Last data update: 24 January 2024 16:39 CET
Plasmid name: pCoofy44 (LMBP 13626)
| New search | Print data sheet |
| Price category: | Cat. 3 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p13626.gb |
| Manual | Download pCoofy manual |
| Sequence analysis results Genecorner: |
Sanger: .fasta |
| Cloned DNA: |
Small ubiquitin-related modifier tag (SUMO tag); variant SUMOstar 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) Histidine tag (His-tag); C-terminal Strep-tag III (Twin-Strep-tag); C-terminal S-tag; C-terminal Calmodulin-binding peptide tag (CBP); C-terminal Protein C HPC4-tag; C-terminal; C-terminal MARTX toxin Cysteine Protease Domain tag (CPD); C-terminal |
| Promoter: | Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter (PH) Escherichia coli plasmid R388 class 1 integron Pc promoter (PcS) Escherichia coli major outer membrane lipoprotein promoter (lpp); mutant lpp5 |
| Ribosome binding site: |
- |
| Terminator: | Simian virus 40 polyadenylation signal (SV40 polyA) |
| Selection marker: | Ampicillin (amp) Gentamicin (Gm) |
| Replicon: | Escherichia coli plasmid pMB1 origin Phage f1 origin |
| Copy number: | High copy number |
| Host range: | Escherichia coli Insect cells; e.g. Sf9 cells |
| Parental clone: | pFastBac1 |
| 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). The SUMOstar tag carries the mutations R64T and R71E (Liu et al., 2008). The S-tag is an oligopeptide derived from pancreatic ribonuclease A. The plasmid contains Tn7 transposon elements, allowing site-directed transposition in the E. coli DH10Bac strain. |
| EMBL Accession number: | - |
| Latest sequence update: | 06/12/2022 |
| Authenticity test: | This plasmid has been fully sequenced but the NGS results still need to be implemented. |
| 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: | - |
| Related plasmid reference: | Liu et al., Protein Expr. Purif. 62 (2008), 21-28 [PMID: 18713650] [DOI: 10.1016/j.pep.2008.07.010] Li and Elledge, Nat. Methods 4 (2007), 251-256 [PMID: 17293868] [DOI: 10.1038/nmeth1010] Scholz et al., BMC Biotechnol. 13 (2013), 12 [PMID: 23410102] [DOI: 10.1186/1472-6750-13-12] |
| 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 + ampicillin (100 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. |
| Other culture collection numbers: | Addgene 55185 |
| Related website: | https://p4eu.org/wiki/pcoofy-slic-cloning/ |
Refer in your Materials and Methods: |
pCoofy44 (LMBP 13626) is available at BCCM/GeneCorner. This plasmid was deposited by J. Scholz and Dr S. Suppmann. |
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.