Last data update: 24 January 2024 16:39 CET
Plasmid name: pSV71BlaHG3f (LMBP 3306)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p3306.gb
(View with Genome Compiler) p3306.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Ampicillin resistance gene (amp) Human immunoglobulin γ3; hinge region and CH2 and CH3 domains of the heavy chain (HG3f) |
| Promoter: | Simian virus 40 late promoter (SV40 late) Simian virus 40 early promoter (SV40 early) |
| Ribosome binding site: |
- |
| Terminator: | Simian virus 40 polyadenylation signal (SV40 polyA) |
| Selection marker: | Chloramphenicol (cam) |
| Replicon: | Escherichia coli plasmid pMB1 origin Simian virus 40 bidirectional origin (SV40) |
| Host range: | Escherichia coli; preferably recombination-deficient strains Mammalian cells; SV40 permissive cells |
| Parental clone: | pSV71BLAHI; pSV51cE6Hy3 |
| Further information: | The plasmid was constructed by substituting the 182 bp ApaI (blunted with T4 DNA polymerase) - SstI(SacI) fragment of pSV71BLAHI encoding the isolated human γ3 hinge region, with the 952 bp HindII-SstI(SacI) fragment of pSV51cE6Hy3 which contains the cDNA of the human γ3 heavy chain fragment (HG3f) comprising the complete hinge region and the CH2 and CH3 domains. As a result, the mutated ampicillin resistance gene in which the termination codon has been replaced by GAG, is directly fused in frame to the start of the first hinge region H1 of the HG3f sequence. The complete fusion gene is placed under control of the SV40 late promoter. After transient expression in COS-1 cells, the BlaHG3f fusion protein was correctly synthesized and efficiently secreted into the culture medium. This indicates that the signal peptide of Bla (β-lactamase) can direct secretion of fusion polypeptides consisting of the complete mature Bla sequence and an adventitious sequence fused at its C-terminus. Furthermore, the BlaHG3f monomers form correctly assembled antibody-like homodimers with two Bla moieties instead of two Fab-arms. The ampicillin resistance gene lacks its own promoter. Nevertheless, the transformants grow on ampicillin selective medium. β-lactamase is also enzymatically active after expression in COS-1 cells. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727461.1. The SacI-NsiI fragment of pOMMB (located between nucleotide 3360 and 4139) was obtained from Genbank 'Release 55; Locus: HUMIGHAF' and differs in 6 nucleotides from the nucleotide sequence of the coding regions of the E6Hy3 SacI-NsiI fragment of pATE6Hy3. The unknown nucleotides in the Genbank-obtained sequence have been sequenced (Dr R. Schoonjans, Department of Biomedical Molecular Biology, Ghent University, Belgium) and correspond to the nucleotides of the genomic E6Hy3 fragment of pATE6Hy3. |
| EMBL Accession number: | J00231, view at EMBL, GenBank, DDBJ LT727461.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 23/01/1997 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: ApaI/HindII, BamHI/BglI, EcoRI/PstI, EcoRV, NaeI and SacI/XmnI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr K. De Sutter(1). (1) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | De Sutter et al., Mol. Immunol. 31 (1994), 261-267 [PMID: 8139581] |
| Restricted distribution: | - BCCM MTA |
| 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 + chloramphenicol (25 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pSV71BlaHG3f (LMBP 3306) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. De Sutter and was published in De Sutter et al., 1994. |
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.