URL: http://www.addgene.org/66618

Proper Citation: RRID:Addgene_66618

Insert Name: TPST2

Organism: Homo sapiens

Bacterial Resistance: Kanamycin

Defining Citation: PMID:18522538

Vector Backbone Description: Backbone Marker:Clontech; Vector Backbone:pEGFP-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin

Comments: Molecular cloning of TPST (tyrosylprotein sulfotransferase)–EGFP (enhanced green fluorescent protein) The TPST1 and TPST2 sequences were obtained from human ESTs (expressed sequence tags) from the RZPD (Deutsches Ressourcenzentrum für Genomforschung, Berlin, Germany; GenBank® accession numbers NM_003596 and AF061254 respectively). TPST1–EGFP and TPST2–EGFP were obtained by PCR using the following primers: plus strand, 5′-GGGGCTCGAGATGGTTGGAAAGCTGAAG-3′, and minus strand, 5′-GGGGCTGCAGCTCCACTTGCTCAGTC-3′ for TPST1; and plus strand, 5′-GGGGCTCGAGATGCGCCTGTCGGTGC-3′, and minus strand, 5′-GGGGCTGCAGCGAGCTTCCTAAGTGG-3′ for TPST2 (XhoI and PstI, restriction sites are underlined). PCR products were purified and ligated into the pGEM-T-Easy vector (Promega) following the manufacturer's instructions. Positive clones were digested with XhoI and PstI, and the fragments obtained were inserted in the corresponding sites of pEGFP-N1 (Clontech). All constructs were verified by double-strand DNA sequencing (MWG Biotech, Ebersburg, Germany). Two differences to the published sequence of TPST2 were identified (276C>T and 1002T>C, relative to the start codon), but neither of which affect the predicted protein sequence. Insert contains T370A relative to reference sequence. The depositing laboratory does not know the effect of this substitution on gene function

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