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. 2017 Aug 10;170(4):800–814.e18. doi: 10.1016/j.cell.2017.07.031

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© 2017 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Figure S7 — List of All DNA Constructs Used to Produce the Different Second-Generation ifgMosaic Constructs, Related to Figures 5–7

(A and B) LoxP- (A) or FRT-containing (B) entry vectors used to clone the desired genes in frame with the upstream FPs and the 2A peptide.

(C and D) Map of LoxP- (C) or FRT-containing (D) donor vector used to clone the 3 cassettes from (A) or (B) in the HindIII/NotI sites.

(E and F) The Triple ORF donor vectors of C and D can be digested with SgraI/PacI or SgraI/ScaI to insert the mosaic by Cas9 recombineering in pre-modified ES cells (E) or by ligation to Rosa26 gene targeting vectors (F). Plasmids LH500 or LH416 are required to express and guide the Cas9 to the pre-modified (E) or wildtype (F) Rosa26 locus.

(G) The large SgraI/PacI fragments generated in C and D can be inserted by recombineering in different acceptor Rosa26 BACs containing the following promoters: CAG, Tre-Tight, or UAS 4x NR (4 UAS elements non-repeated). BAC G256 can be used for transgenesis in zebrafish and contains a marker to directly select transgenic founders (Cmcl2-turquoise) based on turquoise fluorescence in the heart.

(H) Smaller vectors that can be used to directly clone the mosaic constructs downstream of the Tre-Tight or UAS promoters for titratable and reversible induction. These vectors can also be digested with the rare cutters SgraI/PacI and cloned in a plasmid (AG103) containing the Rosa26 homology arms for gene targeting.

LOXP1, LoxN; LOXP2, Lox2272; LOXP3, LoxP; FRT1, F3; FRT2, 5T2; FRT3, 545; 2A, viral peptide allowing equimolar expression of multiple independent proteins from a single ORF; Mb2, second generation membrane tag; HA, V5 and His (small epitopes that can be used for specific antibody detection); H2B, histone tag that targets proteins to the chromatin/nucleus; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element that enhances gene expression; Sv40pA, polyadenylation signal to stop transcription; N-PhiM, non-fluorescent protein that is used as a reporter of promoter expression; CAG, Strong and ubiquitous promoter; PGK-Neo, resistance marker for ES cell selection; INS-INS, Double Chicken B-globin insulator to increase gene expression and minimize regulatory interference.