126 



5 'end 3 'end 



B pBluescripc 2958 bp E 



C c 



o 

 R R 



<-MCR I insert about 2000bp I MCR-> 

 I I I I I I 



1 --> 444 423 <-- 1 



I ., , I I I 



KB S K 



ic a c 



no 1 o 



dR I R 



IV I 



I 

 I 



227 --> 844 603 



1 



H 



1 1 



B E 



1 

 H 



r 



H 



1 

 N 



1 

 E 



i 



a c 



i 



1 



d 



c 



n 



n o 



n 



n 



e 



o 



d 



I R 



d 



d 



I 



R 



I 



I V 



I 



I 





V 



I 





I 



I 







I 





I 



I 







320 --> 1S3S 



T r^ — r 



S B S N 



a a p d 



1 n h e 



I III 

 I 



Plasmid 



B B 



a a 



n n 



I I 



I I 



1376 



E S 



c p 



o h 



R I 

 V 



Fig. 7-3. Sequencing strategy for cDNA encoding the bifunctional 

 protein. Overlapping fragments were obtained when the SP3 cDNA clone 

 was cut with restriction enzymes in the sizes denoted in the Figure. 

 The cDNA clone was first cut with Hind III only, and Hind III plus 

 EcoRV. From this four of six possible fragments were obtained for 

 sequencing, two are shown in the second step of the sequencing strategy. 

 Next the cDNA clone was cut with Ban II and two of three fragment were 

 obtained. Two combinations of RE were used, Ndel plus Sal I, and SphI 

 plus EcdRV. Sequencing was completed for multiple samples of these 

 fragments in both directions . 



