in another host, produces protein dihydrofolate reductase

Construct size (kb): 5.099999904632568

DESCRIPTION OF VECTOR COMPONENT:
Name of vector: pSV2-neo
Intact vector size: 5.729
Type of vector: plasmid
Vector end: HindIII
Vector end: SmaI
Cloning sites: EcoRI BamHI PvuII PstI HindIII
Polylinker sites:
Construction: SV40, Tn5, pBR322
Host range: vertebrate cells; Escherichia coli
Features (with orientation and position when available):
replicon: SV40
marker(s): G418R, <-
restriction site: HindIII
promoter: SV40 early, <-
replicon: pMB1
marker(s): ampR, <-
Cross references: DNA Seq. Acc.: U02434

DNA: cDNA

DESCRIPTION OF INSERT COMPONENT:
Genome: hamster, Chinese
Gene symbol: DHFR
Gene name: dihydrofolate reductase
Contains complete coding sequence?: U
Tissue: lung cell line
Type of DNA: cDNA
Insert 5' end: HindIII
Insert 3' end: BglII
Insert size (kb): 0.71
Cross references:

Insert lengths(kb): 0.7099999785423279

Tissue: lung cell line

Gene product: dihydrofolate reductase [DHFR]

Target Gene: dihydrofolate reductase

Restriction digests of the clone give the following sizes (kb): BamHI--3.5, 1.7; EcoRI--2.7, 2.3; PstI--1.9, 1.4, 0.95, 0.9; PvuII--4.0, 1.1; HindIII/SmaI--5.2.

Transfected cells amplify DHFR sequences.

Encodes the L22F mutant of DHFR that can serve as a dominant selection marker because it retains catalytic activity and also confers methotrexate (MTX) resistance at low MTX concentrations (125 nM).

Constructed by cloning a HindIII/SmaI fragment containing the gene into pSV2-neo from which neoR had been removed. This plasmid was then cut with SmaI and BglII to remove 18 G nucleotides at the 3' end.

The BglII site was filled in, and ligated to the SmaI site, thereby destroying the original BglII and SmaI sites.

Removing the HindIII/SmaI fragment from pSV2-neo removes all but the 3' 171 bp of neoR.