Table 1.

Plasmids and primers used in this study

Expression in Dictyostelium cells
UsagePlasmid backboneSequence, 5′-3′
GxcM-GFP or GxcM-RFP pDM323 or pDM451 F: 5′-GGA​AAG​TCG​ACA​AAG​CTA​GCA​GTA​AAA​TAA​AAA​TGA​AAA​TTA​AAT​TTT​TTC​GAT​ATG-3′ 
  R: 3′-GTT​CTT​CTC​CTT​TAC​CAC​TAC​TAG​ATT​TTC​TTG​GTG​GAA​TGA​TTG​G-5′ 
GxcMN790-GFP pDM323 F: 5′-GGA​GCT​CAA​ATA​AAA​ATG​AAA​ATT​AAA​TTT​TTT​CGA​TAT​G-3′ 
  R: 3′-GGA​CTA​GTA​CTT​CTT​TTA​ATA​TGA​TCT​AAA​AC-5′ 
GxcMN790-RFP pDM451 F: 5′-CCG​GAG​CTC​AAA​TAA​AAA​TGA​AAA​TTA​AAT​TTT​TTC​GAT​ATG-3′ 
  R: 3′-CTA​GCT​AGC​ACT​TCT​TTT​AAT​ATG​ATC​TAA​AAC​TTT​ATC-5′ 
GxcM4A-GFP or GxcM4A-RFP pDM323 or pDM451 F1: 5′-GAG​AGT​TTT​CTA​ATT​AAA​GCA​GCA​GCA​GCA​CTT​TGT​AAA​TAT​CCA​TTA​C-3′ 
  R1: 3′-GTA​ATG​GAT​ATT​TAC​AAA​GTG​CTG​CTG​CTG​CTT​TAA​TTA​GAA​AAC​TCT​C-5′ 
  F: 5′-CCG​GAG​CTC​AAA​TAA​AAA​TGA​AAA​TTA​AAT​TTT​TTC​GAT​ATG-3′ 
  R: 3′-CTA​GCT​AGC​TTT​TCT​TGG​TGG​AAT​GAT​TGG​TTT​TTT​AG-5′ 
GxcMN455-GFP pDM323 F: 5′-GGA​GCT​CAA​ATA​AAA​ATG​AAA​ATT​AAA​TTT​TTT​CGA​TAT​G-3′ 
  R: 3′-CGG​ACT​AGT​CAT​TTT​ATT​TTC​ACC​TTC​TTC​TTT​CAT​TAC-5′ 
GxcMN456-770-GFP pDM323 F: 5′-GGG​GAG​CTC​ATG​AGA​AAC​ATG​GTA​ATT​AAT​G-3′ 
  R: 3′-CGG​ACT​AGT​TTT​AAT​TAA​TTC​ATC​CAA​ATC​AG-5′ 
GxcM pDM358 F: 5′-CCG​GAG​CTC​AAA​TAA​AAA​TGA​AAA​TTA​AAT​TTT​TTC​GAT​ATG-3′ 
  R: 3′-CTA​GCT​AGC​TTT​TCT​TGG​TGG​AAT​GAT​TGG​TTT​TTT​AG-5′ 
GFP-Fbp17 or RFP-Fbp17 pDM317 or pDM449 F: 5′-CCG​GAG​CTC​ATG​AGT​TAT​TCA​ACC​GAT​TTA​TTA​GAT​GG-3′ 
  R: 3′-CTA​GCT​AGC​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GGC​CGG​AAT​G-5′ 
GFP-Fbp17ΔSH3 or RFP-Fbp17ΔSH3 pDM317 or pDM449 F: 5′-CCG​GAG​CTC​ATG​AGT​TAT​TCA​ACC​GAT​TTA​TTA​GAT​GG-3′ 
  R: 3′-CTA​GCT​AGC​CAA​TTC​CTC​CAA​TTG​AGC​AGT​GAT​AAG-5′ 
GFP-Fbp17SH3 or RFP-Fbp17SH3 pDM317 or pDM449 F: 5′-CCG​GAG​CTC​GGT​AAA​ACT​AAT​CTT​TCT​GGT​TCA​AG-3′ 
  R: 3′-CTA​GCT​AGC​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GG-5′ 
GFP-Fbp17F-BAR or RFP-Fbp17F-BAR pDM317 or pDM449 F: 5′-CCG​GAG​CTC​GGT​AAA​ACT​AAT​CTT​TCT​GGT​TCA​AG-3′ 
  R: 3′-CTA​GCT​AGC​GGT​GCC​AAC​TCT​AAA​GTT​TGT​TGG​AGA​G-5′ 
GFP-Fbp17ΔBAR or RFP-Fbp17ΔBAR pDM317 or pDM449 F: 5′-CCG​GAG​CTC​CAG​ATC​GAC​AAG​GAC​GCT​GAT​ATC​CGT​C-3′ 
  R: 3′-CTA​GCT​AGC​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GGC​CG-5′ 
GFP-WASP pDM317 F: 5′-CTA​TCT​AGA​ATG​GGT​AGT​CCA​ACT​ATT​AGT​GAT​C-3′ 
