Last data update: 24 October 2021 17:14 CEST
Plasmid name: pENTR223-ORF (LMBP 9210)
|New search||Print data sheet|
|Price category:||Cat. 2 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
Human ORF cDNA|
|Promoter:||Phage T7 gene 10 promoter (T7g10)|
|Terminator:||Escherichia coli rrnB operon T1 terminator
Escherichia coli rrnB operon T2 terminator
|Selection marker:||Streptomycin (Sm; spectinomycin (Sp))|
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli|
|Further information:||This datasheet is representative for all the hORFeome v8.1 clones developed at the Center for Cancer Systems Biology (USA) and available at BCCM/GeneCorner. Consequently, the accession number 'LMBP 9210' of this template clone is fictitious.
More information on the hORFeome project can be found on www.orfeomecollaboration.org and http://horfdb.dfci.harvard.edu/
The library was based on the Mammalian Gene Collection (MGC), isolating all available ORFs in this resource via PCR, adding the Gateway attenuator sites attB1.1 and attB2.1 to the forward and reverse primer respectively. The integrase binding sites within the attenuator sites attB1.1 and attB2.1 has been altered to more closely resemble the bacteriophage lambda consensus sequence, increasing the efficiency of Gateway recombination. The forward ORF-specific primer includes the ATG start codon, and incorporates a Kozak consensus nucleotide G at position -3 relative to the ATG, thereby allowing efficient translation initiation from the ORF start codon. The reverse ORF-specific primer does not contain the last nucleotide of the termination codon, to allow generation of C-terminal fusion proteins. The resulting ORFs were cloned into pDONR223 (Invitrogen) via Gateway BP recombination.
Please take into account that the reading frame has been shifted by one nucleotide for the creation of C-terminal fusion proteins due to the absense of the last nucleotide of the ORF termination codon.
pENTR223-ORF contains the streptomycin/spectinomycin resistance gene aadA.
|EMBL Accession number:||-|
|Latest sequence update:||15/05/2014|
Primers used for PCR amplification of the human ORFs: <--- attB1.1 ---> Forward primer: 5' GGGGACAACTTTGTACAAAAAAGTTGGC.ATG.NNN.NNN.NNN.NNN.NNN.NNN + ++ *** <--- attB2.1 ---> Reverse primer: 5' GGGGACAACTTTGTACAAGAAAGTTGG.GYA.NNN.NNN.NNN.NNN.NNN.NNN + + ^^^ +: changes compared to standard attB1 and attB2 sequence ***: start codon. ^^^: modified, inactive termination codon. Punctuation indicates reading frame.
|Authenticity test:||The inserts of all clones contained in the hORFeome v8.1 library have been sequenced by the depositor. The clones were sequenced in bulk via Illumina sequencing, which may have a higher error rate than individual Sanger sequencing.|
|History of deposit:||This plasmid was deposited by Dr M. Vidal(1) and Dr D. Hill(1). It was received from Dr I. Lemmens(2).
(1) Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA, USA
(2) Department of Medical Protein Research, VIB, Ghent, Belgium
|Plasmid reference:||Yang et al., Nat. Methods 8 (2011), 659-661 [PMID: 21706014]
|Related plasmid reference:||Rual et al., Genome Res. 14 (2004), 2128-2135 [PMID: 15489335]
|Restricted distribution:||- BCCM/MTA adapted for hORFeome
- The depositor will be informed of the customer's identity upon release of a sample outside the depositor's department or outside the departments in which BCCM/GeneCorner is embedded, namely UGent-DBMB and VIB-IRC.
- RECIPIENT agrees to refer to 'Yang et al., Nat. Methods 8 (2011), 659-661 [PMID: 21706014]' in the first publication making use of the MATERIAL.
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 DH5α|
|Host reference:||Focus 8 (1986), 9
|Related host reference:||Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
|Cultivation medium:||LB-Lennox + spectinomycin (50 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pENTR223-ORF (LMBP 9210) is available at BCCM/GeneCorner. This plasmid was deposited by Dr M. Vidal and Dr D. Hill and was published in Yang et al., 2011.|
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.