Last data update: 23 October 2020 04:27 CEST
Plasmid name: pCAhIFNGSTmE (LMBP 4168)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Human interferon γ gene (IFNG); signal sequence
Human α2,6-sialyltransferase gene (ST6GalI); mutated mature sequence
Phage fd gene 3 (g3); fragment
|Promoter:||Chicken β-actin/rabbit β-globin hybrid promoter (AG)
Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only
Escherichia coli lac operon promoter
|Terminator:||Rabbit β-globin polyadenylation signal (β-globin polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
|Host range:||Escherichia coli
Mammalian cells; SV40 permissive cells
|Parental clone:||pCAhIFNGSTm; pMa58; pCANTAB5E-PLAP|
|Further information:||The plasmid was constructed as follows: 1) pMa58 was BamHI opened and the BglII fragment from pCAhIFNGSTm, containing part of the mature human α2,6-sialyltransferase gene (hST6GalI), was inserted, resulting in the intermediate construct pMa58STfr2; 2) The termination codon at the 3' end of the hST6GalI coding sequence was deleted and a unique FspI site was introduced via site-specific mutagenesis using the synthetic oligonucleotide 5'-GGAGCCTGTGCTGCGCAGTGAATGG-3', leading to pMa58STfr2deltastop; 3) To ligate an E-tag at the 3' end of the hST6GalI gene, the pCANTAB5E-PLAP vector was NotI (treated with S1 nuclease)/AflIII opened and part of the coding sequence of the hST6GalI gene was inserted as a NcoI/FspI fragment; 4) The EcoRI/AccI (filled in with Klenow DNA polymerase) fragment from the resulting pCANTAB-STE vector, containing part of the coding sequence of hST6GalI ligated to the E-tag and the phage fd gene 3 (g3), was inserted into the SmaI opened pMa58. The orientation of the inserted fragment in this pMa58ST intermediate vector is sense relative to the ampicillin resistance gene; 5) At the 3' end of the hST6GalI gene, a unique MfeI site was introduced by site-specific mutagenesis using the synthetic oligonucleotide 5'-CCATTCACTGCTGCCAATTGGGTGCGCCGGTGCCG-3', resulting in pMa58STb; 6) Finally, the mature hST6GalI gene was restored by inserting the KpnI/BstBI (AsuII, Csp45I) fragment from pMa58STb into the KpnI/BstBI (AsuII, Csp45I) opened pCAhIFNGSTm vector, leading to pCAhIFNGSTmE.
pCAhIFNGSTmE is useful for highly efficient expression of human α2,6-sialyltransferase, C-terminally fused to an E-tag, under the control of the chicken β-actin/rabbit β-globin hybrid promoter (AG) and the human CMV-IE enhancer in various mammalian cells.
The AG promoter sequence consists of the chicken β-actin promoter, the first exon and part of the first intron (that seems to have a strong enhancer-like activity) linked to a rabbit β-globin fragment, consisting of a 3' part of the second intron (inclusive a branch point which is required for normal splicing reactions) and a 5' part of the third exon.
When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
The nucleotide sequence of hST6GalI corresponds with the Genbank accession number X62822.1.
Other name of the plasmid is pCAIFNSTE.
|EMBL Accession number:||X62822.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||25/08/2000|
Nucleotide sequence at the fusion between the hIFNG signal sequence and the mutated mature hST6GalI gene: -->hIFNG signal seq. hST6GalI--> -------------------- ----------------- Leu Gly Cys Tyr Cys Gln Asp Pro Ala Ala Ala Met Gly 5' … CTT.GGC.TGT.TAC.TGC.CAG.GAC.CCA.GCG.GCC.GCC.ATG.GGG … 3' ^^ *** NotI ^: Mutated nucleotide. *: Nucleotide inserted. Punctuation indicates reading frame. Nucleotide sequence at the end of the hST6GalI gene: 5'… ACC.ATT.CAC.TGC.TGC.CAA.TTG|GGT.GCG.CCG.GTG … 3' MfeI hST6GalI-->|-->E-tag Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: EcoRI, MfeI/XbaI and NotI.|
|History of deposit:||This plasmid was deposited by Dr W. Laroy(1) (2) and Prof. Dr R. Contreras(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Plasmid reference:||Laroy et al., Glycobiology 11 (2001), 175-182 [PMID: 11320056]
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 MC1061|
|Host reference:||Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
|Related host reference:||Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pCAhIFNGSTmE (LMBP 4168) is available at BCCM/GeneCorner. This plasmid was deposited by Dr W. Laroy and Prof. Dr R. Contreras and was published in Laroy et al., 2001.|
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.