Mammalian Expression of Recombinant Enzymes

The coding sequence of FLPo and ΦC31o was commercially synthesized de novo (Geneart GmbH, Regensburg, Germany) based on the published FLPe and ΦC31 coding sequence [8] (link), [11] (link). The coding sequence of endogenous ΦC31 was PCR-amplified from phage lysate (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Braunschweig, Germany). A C-terminal SV40 nuclear localization signal was also added to the endogenous ΦC31 coding sequence as previously described [11] (link). The FLPo, ΦC31o, and ΦC31 coding sequences were blunt cloned into mammalian expression vectors driven by the high expressing phosphoglycerate kinase 1 (PGK) promoter and containing the bovine growth hormone polyadenylation (bpA) sequence.

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Raymond C.S, & Soriano P. (2007). High-Efficiency FLP and ΦC31 Site-Specific Recombination in Mammalian Cells. PLoS ONE, 2(1), e162.

Publication 2007

Bovine growth hormone Coding sequence Coding sequences Flpe Mammalian Nuclear localization signal Phage Phosphoglycerate kinase Polyadenylation Sv40 Vectors

Corresponding Organization :

Other organizations : Fred Hutch Cancer Center

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Protocol cited in 54 other protocols

Variable analysis

independent variables

FLPo coding sequence
ΦC31o coding sequence
Endogenous ΦC31 coding sequence

dependent variables

Not explicitly mentioned

control variables

Addition of C-terminal SV40 nuclear localization signal to the endogenous ΦC31 coding sequence
Cloning of FLPo, ΦC31o, and ΦC31 coding sequences into mammalian expression vectors driven by the PGK promoter and containing the bpA sequence

controls

Positive control: Published FLPe and ΦC31 coding sequences used as reference for commercial synthesis of FLPo and ΦC31o
Negative control: Not explicitly mentioned

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