  R: 3′-CTA​GTC​GAC​ATA​ATC​TGA​CCA​TTC​ATC​ATC​GTC-5′ 
GFP-ArpC4 pDM317 F: 5′-GGG​GTA​CCA​TGT​CCA​CAG​CTC​AAG​TTC​CAT​ATT​TAA​ATT​G-3′ 
  R: 3′-CGA​GCG​GCC​GCA​GCA​AAG​TTC​TTT​AAG​TAT​TCA​GAA​GCA​AC-5′ 
RFP-ArpC4 pDM449 F: 5′-CGG​GAT​CCA​TGT​CCA​CAG​CTC​AAG​TTC​CAT​ATT​TAA​ATT​G-3′ 
  R: 3′-GCT​CTA​GAA​GCA​AAG​TTC​TTT​AAG​TAT​TCA​GAA​GCA​AC-5′ 
GFP-Coronin A pDM317 F: 5′-GGT​CTA​GAA​TGT​CTA​AAG​TAG​TCC​GTA​GTA​G-3′ 
  R: 3′-CCG​GCG​GCC​GCG​TTG​GTG​AGT​TCT​TTG​ATT​TTG​GC-5′ 
GFP-RacC pDM317 F: 5′-CCG​GAG​CTC​ATG​TCA​GCA​GCA​GAA​GTT​ATT​AAA​TTA​G-3′ 
  R: 3′-CTA​GCT​AGC​TTA​CAT​AAC​AAT​ACA​CTT​GGA​TTT​CTT​TTT​TTT​G-5′ 
GFP-RacCG15V pDM317 F: 5′-GTT​GCT​GTA​GGT​AAA​ACT​TGT​TTA​TTG​ATT​AG-3′ 
  R: 3′-ATC​ACC​AAT​AAC​GAC​TAA​TTT​AAT​AAC​TTC-5′ 
tet on GFP-RacCG15V pDM371 F: 5′-CGC​GGA​TCC​ATG​TCA​GCA​GCA​GAA​GTT​ATT​AAA​TTA​G-3′ 
  R: 3′-CGG​ACT​AGT​TTA​CAT​AAC​AAT​ACA​CTT​GGA​TTT​CTT​TTT​TTT​G-5′ 
tet on GFP-RacCQ64L pDM371 F: 5′-CTT​TGG​GAT​ACT​GCA​GGT​TTA​GAA​GAG​TAC​GAT​AAA​TTA​AG-3′ 
  R: 3′-CTT​AAT​TTA​TCG​TAC​TCT​TCT​AAA​CCT​GCA​GTA​TCC​CAA​AG-5′ 
tet on RFP-RacCG15V pDM359 F: 5′-CGC​GGA​TCC​ATG​GCA​TCA​TCA​GAA​GAT​GTT​ATT​AAA​GAA​TTT​ATG-3′ 
  R: 3′-CGG​ACT​AGT​TTA​CAT​AAC​AAT​ACA​CTT​GGA​TTT​CTT​TTT​TTT​G-5′ 
GFP-Rac1A pDM317 F: 5′-CCG​GAG​CTC​ATG​CAA​GCA​ATT​AAA​TGT​GTC​GTT​GTC​GGT-3′ 
  R: 3′-CTA​GCT​AGC​TAA​AAT​GTT​GCA​ACC​ACC​TGA​ACT​TTT​CTT​CTT​TTT​GC-5′ 
GFP-ABD pCV5 F: 5′-CCG​GAG​CTC​ATG​GCT​GCT​GCT​CCA​AGT​GGA​AAA​AC-3′ 
  R: 3′-CAG​GCT​CGA​GGG​CAT​CTG​AAG​TTT​CAC​GAC​GTT​TCT​TTT​C-5′ 
GFP-Myosin II pCV5 F: 5′-CCG​GAG​CTC​AAT​CCA​ATT​CAT​GAT​AGA​ACT​TCA​GAT​TAT​C-3′ 
  R: 3′-CAG​GCT​CGA​GTT​AAG​CTT​TGA​AAC​CAC​CAA​AGA​AAT​CGG​C-5′ 
GFP-CLC pDM317 F: 5′-CAG​GAG​CTC​ATG​TCA​GAT​CCA​TTT​GGT​GAA​G-3′ 
  R: 3′-CTA​GCT​AGC​AAC​AAT​TGG​TTG​ATT​TTT​TAA​ACG-5′ 
Generation of knockout cells 
gxcM knockout pBluescript-BSR Insert 1 F: 5′-CAC​GGT​ACC​GAT​ACC​ATC​ATC​GAT​GAT​ATC​AC-3′ 
  Insert 1 R: 3′-GCC​GGA​ATT​CGT​GGT​GGT​ATT​ATT​TGA​TGA​TG-5′ 
  Insert 2 F: 5′-CGG​ACT​AGT​GTA​AAG​TTA​CAA​CAC​CAA​TTT​CTA​CAC-3′ 
  Insert 2 R: 3′-CGG​CGG​CCG​CTT​ATT​TTC​TTG​GTG​GAA​TGA​TTG​G-5′ 
fbp17 knockout pBluescript-BSR Insert 1 F: 5′-CAC​GGT​ACC​CGT​GTA​TTT​CTA​AAC​TAT​TTT​ATA​C-3′ 
  Insert 1 R: 3′-GAG​AAG​CTT​CAT​CTA​ATA​AAT​CGG​TTG​AAT​AAC​TCA​T-5′ 
