Products

pTagGFP2-H2B vector, FP196

* The vector sequence has been compiled using the information from sequence databases, published

   literature, and other sources, together with partial sequences obtained by Evrogen. This vector has not

   been completely sequenced.

Vector description

pTagGFP2-H2B is a mammalian expression vector encoding TagGFP2-H2B fusion protein. The vector can be

used for fluorescent labeling of histone H2B in living cells.

Transiently transfected HeLa cells expressing TagGFP2 fusion with human histone H2B.

TagGFP2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al. 1996].

Human histone H2B is fused to the TagGFP2 N-terminus.

pTagGFP2-H2B vector can be used as a source of TagGFP2-H2B hybrid sequence. The vector backbone

contains unique restriction sites that permit its excision and further insertion into expression vector of choice.

Note: The plasmid DNA was isolated from dam+-methylated E.coli. Therefore some restriction sites are

blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector

into a dam- host and make fresh DNA.

The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein

expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of

replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation

signals (SV40 poly A) direct proper processing of the 3’-end of the reporter mRNA.

SV40 early promoter (PSV40) provides neomycin resistance gene (Neor ) expression to select stably

transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene

expression (Kanr ) in E. coli. Kanr/Neor gene is linked with herpes simplex virus (HSV) thymidine kinase

(TK) polyadenylation signals.

Expression in mammalian cells

pTagGFP2-H2B vector can be transfected into mammalian cells by any known transfection method. CMV

promoter provides strong, constitutive expression of the TagGFP2-H2B fusion in eukaryotic cells. If required,

stable transformants can be selected using G418 [Gorman, 1985].

Propagation in E. coli

Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose

strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μ

g/ml) to E. coli hosts. Copy number in E. coli is about 500.