted DNA methylation in mammalian cells； cloning backbone for sgRNA
Backbone
Vector backbone：PX462
Backbone size w/o insert (bp)：4246
Total vector size (bp)：10152
Vector type：Mammalian Expression, CRISPR
Growth in Bacteria
Bacterial Resistance(s)：Ampicillin, 100 μg/mL
Growth Temperature：37℃
Growth Strain(s)：DH5alpha
Copy number：Unknown
Gene/Insert
Gene/Insert name：S. pyogenes dCas9 fused with the catalytic domain of human DNMT3A (amino acids P602-V912) and T2A-PuroR
Alt name：dCas9-DNMT3A-PuroR
Alt name：SpdCas9-DNMT3A-T2A-PuroR
Alt name：dCas9-DNMT3A
Species：H. sapiens (human), Synthetic； S. pyogenes
Insert Size (bp)：5906
Mutation：D10A and H840A in S.pyogenes Cas9
GenBank ID：AKA60242.1 NP_072046.2
Entrez Gene：DNMT3A (a.k.a. DNMT3A2, HESJAS, M.HsaIIIA, TBRS)
Promoter：CBh
Tags/ Fusion Proteins：
3xFLAG (N terminal on insert)
SV40 NLS (N terminal on insert)
T2A-PuroR (C terminal on insert)
Cloning Information
Cloning method：Restriction Enzyme
5′ cloning site：BamHI (not destroyed)
3′ cloning site：FseI (not destroyed)
5′ sequencing primer：GS-linker For 5' GAAGAGGTACACCAGCACCAAAG 3'
3′ sequencing primer：BGH Rev