  Insert 2 F: 5′-CGG​ACT​AGT​CCA​ATT​TTG​TTG​ACA​TCA​TCG​ATG-3′ 
  Insert 2 R: 3′-CGG​CGG​CCG​CGA​TTA​AGG​TTG​GTA​TTT​TTG​AAA​G-5′ 
racC knockout pBluescript-BSR Insert 1 F: 5′-GAC​GTC​GAC​CCC​CCC​TAA​CAT​TAA​AAG​TAT​TAA​TAA​TAG-3′ 
  Insert 1 R: 3′-GAG​AAG​CTT​CCC​TAC​AGC​ACC​ATC​ACC​AAT​AAC​G-5′ 
  Insert 2 F: 5′-CGG​ACT​AGT​GGA​ATG​TTC​TGC​CAA​AAC​CTC​ACA​AAA​TCT​C-3′ 
  Insert 2 R: 3′-CGG​CGG​CCG​CGG​TGG​CTT​CCT​AAT​TTG​CCT​TTT​CTC​AAA​G-5′ 
wasA knockout pBluescript-BSR Insert 1 F: 5′-GAC​GTC​GAC​GGT​ATT​ATA​TAT​TGT​TCA​TTT​GCA​TTG​GTT​T-3′ 
  Insert 1 R: 3′-GAG​AAG​CTT​CAT​ACA​ATC​TTG​CAA​CAG​TAG​TTG​AAT​G-5′ 
  Insert 2 F: 5′-CGG​ACT​AGT​CTA​TGG​CTA​ATC​GTA​GAG​GTG​GTA​TGA​GA-3′ 
  Insert 2 R: 3′-CGG​CGG​CCG​CCT​GGT​AAT​GTT​TTA​TGA​CTT​GAT​GTA​AAT​G-5′ 
Expression in bacteria or insect cells 
GST-Fbp17 pFastBac1-GST F: 5′-GAG​CAT​ATG​ATG​AGT​TAT​TCA​ACC​GAT​TTA​TTA​GAT​GGA​TTT​GAA​AGA​TTA​TAT​AAA​AGA​ACt​TAT​GGT​GTT​TTA​AAA​TGT​AAT​C-3′ 
  R: 3′-TGC​TCT​AGA​TTA​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GGC-5′ 
GST-WASP pFastBac1-GST F: 5′-GAG​CAT​ATG​ATG​GGT​AGT​CCA​ACT​ATT​AGT​GAT​CAA​G-3′ 
  R: 3′-TGC​TCT​AGA​ATA​ATC​TGA​CCA​TTC​ATC​ATC​GTC​ATC​ATC​ATC-5′ 
GST-WASPVCA pGEX-6P-1 F: 5′-CGC​GGA​TCC​CCA​TCA​TCA​GGA​GGT​GGT​GGT​GCC-3′ 
  R: 3′-TGC​TCT​AGA​TTA​ATA​ATC​TGA​CCA​TTC​ATC​ATC​GTC​ATC-5′ 
GST-Fbp17SH3 pGEX-6P-1 F: 5′-CGC​GGA​TCC​GCT​GGT​GCA​ACT​GGT​GGT​GCC​ACT​AGA​G-3′ 
  R: 3′-CAG​GCT​CGA​GAC​CAT​CGA​TGA​TGT​CAA​CAA​AAT​TGG​C-5′ 
His-MBP-Fbp17SH3 pET-MBP-3C F: 5′-CGC​GGA​TCC​GCT​GGT​GCA​ACT​GGT​GGT​GCC​ACT​AGA​G-3′ 
  R: 3′-CAG​GCT​CGA​GAC​CAT​CGA​TGA​TGT​CAA​CAA​AAT​TGG​C-5′ 
Expression in yeast cells 
Fbp17 pGADT7 F: 5′-GAG​CAT​ATG​ATG​AGT​TAT​TCA​ACC​GAT​TTA​TTA​GAT​GG-3′ 
  R: 3′-CGC​GGA​TCC​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GGC​CGG-5′ 
WASPN126-230 pGADT7 F: 5′-GAG​CAT​ATG​ATT​GGT​AAA​ACT​GCT​GCA​ATT​AAT​CCA​AAT​AC-3′ 
  R: 3′-GG AGA​TCT​ACC​AAT​GAT​ATT​GAC​AAT​GAA​TTG​AGC​AG-5′ 
Rac1AG12V pGBKT7 F: 5′-GTT​GCT​GTA​GGT​AAA​ACC​TGT​CTT​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​GAC​AAC​GAC​ACA​TTT​AAT​TGC​TTG-5′ 
Rac1BG12V pGBKT7 F: 5′-GTT​GCA​GTT​GGT​AAA​ACA​TGT​CTT​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​AAC​AAC​TAC​ACA​TTT​AAT​TGC-5′ 
