Table S3.

pBIG1 constructs in this study

pBIG1 constructAliasVectorCasICasIICasIIICasIVCasVConstruct size, kbShown in figureClones correct/analyzed
FW_I-1pBIG1aAPC1APC2APC5APC11APC1520.0Fig. 2A1/3
FW_I-2pBIG1bAPC4-Strep8.9Fig. 2A3/3
FW_I-3pBIG1cAPC6APC7APC12(CDC26)APC13APC1613.2Fig. 2A1/7
FW_I-4pBIG1dAPC3APC8APC1012.5Fig. 2A2/3
FW_I-5pBIG1eCDH1EMI1SKP110.9Fig. 2A4/12
FW_I-6pBIG1bAPC4-StrepAPC811.42/7
FW_I-7pBIG1cAPC6-StrepAPC7APC12(CDC26)APC13APC1613.32/13
FW_I-8pBIG1dAPC3mAPC8APC1012.51/3
FW_I-9pBIG1dAPC3APC8mAPC1012.51/3
FW_I-10pBIG1dAPC3APC8APC10m12.51/3
FW_I-11pBIG1dAPC3mAPC8mAPC1012.51/3
FW_I-12pBIG1dAPC3mAPC8APC10m12.52/3
FW_I-13pBIG1dAPC3APC8mAPC10m12.51/3
FW_I-14pBIG1dAPC3mAPC8mAPC10m12.52/3
FW_I-15pBIG1dAPC3APC811.32/3
FW_I-16Cohesin wtpBIG1cSMC1ASMC3-FlagSCC1His-SA121.2Fig. 3F1/6*
FW_I-17Cohesin KApBIG1cSMC1A(KA)SMC3(KA)-FlagSCC1His-SA121.2Fig. 3F1/42*
FW_I-18Cohesin EQpBIG1cSMC1A(EQ)SMC3(EQ)-FlagSCC1His-SA121.2Fig. 3F4/30*
FW_I-19Kinetochore NpBIG1aNdc80-HisNuf2Spc24Spc2512.9Fig. 3K2/12
FW_I-20Kinetochore MpBIG1bHis-Dsn1Mtw1Nnf1Nsl112.0Fig. 3K1/6
FW_I-21pBIG1bDsn1Mtw1Nnf1Nsl112.03/6
FW_I-22pBIG1aNdc80-HisNuf2Spc24Spc25Kre2814.62/10
FW_I-23pBIG1bDsn1Mtw1Nnf1Nsl1Spc105-Flag15.3Fig. S5A2/12
  • * Although the assembly of APC/C and kinetochore biGBac constructs occurred with high efficiency, more transformed E. coli clones needed to be analyzed when we assembled the four cohesin core subunits into pBIG1 vectors, possibly due to the relatively large size of the cohesin cDNAs, resulting in pBIG1 constructs of 21.2 kb. We assume this is due to difficulties generating pure PCR products of large GECs. We therefore also generated cohesin expression vectors in two steps with the two SMC subunits on pBIG1a and SCC1 and SA1 on pBIG1b. In the second step, we combined the four subunits into a pBIG2 expression vector. Using this strategy, most analyzed clones were correct. If several subunits of a protein complex are large, it may therefore be advantageous to distribute the cDNAs encoding the large subunits onto more than one pBIG1 vector.