pFusionRed-H2B vector

cat.# FP421

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.

Download vector information: Data sheet (pdf) Sequence (txt) Restriction map (pdf) Restriction table (pdf)

Product Cat.# Size Price pFusionRed-H2B FP421 20 μg € 480 / 240* *50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer. The price does not include delivery. The price varies in different countries. Please contact your local distributor for exact prices and delivery information. Vector type mammalian expression vector Reporter FusionRed Reporter codon usage mammalian Promoter for FusionRed PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use red fluorescent labeling of histone H2B

Vector description

pFusionRed-H2B is a mammalian expression vector encoding FusionRed-H2B fusion protein (see reporter description). The vector can be used for fluorescent labeling of histone H2B in living cells.

Pig kidney fibroblast cells expressing FusionRed fused to human histone H2B. The cell proceeds from early prophase through an entire cell division cycle and cytokinesis. Movie from Shemiakina et al., 2012.

FusionRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Human histone H2B is fused to the FusionRed N-terminus.

pFusionRed-H2B vector can be used as a source of FusionRed-H2B hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.

The vector backbone contains immediate early promoter of cytomegalovirus (P_{CMV 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 (P_SV40) provides neomycin resistance gene (Neo^r) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kan^r) in E. coli. Kan^r/Neo^r gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.

Expression in mammalian cells

pFusionRed-H2B vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-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 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.

Location of features

P_{CMV IE}: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Histone H2B: 657-1034
FusionRed: 1053-1748
Stop codon: 1749-1751
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1904-1909 & 1933-1938
mRNA 3' ends: 1942 & 1954
f1 single-strand DNA origin: 2001-2456
Bacterial promoter for expression of Kan^r gene
-35 region: 2518-2523
-10 region: 2541-2546
Transcription start point: 2553
SV40 origin of replication: 2797-2932
SV40 early promoter
Enhancer (72-bp tandem repeats): 2630-2701 & 2702-2773
21-bp repeats: 2777-2797, 2798-2818 & 2820-2840
Early promoter element: 2853-2859
Major transcription start points: 2849, 2887, 2893 & 2898
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2981-2983
Stop codon: 3773-3775
G->A mutation to remove Pst I site: 3163
C->A (Arg to Ser) mutation to remove BssH II site: 3509
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4011-4016 & 4024-4029
pUC plasmid replication origin: 4360-5003