Rac1CG12V pGBKT7 F: 5′-GTT​GCG​GTT​GGT​AAA​ACA​TGT​CTT​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​TAC​AAC​TAC​ACA​TTT​AAT​TGC-5′ 
RacAG12V pGBKT7 F: 5′-GTTGCTGTAGGTAAAAGTTGTTTATTAATTGC-3′
R: 3′-ATCACCTACTACTACTAATTTAATTGC-5′ 
RacBG12V pGBKT7 F: 5′-GTT​GCT​GTT​GGT​AAA​ACT​TGT​TTA​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​TAC​TAC​CAC​CAA​TTT​AAT​TGA​TTG-5′ 
RacCG15V pGBKT7 F: 5′-GTT​GCT​GTA​GGT​AAA​ACT​TGT​TTA​TTG​ATT​AG-3′ 
  R: 3′-ATC​ACC​AAT​AAC​GAC​TAA​TTT​AAT​AAC​TTC-5′ 
RacDG17V pGBKT7 F: 5′-GTT​GCT​GTT​GGT​AAA​ACT​TCA​CTT​TTA​ATT​TTA​TAT​AC-3′ 
  R: 3′-ATC​ACC​TAC​AAC​TAC​TAC​TTT​AAC​AG-5′ 
RacEG20V pGBKT7 F: 5′-GTT​GCT​GTT​GGT​AAA​ACA​TGT​CTT​TTA​ATT​TG-3′ 
  R: 3′-ATC​ACC​GAC​AAC​TAC​TAA​TTT​AAC​TCT​TG-5′ 
RacF1G12V pGBKT7 F: 5′-GTT​GCA​GTT​GGT​AAA​ACC​TGT​ATG​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​AAC​AAC​AAC​ACA​TTT​AAT​ATT​TTG-5′ 
RacF2G12V pGBKT7 F: 5′-GTT​GCA​GTT​GGT​AAA​ACT​TGT​ATG​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​AAC​AAC​AAC​ACA​TTT​AAT​ATT​TTG-5′ 
RacGG12V pGBKT7 F: 5′-GTT​GGA​ATT​GGT​AAA​ACA​TCA​ATG​TTA​TTA​AG-3′ 
  R: 3′-TTC​GCC​AAC​AAC​ACA​AAC​TTT​AAT​ACT​TTT​C-5′ 
RacHM13V pGBKT7 F: 5′-GTT​AGT​GTA​GGT​AAG​ACA​TGT​TTA​CTC​ATT​TC-3′ 
  R: 3′-ATC​ACC​TAC​AAC​CAT​TAC​TTT​AAT​ATC​TTT​TAC-5′ 
RacIS14V pGBKT7 F: 5′-GTT​AAA​ACT​GGA​AAA​ACT​ACA​ATG​ATG​ATG​AC-3′ 
  R: 3′-ATC​ACC​TAA​TAC​TAA​TAA​CTT​TAT​ATA​TG-5′ 
RacJD18V pGBKT7 F: 5′-GTT​GGA​GTT​GGT​AAA​TCA​TGT​GTA​ATG​AAC​TC-3′ 
  R: 3′-ATC​TCC​AAG​ACA​AAA​TAT​TTT​AAC-5′ 
RacLG12V pGBKT7 F: 5′-GTT​GCA​GTT​GGT​AAA​ACA​AGT​TTA​TTA​ATT​GC-3′ 
  R: 3′-ATC​ACC​ACA​AAT​TAC​CAT​TTT​TAT​ATA​TTG​C-5′ 
RacMY14V pGBKT7 F: 5′-GTT​GGT​GTT​GGT​AAA​ACC​ACC​CTA​TTA​ATG​AC-3′ 
  R: 3′-ATC​GCC​AAT​CGT​AAC​TAT​TTT​AAT​AG-5′ 
RacNG14V pGBKT7 F: 5′-TTA​GAG​GAT​TAT​CCT​CGT​ATA​AGA​CCA​TTA​TC-3′ 
  R: 3′-ACC​ACC​AGT​ATC​CCA​ACA​ACC​AAT​TCT​C-5′ 
RacOG13V pGBKT7 F: 5′-GTT​TTA​ATT​GGT​AAA​ACT​ACA​TTA​TTA​ATG​AC-3′ 
  R: 3′-ATC​ACC​AAC​TGT​CAC​TAT​CTT​TAC​TTC-5′ 
RacPG58V pGBKT7 F: 5′-GTT​TAC​TGT​TGG​AAA​AAC​AAC​ACT​CTT​ATT​ATC-3′ 
  R: 3′-ATC​ACC​TAC​AAC​ACC​ACA​TTT​AAT​AAC​CTT​TG-5′ 
Other plasmids for expression in Dictyostelium cells 
PHcrac-GFP Peter Devreotes Laboratory, Johns Hopkins University 
LimEΔcoil-GFP Peter Devreotes Laboratory, Johns Hopkins University 
LimEΔcoil-RFP Douglas Robinson Laboratory, Johns Hopkins University 
