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LMBP plasmid details

Last data update: 05 March 2021 04:23 CET

Plasmid name: SHC005 (sh-eGFP) (LMBP 7275)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: -
Sequence
analysis results
Genecorner:

-

Cloned DNA: Short hairpin RNA sequence targeting the EGFP transcript (sh-EGFP)
Human immunodeficiency virus pol gene; central polypurine tract (HIV-1 cPPT)
Promoter: Human U6 small nuclear RNA gene promoter
Human phosphoglycerate kinase 1 promoter (PGK1)
Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 5' LTR)
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Escherichia coli lac operon promoter
Ribosome
binding site:
-
Terminator: Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 3' LTR); modified HIV-1 3' LTR with deleted U3 region (ΔU3/3' LTR)
Simian virus 40 polyadenylation signal (SV40 polyA)
Selection marker: Ampicillin (amp)
Puromycin (puro)
Replicon: Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
Phage f1 origin
Host range: Escherichia coli
Mammalian cells; SV40 permissive cells
Parental clone: SHC001 (empty vector)
Further information: Product No: SHC005
www.sigmaaldrich.com/catalog/product/sigma/shc005
SHC005 (sh-eGFP) is a 7086 base pair lentiviral plasmid that contains a shRNA sequence targeting the EGFP transcript (EMBL Accession Number U55761).
The shRNA insert is expressed under control of the human U6 promoter.
EGFP is an enhanced red-shifted variant of the Aequorea victoria green fluorescent protein GFP.
SHC005 (sh-eGFP) is useful as a positive knockdown control in experiments using cell lines expressing EGFP.

MEDIUM INFORMATION

Grow for 20-22 hours in TB-8gly medium (modified Terrific Broth medium, containing 8 ml/l glycerol instead of 4 ml/l) with 100 μg/ml carbenicillin, resulting in an increased plasmid stability as compared to ampicillin.
BCCM/GeneCorner uses the 'Terrific Broth, modified' from Sigma (cat. nr. T0918, Fluka). You can also prepare the medium yourself:
1. add the following to 800 ml distilled H2O:
- 12,0 g tryptone (enzymatic casein digest)
- 24,0 g yeast extract
- 8 ml glycerol
2. adjust to 900 ml with distilled H2O
3. sterilize by autoclaving (20 minutes at 121°C)
4. allow to cool to room temperature
5. adjust volume to 1000 ml with 100 ml of a filter-sterilized solution of 0,17M KH2PO4 and 0,72M K2HPO4

HOST STRAIN INFORMATION

Sigma recommends the host strain Escherichia coli K12 GC5 (catalogue number: G3169) for transformation with this shRNA control vector.
According to Sigma, the genotype of this GC5 strain is identical to the genotype of the Escherichia coli K12 DH5αT1R strain, which is a T1 bacteriophage resistant DH5α derivative.
Genotype: Φ80lacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 relA1 gyrA96 hsdR17(rk-, mk+) phoA supE44 thi-1 tonA (Ref.: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/shclngbul.Par.0001.File.tmp/shclngbul.pdf)
EMBL Accession number: U55761, view at EMBL, GenBank, DDBJ
Latest sequence update: 10/06/2011
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: EcoRI, HindIII and PvuII.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was purchased from Sigma-Aldrich(1) and deposited in the frame of the Hercules project 'RNAi collection and associated facilities' by Prof. Dr R. Beyaert(2)(3).
(1) Schnelldorf, Germany, www.sigmaaldrich.com
(2) Inflammation Research Center, VIB, Ghent, Belgium
(3) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
Related plasmid reference: Stewart et al., RNA 9 (2003), 493-501 [PMID: 12649500]
Zufferey et al., Nat. Biotechnol. 15 (1997), 871-875 [PMID: 9306402]
Zufferey et al., J. Virol. 72 (1998), 9873-9880 [PMID: 9811723]
Restricted distribution: - Acknowledge BCCM/GeneCorner (supplier) and the Hercules Foundation (funder) in any publication resulting from the use of a shRNA clone
- Restricted to project-related users
- The buyer cannot sell or transfer this product, its components nor materials made using this product or its components to another research group.
- Visit www.sigma.com/shRNA for more, up-to-date info on the limited use of this product.
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 GC5
Host reference: -
Cultivation medium: TB-8gly + ampicillin (100 μg/ml)
Cultivation temperature: 37°C
Biosafety level: L1 in E. coli; L2 in mammalian cells
Other culture collection numbers: LMBP 07135
Related website: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/shc001bul.Par.0001.File.tmp/shc001bul.pdf

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.

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