163 pT7A3-MBP-(Tev)-GST-(3C)-sfGFP

PCR fragment 1 from 2369 pT7A3-lc-MBP-(TEV)
2094-T
CTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAT
    TTTATTAAAACAAATTGAAATTCTTCCTCTATATAGTAC

MBP-seq-R (CGGTTCTTGCAGGTTGAAC)
gttcaacctgcaagaaccg

PCR fragment 2 from 2369 pT7A3-lc-MBP-(TEV)

pET-seq-F2  ATCTCGACGCTCTCCCTTATG
2094-B
CATGATATATCTCCTTCTTAAAGTTAAACAAAATTATTT

Purify on gel, PCR again 
pET-seq-F2  ATCTCGACGCTCTCCCTTATG
MBP-seq-R (CGGTTCTTGCAGGTTGAAC)
gttcaacctgcaagaaccg

Digest with BsaI + SphI
clone into  BsaI + SphI 161 pT7A3-MBP-(Tev)-GST-(3C)-sfGFP

1. 1, #5 looks good

Sequencing result: it does not have T7A3 promoter, It is cold promoter.. it is clone 160

http://www.excellgen.net/eln/images/2/2c/163_164_digestion.JPG

Redo

1/1/19: The above methods were not working

use subcloning method:

digest 167 pT7A3-MBP-TRX-6H #2 with Sph1+ BsaI
digest 161 pT7A3-MBP-(Tev)-GST-(3C)-sfGFP  with Sph1+ BsaI

163-1 from 54ng/ul digestion

163-2 from 34ng/ul digestion

163-2 #1, pellet not green, all other clones are green