Expression in Dictyostelium cells
UsagePlasmid backboneSequence, 5′-3′
GxcM-GFP or GxcM-RFP pDM323 or pDM451 F: 5′-GGA​AAG​TCG​ACA​AAG​CTA​GCA​GTA​AAA​TAA​AAA​TGA​AAA​TTA​AAT​TTT​TTC​GAT​ATG-3′ 
  R: 3′-GTT​CTT​CTC​CTT​TAC​CAC​TAC​TAG​ATT​TTC​TTG​GTG​GAA​TGA​TTG​G-5′ 
GxcMN790-GFP pDM323 F: 5′-GGA​GCT​CAA​ATA​AAA​ATG​AAA​ATT​AAA​TTT​TTT​CGA​TAT​G-3′ 
  R: 3′-GGA​CTA​GTA​CTT​CTT​TTA​ATA​TGA​TCT​AAA​AC-5′ 
GxcMN790-RFP pDM451 F: 5′-CCG​GAG​CTC​AAA​TAA​AAA​TGA​AAA​TTA​AAT​TTT​TTC​GAT​ATG-3′ 
  R: 3′-CTA​GCT​AGC​ACT​TCT​TTT​AAT​ATG​ATC​TAA​AAC​TTT​ATC-5′ 
GxcM4A-GFP or GxcM4A-RFP pDM323 or pDM451 F1: 5′-GAG​AGT​TTT​CTA​ATT​AAA​GCA​GCA​GCA​GCA​CTT​TGT​AAA​TAT​CCA​TTA​C-3′ 
  R1: 3′-GTA​ATG​GAT​ATT​TAC​AAA​GTG​CTG​CTG​CTG​CTT​TAA​TTA​GAA​AAC​TCT​C-5′ 
  F: 5′-CCG​GAG​CTC​AAA​TAA​AAA​TGA​AAA​TTA​AAT​TTT​TTC​GAT​ATG-3′ 
  R: 3′-CTA​GCT​AGC​TTT​TCT​TGG​TGG​AAT​GAT​TGG​TTT​TTT​AG-5′ 
GxcMN455-GFP pDM323 F: 5′-GGA​GCT​CAA​ATA​AAA​ATG​AAA​ATT​AAA​TTT​TTT​CGA​TAT​G-3′ 
  R: 3′-CGG​ACT​AGT​CAT​TTT​ATT​TTC​ACC​TTC​TTC​TTT​CAT​TAC-5′ 
GxcMN456-770-GFP pDM323 F: 5′-GGG​GAG​CTC​ATG​AGA​AAC​ATG​GTA​ATT​AAT​G-3′ 
  R: 3′-CGG​ACT​AGT​TTT​AAT​TAA​TTC​ATC​CAA​ATC​AG-5′ 
GxcM pDM358 F: 5′-CCG​GAG​CTC​AAA​TAA​AAA​TGA​AAA​TTA​AAT​TTT​TTC​GAT​ATG-3′ 
  R: 3′-CTA​GCT​AGC​TTT​TCT​TGG​TGG​AAT​GAT​TGG​TTT​TTT​AG-5′ 
GFP-Fbp17 or RFP-Fbp17 pDM317 or pDM449 F: 5′-CCG​GAG​CTC​ATG​AGT​TAT​TCA​ACC​GAT​TTA​TTA​GAT​GG-3′ 
  R: 3′-CTA​GCT​AGC​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GGC​CGG​AAT​G-5′ 
GFP-Fbp17ΔSH3 or RFP-Fbp17ΔSH3 pDM317 or pDM449 F: 5′-CCG​GAG​CTC​ATG​AGT​TAT​TCA​ACC​GAT​TTA​TTA​GAT​GG-3′ 
  R: 3′-CTA​GCT​AGC​CAA​TTC​CTC​CAA​TTG​AGC​AGT​GAT​AAG-5′ 
GFP-Fbp17SH3 or RFP-Fbp17SH3 pDM317 or pDM449 F: 5′-CCG​GAG​CTC​GGT​AAA​ACT​AAT​CTT​TCT​GGT​TCA​AG-3′ 
  R: 3′-CTA​GCT​AGC​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GG-5′ 
GFP-Fbp17F-BAR or RFP-Fbp17F-BAR pDM317 or pDM449 F: 5′-CCG​GAG​CTC​GGT​AAA​ACT​AAT​CTT​TCT​GGT​TCA​AG-3′ 
  R: 3′-CTA​GCT​AGC​GGT​GCC​AAC​TCT​AAA​GTT​TGT​TGG​AGA​G-5′ 
GFP-Fbp17ΔBAR or RFP-Fbp17ΔBAR pDM317 or pDM449 F: 5′-CCG​GAG​CTC​CAG​ATC​GAC​AAG​GAC​GCT​GAT​ATC​CGT​C-3′ 
  R: 3′-CTA​GCT​AGC​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GGC​CG-5′ 
GFP-WASP pDM317 F: 5′-CTA​TCT​AGA​ATG​GGT​AGT​CCA​ACT​ATT​AGT​GAT​C-3′ 
