GREAT AT SMALL THINGS

0

LMBP plasmid details

Last data update: 04 December 2020 04:17 CET

Plasmid name: pLGM-CSF12 (LMBP 2746)

Add to cart

New search

Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p2746.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse granulocyte macrophage colony stimulating factor genomic DNA (mGM-CSF)
Promoter: Mouse granulocyte macrophage colony stimulating factor promoter (GM-CSF)
Escherichia coli lac operon promoter
Ribosome
binding site:
-
Terminator: -
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Parental clone: pUC13; λGM-CSF12
Further information: The plasmid was constructed as follows: the mouse GM-CSF gene was isolated from a sperm DNA library in the λ phage vector Charon 4A and the 12 kb EcoRI insert of one clone, λGM-CSF12, was subcloned into the unique EcoRI site of pUC13. The orientation of this insert was experimentally verified with a SalI-SphI digestion.
This plasmid was used to analyze the organization of the mouse GM-CSF gene, in order to compare it with the structure of human GM-CSF genes as well as with the structure of other inducible T-cell lymphokine genes such as IL2, IL3 and IFNγ.
Strong conservation in the 5' flanking regions of mouse and human GM-CSF genes suggests that these sequences may play a role in regulating expression of the genes during T-cell activation.
The mouse GM-CSF promoter is excisable as an EcoRI-ScaI fragment.
Experimentally verified restriction sites indicated on the circular map: BamHI, EcoRI, EcoRV, HindIII, NdeI, SacI, SalI, ScaI, SphI and XbaI.
Other experimentally verified restriction sites:
Asp700 (XmnI): cutting frequency = 6 or 7; one site is located in the mouse GM-CSF promoter region; a second site is located in the third exon of the gene; a third site is located in the pUC13 part of the vector; the positions of the other sites are unknown.
BglII: cutting frequency = 2; one site is located in the second intron of the mouse GM-CSF gene; the second site must be located either in the 5' or 3' flanking region of the gene.
HindII: cutting frequency = 7; one site is located in the multicloning site of the pUC13 part of the vector; the positions of the other sites are unknown.
KpnI: cutting frequency is probably 2; one site is located in the second intron of the mouse GM-CSF gene.
StuI: cutting frequency = 5; two sites are located in the mouse GM-CSF promoter region; a third site is located in the 3' flanking region of the gene; the positions of the other two sites are unknown.
When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
The nucleotide sequence of the 5' and 3' flanking regions of the mouse GM-CSF gene is not known.
EMBL Accession number: -
Latest sequence update: 11/09/1992
Authenticity test: The plasmid still needs to be subjected to the authenticity test.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr R. Kastelein(1).
(1) DNAX, Palo Alto, USA
Plasmid reference: Miyatake et al., EMBO J. 4 (1985), 2561-2568 [PMID: 3876930]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 MC1061
Host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Related host reference: Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml)
Cultivation temperature: 37°C
Biosafety level: L1
Other culture collection numbers: -

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.

New search