Last data update: 24 January 2024 16:39 CET
Plasmid name: pSV71BlaMG2fmRK (LMBP 3604)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p3604.gb
(View with Genome Compiler) p3604.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Ampicillin resistance gene (amp) Mouse anti-hPLAP monoclonal antibody E6(IgG2b,κ); hinge region and CH2 and mutated CH3 domains of the heavy chain (MG2f) |
| 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: | pSV71BlaHG3f; pSV51E6HmRK; pMac254mSA |
| Further information: | The plasmid was constructed by ligating the following three fragments: (i) the EcoRI-ApaI fragment from pSV51E6HmRK, containing the SV40 small and large T-antigen and the hinge, CH2 and mutated CH3 domains of the mouse anti-hPLAP monoclonal antibody E6(IgG2b,κ) heavy chain (E6H), (ii) the ApaI-AhaII (AcyI) fragment of the mutated ampicillin resistance gene from pMac254mSA, (iii) the AhaII (AcyI)-EcoRI fragment from pSV71BlaHG3f, containing the completing 5' coding region of the ampicillin resistance gene, the SV40 late promoter, part of the SV40 large T-antigen, the origin of replication and the chloramphenicol resistance gene. As a result, the mutated ampicillin resistance gene in which the termination codon has been replaced by GAG, is fused in frame to the start of the hinge region of the mutated MG2f sequence. The complete fusion gene is placed under control of the SV40 late promoter. The hinge region comprises 22 amino acids. The Leu at position 2 is replaced by Pro. As compared to the wt E6H cDNA, the codons at position 406 (Ile) and 407 (Tyr) were replaced, respectively by a Val and an Arg codon. Furthermore, a unique SnaBI site as well as an extra RsaI site were introduced. Since the amino acids at positions 407 and 409 are positively charged, the expressed heavy chain will preferentially associate with a negatively charged heavy chain (e.g. pSV51E6HmDE) and form heterodimers. In combination with pSV51E6HmDE and pSV51E6L1, transient expression of the mutated BlaMG2f fusion gene in COS-1 cells resulted in the synthesis and secretion of bifunctional anti-hPLAP/Bla-immunoconjugates. 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. 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 LT727361.1. The hinge fusion region, as well as the amino acid substitutions and the introduction of the RsaI site have been verified by DNA sequence analysis at the Department of Biomedical Molecular Biology (Ghent University, Belgium). Other name of the plasmid is pSV71BlaMG2fm407R409K. |
| EMBL Accession number: | LT727361.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 02/08/2000 |
| Sequence detail: | Nucleotide sequence at the hinge fusion:
CH1 ->|-> hinge
wt E6H 5' … CTT|GAG.CCC.AGC.GGG.CCC.ACT.TCA.ACA.ATC. … 3'
Leu|Glu Pro Ser Gly Pro Thr Ser Thr Ile
ApaI
Bla ->|-> hinge
mutated E6H 5' … TGG|GAG.CTC.AGC.GGG.CCC.ACT.TCA.ACA.ATC. … 3'
Trp|Glu Leu Ser Gly Pro Thr Ser Thr Ile
SacI ApaI
Nucleotide sequence of the mutated CH3 cDNA:
406 407 409
wt E6H 5'…TCT.TAC.TTC.ATA.TAT.AGC.AAG.CTC.…3'
Ser Tyr Phe Ile Tyr Ser Lys Leu
mutated E6H 5'…TCT.TAC.TTC.GTA.CGT.AGC.AAG.CTC.…3'
Ser Tyr Phe Val Arg Ser Lys Leu
RsaI
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: ApaI/SnaBI, BamHI/BglI, EcoRI/PstI, EcoRV and SacI/XmnI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr K. De Sutter(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | - |
| Related 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 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pSV71BlaMG2fmRK (LMBP 3604) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. De Sutter . |
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.