  R: 3′-CTA​GTC​GAC​ATA​ATC​TGA​CCA​TTC​ATC​ATC​GTC-5′ 
GFP-ArpC4 pDM317 F: 5′-GGG​GTA​CCA​TGT​CCA​CAG​CTC​AAG​TTC​CAT​ATT​TAA​ATT​G-3′ 
  R: 3′-CGA​GCG​GCC​GCA​GCA​AAG​TTC​TTT​AAG​TAT​TCA​GAA​GCA​AC-5′ 
RFP-ArpC4 pDM449 F: 5′-CGG​GAT​CCA​TGT​CCA​CAG​CTC​AAG​TTC​CAT​ATT​TAA​ATT​G-3′ 
  R: 3′-GCT​CTA​GAA​GCA​AAG​TTC​TTT​AAG​TAT​TCA​GAA​GCA​AC-5′ 
GFP-Coronin A pDM317 F: 5′-GGT​CTA​GAA​TGT​CTA​AAG​TAG​TCC​GTA​GTA​G-3′ 
  R: 3′-CCG​GCG​GCC​GCG​TTG​GTG​AGT​TCT​TTG​ATT​TTG​GC-5′ 
GFP-RacC pDM317 F: 5′-CCG​GAG​CTC​ATG​TCA​GCA​GCA​GAA​GTT​ATT​AAA​TTA​G-3′ 
  R: 3′-CTA​GCT​AGC​TTA​CAT​AAC​AAT​ACA​CTT​GGA​TTT​CTT​TTT​TTT​G-5′ 
GFP-RacCG15V pDM317 F: 5′-GTT​GCT​GTA​GGT​AAA​ACT​TGT​TTA​TTG​ATT​AG-3′ 
  R: 3′-ATC​ACC​AAT​AAC​GAC​TAA​TTT​AAT​AAC​TTC-5′ 
tet on GFP-RacCG15V pDM371 F: 5′-CGC​GGA​TCC​ATG​TCA​GCA​GCA​GAA​GTT​ATT​AAA​TTA​G-3′ 
  R: 3′-CGG​ACT​AGT​TTA​CAT​AAC​AAT​ACA​CTT​GGA​TTT​CTT​TTT​TTT​G-5′ 
tet on GFP-RacCQ64L pDM371 F: 5′-CTT​TGG​GAT​ACT​GCA​GGT​TTA​GAA​GAG​TAC​GAT​AAA​TTA​AG-3′ 
  R: 3′-CTT​AAT​TTA​TCG​TAC​TCT​TCT​AAA​CCT​GCA​GTA​TCC​CAA​AG-5′ 
tet on RFP-RacCG15V pDM359 F: 5′-CGC​GGA​TCC​ATG​GCA​TCA​TCA​GAA​GAT​GTT​ATT​AAA​GAA​TTT​ATG-3′ 
  R: 3′-CGG​ACT​AGT​TTA​CAT​AAC​AAT​ACA​CTT​GGA​TTT​CTT​TTT​TTT​G-5′ 
GFP-Rac1A pDM317 F: 5′-CCG​GAG​CTC​ATG​CAA​GCA​ATT​AAA​TGT​GTC​GTT​GTC​GGT-3′ 
  R: 3′-CTA​GCT​AGC​TAA​AAT​GTT​GCA​ACC​ACC​TGA​ACT​TTT​CTT​CTT​TTT​GC-5′ 
GFP-ABD pCV5 F: 5′-CCG​GAG​CTC​ATG​GCT​GCT​GCT​CCA​AGT​GGA​AAA​AC-3′ 
  R: 3′-CAG​GCT​CGA​GGG​CAT​CTG​AAG​TTT​CAC​GAC​GTT​TCT​TTT​C-5′ 
GFP-Myosin II pCV5 F: 5′-CCG​GAG​CTC​AAT​CCA​ATT​CAT​GAT​AGA​ACT​TCA​GAT​TAT​C-3′ 
  R: 3′-CAG​GCT​CGA​GTT​AAG​CTT​TGA​AAC​CAC​CAA​AGA​AAT​CGG​C-5′ 
GFP-CLC pDM317 F: 5′-CAG​GAG​CTC​ATG​TCA​GAT​CCA​TTT​GGT​GAA​G-3′ 
  R: 3′-CTA​GCT​AGC​AAC​AAT​TGG​TTG​ATT​TTT​TAA​ACG-5′ 
Generation of knockout cells 
gxcM knockout pBluescript-BSR Insert 1 F: 5′-CAC​GGT​ACC​GAT​ACC​ATC​ATC​GAT​GAT​ATC​AC-3′ 
  Insert 1 R: 3′-GCC​GGA​ATT​CGT​GGT​GGT​ATT​ATT​TGA​TGA​TG-5′ 
  Insert 2 F: 5′-CGG​ACT​AGT​GTA​AAG​TTA​CAA​CAC​CAA​TTT​CTA​CAC-3′ 
  Insert 2 R: 3′-CGG​CGG​CCG​CTT​ATT​TTC​TTG​GTG​GAA​TGA​TTG​G-5′ 
fbp17 knockout pBluescript-BSR Insert 1 F: 5′-CAC​GGT​ACC​CGT​GTA​TTT​CTA​AAC​TAT​TTT​ATA​C-3′ 
  Insert 1 R: 3′-GAG​AAG​CTT​CAT​CTA​ATA​AAT​CGG​TTG​AAT​AAC​TCA​T-5′ 
