Last data update: 24 January 2024 16:39 CET
Plasmid name: pCoofy8 (LMBP 13598)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p13598.gb |
| Manual | Download pCoofy manual |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Halo-tag; N-terminal Human rhinovirus 3C protease cleavage site (PreScission site, PRS) B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene) Escherichia coli DNA-binding transcriptional repressor LacI cDNA (lacI, GeneID 945007) |
| Promoter: | Phage T7 gene 10 promoter (T7g10) with lac operator (T7lac) Escherichia coli major outer membrane lipoprotein promoter (lpp); mutant lpp5 |
| Ribosome binding site: |
- |
| Terminator: | Phage T7 gene 10 terminator (T7g10) |
| Selection marker: | Kanamycin (kan) |
| Replicon: | Phage f1 origin Escherichia coli plasmid pMB1 origin |
| Copy number: | Low copy number |
| Host range: | Escherichia coli |
| Parental clone: | pETM14 |
| Further information: | The plasmid has been generated for SLIC cloning, parallel Sequence and Ligation Independent Cloning, which is based on homologous recombination. For parallel SLIC cloning with negative ccdB selection, the vector is PCR linearized with an LP1 forward, corresponding to the PreScission site for tag removal, and an LP2 reverse primer either located at the C-terminus of ccdB or corresponding to a C-terminal tag. In both cases the ccdB gene is deleted upon PCR amplification thereby allowing counterselection of parental empty vector in ccdB sensitive cells. The gene of interest needs to be PCR amplified with primers composed of 5’ and 3’ gene specific sequences plus 15 bp-25 bp extensions complementary to LP1 and LP2 vector primers, respectively. Primers can be found in Scholz et al. (2013) and in the P4EU document (see Related website). |
| EMBL Accession number: | - |
| Latest sequence update: | 01/12/2022 |
| 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 J. Scholz(1) and Dr S. Suppmann(1). (1) Protein Production Core Facility, Max-Planck Institute of Biochemistry, Martinsried, Germany |
| Plasmid reference: | Scholz et al., BMC Biotechnol. 13 (2013), 12 [PMID: 23410102] [DOI: 10.1186/1472-6750-13-12] |
| Related plasmid reference: | Li and Elledge, Nat. Methods 4 (2007), 251-256 [PMID: 17293868] [DOI: 10.1038/nmeth1010] |
| Restricted distribution: | - BCCM MTA adapted by Max Planck - The depositor will be informed of the customer's identity upon release of a sample outside the depositor's department. - Restricted to academic users |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12xB DB3.1 |
| Host reference: | - |
| Related host reference: | Bernard et al., J. Mol. Biol. 226 (1992), 735-745 [PMID: 1324324] |
| Cultivation medium: | LB-Lennox + kanamycin (50 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | For growth more than 24h may be needed. SOB medium instead of LB-Lennox may be required for DNA isolation of this low copy construct. |
| Other culture collection numbers: | Addgene 43984 |
| Related website: | https://p4eu.org/wiki/pcoofy-slic-cloning/ |
Refer in your Materials and Methods: |
pCoofy8 (LMBP 13598) is available at BCCM/GeneCorner. This plasmid was deposited by J. Scholz and Dr S. Suppmann and was published in Scholz et al., 2013. |
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.