Last data update: 16 January 2021 04:19 CET
Plasmid name: pAdD26SV(A)-3 (LMBP 729)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
Mouse dihydrofolate reductase cDNA (DHFR, Dhfr)|
|Promoter:||Adenovirus 2 major late promoter
Simian virus 40 early promoter (SV40 early)
Simian virus 40 late promoter (SV40 late)
|Terminator:||Simian virus 40 polyadenylation signal (SV40 polyA)|
|Selection marker:||Tetracycline (tet)
|Replicon:||Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
|Host range:||Escherichia coli
Mammalian cells; CHO/Dhfr(-)
|Further information:||The construction of the plasmid is described in Kaufman et al. (1982).
This vector carries an mDhfr cDNA as a directly selectable marker, under control of the strong adenovirus 2 major late promoter. Between this promoter and the mDhfr coding sequence, an intervening sequence is present: a hybrid intron with a 5' donor splice site included in the adenovirus DNA and a 3' acceptor splice site derived from an Ig variable region. The SV40 early polyadenylation signal is located downstream from the Dhfr coding sequence.
This plasmid is a convenient vector for the introduction of exogenous DNA into mammalian cells (preferably CHO/Dhfr(-) cells) using the method of cotransformation: transformants selected by means of the marker gene can then be examined for their ability to express the introduced gene. Growth in increasing metothrexate concentration allows amplification of the Dhfr gene as well as linked genes.
The prokaryotic-derived part of the vector contains a (+-) 1.1 kb deletion in pBR322 (between the AvaI site (nucleotide position 1425 on the LMBP pBR322 map) and the Eco-pMB1 origin), removing sequences inhibitory to replication in mammalian cells.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726852.1.
The nucleotide sequence of the mDhfr cDNA corresponds with the EMBL Nucleotide Sequence Database accession number L26316.1.
Since the nucleotide sequence of pAdD26SV(A)-3 could not completely be traced back, there is uncertainty as to the cutting frequency of the restriction enzymes, except for the sites that were analysed in the authenticity test.
|EMBL Accession number:||L26316.1, view at EMBL, GenBank, DDBJ
LT726852.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||24/02/2010|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: AccI, AflIII, AvaI, BglI, EcoRI, HindIII, NcoI, PstI and XhoI.
As compared to the fragments to be expected from the compiled nucleotide sequence, the 1264 bp BglI fragment is slightly larger (located in the agarose gel above the 1323 bp AccI fragment). Furthermore, the 2785 bp AccI fragment is rather approximately 2900 bp long (located above the 2872 bp BglI fragment), assuming that the plasmid is approximately 150 bp larger at the region between the AvaI site (position 1394) and the pMB1 origin.
|History of deposit:||This plasmid was deposited by R.J. Kaufman(1).
(1) Genetics Institute Inc., Cambridge, Massachusetts, USA
|Plasmid reference:||Kaufman et al., Mol. Cell. Biol. 2 (1982), 1304-1319 [PMID: 6131378]
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12xB HB101|
|Host reference:||Boyer and Roulland-Dussoix, J. Mol. Biol. 41 (1969), 459-472 [PMID: 4896022]
|Related host reference:||Sambrook et al. (eds), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989) [ISSN/ISBN: 0879693096]
|Cultivation medium:||LB-Lennox + tetracycline (10 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pAdD26SV(A)-3 (LMBP 729) is available at BCCM/GeneCorner. This plasmid was deposited by R.J. Kaufman and was published in Kaufman et al., 1982.|
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.