  Insert 2 F: 5′-CGG​ACT​AGT​CCA​ATT​TTG​TTG​ACA​TCA​TCG​ATG-3′ 
  Insert 2 R: 3′-CGG​CGG​CCG​CGA​TTA​AGG​TTG​GTA​TTT​TTG​AAA​G-5′ 
racC knockout pBluescript-BSR Insert 1 F: 5′-GAC​GTC​GAC​CCC​CCC​TAA​CAT​TAA​AAG​TAT​TAA​TAA​TAG-3′ 
  Insert 1 R: 3′-GAG​AAG​CTT​CCC​TAC​AGC​ACC​ATC​ACC​AAT​AAC​G-5′ 
  Insert 2 F: 5′-CGG​ACT​AGT​GGA​ATG​TTC​TGC​CAA​AAC​CTC​ACA​AAA​TCT​C-3′ 
  Insert 2 R: 3′-CGG​CGG​CCG​CGG​TGG​CTT​CCT​AAT​TTG​CCT​TTT​CTC​AAA​G-5′ 
wasA knockout pBluescript-BSR Insert 1 F: 5′-GAC​GTC​GAC​GGT​ATT​ATA​TAT​TGT​TCA​TTT​GCA​TTG​GTT​T-3′ 
  Insert 1 R: 3′-GAG​AAG​CTT​CAT​ACA​ATC​TTG​CAA​CAG​TAG​TTG​AAT​G-5′ 
  Insert 2 F: 5′-CGG​ACT​AGT​CTA​TGG​CTA​ATC​GTA​GAG​GTG​GTA​TGA​GA-3′ 
  Insert 2 R: 3′-CGG​CGG​CCG​CCT​GGT​AAT​GTT​TTA​TGA​CTT​GAT​GTA​AAT​G-5′ 
Expression in bacteria or insect cells 
GST-Fbp17 pFastBac1-GST F: 5′-GAG​CAT​ATG​ATG​AGT​TAT​TCA​ACC​GAT​TTA​TTA​GAT​GGA​TTT​GAA​AGA​TTA​TAT​AAA​AGA​ACt​TAT​GGT​GTT​TTA​AAA​TGT​AAT​C-3′ 
  R: 3′-TGC​TCT​AGA​TTA​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GGC-5′ 
GST-WASP pFastBac1-GST F: 5′-GAG​CAT​ATG​ATG​GGT​AGT​CCA​ACT​ATT​AGT​GAT​CAA​G-3′ 
  R: 3′-TGC​TCT​AGA​ATA​ATC​TGA​CCA​TTC​ATC​ATC​GTC​ATC​ATC​ATC-5′ 
GST-WASPVCA pGEX-6P-1 F: 5′-CGC​GGA​TCC​CCA​TCA​TCA​GGA​GGT​GGT​GGT​GCC-3′ 
  R: 3′-TGC​TCT​AGA​TTA​ATA​ATC​TGA​CCA​TTC​ATC​ATC​GTC​ATC-5′ 
GST-Fbp17SH3 pGEX-6P-1 F: 5′-CGC​GGA​TCC​GCT​GGT​GCA​ACT​GGT​GGT​GCC​ACT​AGA​G-3′ 
  R: 3′-CAG​GCT​CGA​GAC​CAT​CGA​TGA​TGT​CAA​CAA​AAT​TGG​C-5′ 
His-MBP-Fbp17SH3 pET-MBP-3C F: 5′-CGC​GGA​TCC​GCT​GGT​GCA​ACT​GGT​GGT​GCC​ACT​AGA​G-3′ 
  R: 3′-CAG​GCT​CGA​GAC​CAT​CGA​TGA​TGT​CAA​CAA​AAT​TGG​C-5′ 
Expression in yeast cells 
Fbp17 pGADT7 F: 5′-GAG​CAT​ATG​ATG​AGT​TAT​TCA​ACC​GAT​TTA​TTA​GAT​GG-3′ 
  R: 3′-CGC​GGA​TCC​ACC​ATC​GAT​GAT​GTC​AAC​AAA​ATT​GGC​CGG-5′ 
WASPN126-230 pGADT7 F: 5′-GAG​CAT​ATG​ATT​GGT​AAA​ACT​GCT​GCA​ATT​AAT​CCA​AAT​AC-3′ 
  R: 3′-GG AGA​TCT​ACC​AAT​GAT​ATT​GAC​AAT​GAA​TTG​AGC​AG-5′ 
Rac1AG12V pGBKT7 F: 5′-GTT​GCT​GTA​GGT​AAA​ACC​TGT​CTT​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​GAC​AAC​GAC​ACA​TTT​AAT​TGC​TTG-5′ 
Rac1BG12V pGBKT7 F: 5′-GTT​GCA​GTT​GGT​AAA​ACA​TGT​CTT​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​AAC​AAC​TAC​ACA​TTT​AAT​TGC-5′ 
Rac1CG12V pGBKT7 F: 5′-GTT​GCG​GTT​GGT​AAA​ACA​TGT​CTT​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​TAC​AAC​TAC​ACA​TTT​AAT​TGC-5′ 
