Last data update: 24 January 2024 16:39 CET
Plasmid name: pEntry attL4pCAGG-loxP-Bgeo-3xpA-loxP-attR1 (LMBP 6455)
| New search | Print data sheet |
| Price category: | Cat. 3 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p6455.gb
(View with Genome Compiler) p6455.txt p6455.pdf |
| Sequence analysis results Genecorner: |
Sanger: 79540.fasta Sanger: 105628.fasta NGS: c08-gc-dec2018.fasta |
| Cloned DNA: |
Escherichia coli lac Z gene (lacZ); with modified 5' end Transposon Tn5 neomycin phosphotransferase gene (neo); modified 5' end |
| Promoter: | Chicken β-actin/rabbit β-globin hybrid promoter (AG) Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only Phage T7 gene 10 promoter (T7g10) |
| Ribosome binding site: |
- |
| Terminator: | Escherichia coli rrnB operon T1 terminator Escherichia coli rrnB operon T2 terminator Simian virus 40 polyadenylation signal (SV40 polyA) |
| Selection marker: | Neomycin (neo; kanamycin (kan)) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pCall2-IRES-eGFP; pEntry L4-R1 |
| Further information: | The plasmid was created by cloning the ScaI/XhoI pCAGG-loxP-βgeo-3xpA+ fragment from the pCall2-IRES-eGFP vector into the BamHI (blunted)/XhoI pEntry L4-R1 opened vector. This Gateway entry vector is to be used with the pROSA26-DV2 or pROSA26-DV3 multisite plasmids. The plasmid contains the E. coli lac Z gene fused to the C-terminal Tn5 neomycin phosphotransferase gene. The fusion gene is called the β-galactosidase-neomycin fusion gene (Bgeo). The Tn5 neomycin phosphotransferase gene contains a E182D mutation, reducing the activity of the phosphotransferase to 20% of the wild type enzyme as described in Yenofsky et al. (1990). Taking the modified 5' end of the Tn5 neo gene into account, this mutation is located at codon 185. The depositor advises a G418 concentration of 200 μg/ml when selecting for transfected ES cells, although this may vary depending on the batch of G418 antibiotic and of the ES cells. The regions downstream from the T7 promoter and downstream from the T1 terminator were analysed at the Department for Molecular Biomedical Research (VIB, Ghent, Belgium). The depositor suggests that the products of the LR reactions using this vector should be sequenced at their junctions as is outlined in Nyabi et al. (2009) to ensure correct plasmid recombination before final electroporation of the targeting constructs into ES cells. The attR1 region was sequenced at the Department for Molecular Biomedical Research (VIB, Ghent, Belgium). The attL4 region was also sequenced at the Department for Molecular Biomedical Research (VIB, Ghent, Belgium) and revealed the presence of an insert between the attL4 site and the human CMV-IE enhancer, carrying a fragment of the ampicillin resistance gene. The depositor confirmed the presence of the insert: In theory, the presence of this fragment could lead to the silencing of the promoter in vivo although I have no direct proof that this is the case. This plasmid does work as how we published it. The Tn5 neomycin resistance gene was sequenced by a LMBP customer (December 2013). LMBP could confirm these sequence analysis results. The region downstream from the AG promoter up to nucleotide 6048 was sequenced by LMBP. Other name of the plasmid is pEntry attL4pCAGG-loxP-βgeo-3xpA-loxP-attR1. |
| EMBL Accession number: | - |
| Latest sequence update: | 13/12/2013 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BstXI/EcoRV, BstXI/NheI, Bsu36I/HindIII, EcoRI, HindIII, HpaI, NcoI, NcoI/XmaI, PvuII, SacII and XmaI. This plasmid has also been fully sequenced but the NGS sequence data still need to be implemented. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr J. Haigh(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Nyabi et al., Nucleic Acids Res. 37 (2009), e55 [PMID: 19279185] [DOI: 10.1093/nar/gkp112] |
| Related plasmid reference: | Yenofsky et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 3435-3439 [PMID: 2159150] |
| Restricted distribution: | - VIB/BCCM MTA - When appropriate in accordance with academic customs, RECIPIENT agrees to include the depositor(s) as co-author(s) 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] [DOI: 10.1016/s0022-2836(83)80284-8] 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 + kanamycin (50 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | LMBP 06354 |
Refer in your Materials and Methods: |
pEntry attL4pCAGG-loxP-Bgeo-3xpA-loxP-attR1 (LMBP 6455) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr J. Haigh and was published in Nyabi et al., 2009. |
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.