pNEBR-X1Hygro plasmid vector质粒载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pNEBR-X1Hygro plasmid vector质粒载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pNEBR-X1Hygro is a plasmid cloning vector capable both of
replication in E. coli and stable transfection of mammalian cells.
It is designed for inducible expression of recombinant proteins
in mammalian cells using the RheoSwitch Mammalian Inducible
Expression System (NEB #E3000).
In E. coli, it replicates using the pMB1 origin of replication from
pBR322 (although the rop gene is missing) and carries the bla
(ApR
) marker for selection with ampicillin. It also carries the hpt
(HygR
) marker under control of the thymidine kinase promoter;
thus, following transfection into mammalian cells, it can be used
to form stable cell lines by selection with hygromycin.
The multiple cloning site (MCS) is positioned downstream of a
promoter apparatus consisting of 5 tandem copies of the yeast
GAL4 response element (5XRE) followed by a minimal TATA
box and a short leader sequence, and upstream of the SV40
polyadenylation (polyA) sequence. When co-transfected in mammalian cells with pNEBR-R1 (which encodes the RheoReceptor-1
protein), expression from the GAL4-derived promoter can be
induced by the synthetic RSL1 ligand and controlled in a RSL1
concentration-dependent manner. A transcription terminator upstream of the 5XRE (not shown) prevents read-through
transcription from other sources.
pNEBR-X1 is identical to pNEBR-X1Hygro except that it does
not contain the hpt (HygR
) marker, so it is intended to be used
for transient transfection. pNEBR-X1GLuc is a control plasmid
with the reporter gene GLuc (the humanized coding sequence
for the secreted Gaussia princeps luciferase) (1) cloned into the
HindIII-NotI sites of pNEBR-X1.
Enzymes with unique restriction sites are shown in bold type.
Location of sites of all NEB restriction enzymes can be found on
the NEB web site (choose Technical Reference > DNA Sequences
and Maps). Restriction site coordinates refer to the position of
the 5’-most base on the top strand in each recognition sequence.
Open reading frame (ORF) coordinates are in the form “translational start – translational stop”; numbers refer to positions
on the top (clockwise) strand, regardless of the direction of
transcription and include the start and stop codons. Components
of coordinated regions are indented below the region itself.
pMB1 origin of replication coordinates include the region
from the –35 promoter sequence of the RNAII transcript to the
RNA/DNA switch point. SV40 transcription terminator and polyA
coordinates represent cloned regions and not necessarily the
precise functional elements.

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