RacAG12V pGBKT7 F: 5′-GTTGCTGTAGGTAAAAGTTGTTTATTAATTGC-3′
R: 3′-ATCACCTACTACTACTAATTTAATTGC-5′ 
RacBG12V pGBKT7 F: 5′-GTT​GCT​GTT​GGT​AAA​ACT​TGT​TTA​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​TAC​TAC​CAC​CAA​TTT​AAT​TGA​TTG-5′ 
RacCG15V pGBKT7 F: 5′-GTT​GCT​GTA​GGT​AAA​ACT​TGT​TTA​TTG​ATT​AG-3′ 
  R: 3′-ATC​ACC​AAT​AAC​GAC​TAA​TTT​AAT​AAC​TTC-5′ 
RacDG17V pGBKT7 F: 5′-GTT​GCT​GTT​GGT​AAA​ACT​TCA​CTT​TTA​ATT​TTA​TAT​AC-3′ 
  R: 3′-ATC​ACC​TAC​AAC​TAC​TAC​TTT​AAC​AG-5′ 
RacEG20V pGBKT7 F: 5′-GTT​GCT​GTT​GGT​AAA​ACA​TGT​CTT​TTA​ATT​TG-3′ 
  R: 3′-ATC​ACC​GAC​AAC​TAC​TAA​TTT​AAC​TCT​TG-5′ 
RacF1G12V pGBKT7 F: 5′-GTT​GCA​GTT​GGT​AAA​ACC​TGT​ATG​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​AAC​AAC​AAC​ACA​TTT​AAT​ATT​TTG-5′ 
RacF2G12V pGBKT7 F: 5′-GTT​GCA​GTT​GGT​AAA​ACT​TGT​ATG​TTA​ATT​TC-3′ 
  R: 3′-ATC​ACC​AAC​AAC​AAC​ACA​TTT​AAT​ATT​TTG-5′ 
RacGG12V pGBKT7 F: 5′-GTT​GGA​ATT​GGT​AAA​ACA​TCA​ATG​TTA​TTA​AG-3′ 
  R: 3′-TTC​GCC​AAC​AAC​ACA​AAC​TTT​AAT​ACT​TTT​C-5′ 
RacHM13V pGBKT7 F: 5′-GTT​AGT​GTA​GGT​AAG​ACA​TGT​TTA​CTC​ATT​TC-3′ 
  R: 3′-ATC​ACC​TAC​AAC​CAT​TAC​TTT​AAT​ATC​TTT​TAC-5′ 
RacIS14V pGBKT7 F: 5′-GTT​AAA​ACT​GGA​AAA​ACT​ACA​ATG​ATG​ATG​AC-3′ 
  R: 3′-ATC​ACC​TAA​TAC​TAA​TAA​CTT​TAT​ATA​TG-5′ 
RacJD18V pGBKT7 F: 5′-GTT​GGA​GTT​GGT​AAA​TCA​TGT​GTA​ATG​AAC​TC-3′ 
  R: 3′-ATC​TCC​AAG​ACA​AAA​TAT​TTT​AAC-5′ 
RacLG12V pGBKT7 F: 5′-GTT​GCA​GTT​GGT​AAA​ACA​AGT​TTA​TTA​ATT​GC-3′ 
  R: 3′-ATC​ACC​ACA​AAT​TAC​CAT​TTT​TAT​ATA​TTG​C-5′ 
RacMY14V pGBKT7 F: 5′-GTT​GGT​GTT​GGT​AAA​ACC​ACC​CTA​TTA​ATG​AC-3′ 
  R: 3′-ATC​GCC​AAT​CGT​AAC​TAT​TTT​AAT​AG-5′ 
RacNG14V pGBKT7 F: 5′-TTA​GAG​GAT​TAT​CCT​CGT​ATA​AGA​CCA​TTA​TC-3′ 
  R: 3′-ACC​ACC​AGT​ATC​CCA​ACA​ACC​AAT​TCT​C-5′ 
RacOG13V pGBKT7 F: 5′-GTT​TTA​ATT​GGT​AAA​ACT​ACA​TTA​TTA​ATG​AC-3′ 
  R: 3′-ATC​ACC​AAC​TGT​CAC​TAT​CTT​TAC​TTC-5′ 
RacPG58V pGBKT7 F: 5′-GTT​TAC​TGT​TGG​AAA​AAC​AAC​ACT​CTT​ATT​ATC-3′ 
  R: 3′-ATC​ACC​TAC​AAC​ACC​ACA​TTT​AAT​AAC​CTT​TG-5′ 
Other plasmids for expression in Dictyostelium cells 
PHcrac-GFP Peter Devreotes Laboratory, Johns Hopkins University 
LimEΔcoil-GFP Peter Devreotes Laboratory, Johns Hopkins University 
LimEΔcoil-RFP Douglas Robinson Laboratory, Johns Hopkins University 
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