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The first step was to clone individual sgRNA into these entry vectors by BpiI digestion and ligation.
c and d Target cloning to the entry vectors.
Resulting entry vectors were LR recombined with pYES-DEST52.
With a wide variety of destination vectors, ORFs cloned into the entry vectors can be rapidly transferred for different applications.
Transfer of inserts from entry vectors into destination vectors was performed using LR reaction as described by the manufacturer (Invirogen).
Other Entry vectors that contain attL1 and attL2 and do not encode ampicillin resistance are compatible with our Destination vectors.
All Entry vectors were propagated in the E.coli strain TOP10F' (Invitrogen) under kanamycin selection.
The coding regions of MKK4 on all the Entry vectors were verified by DNA sequencing.
cDNAs, shRNAs or miRNAs are initially cloned into Entry vectors (Figure 1).
All entry vectors were sequenced and verified.
OFP-containing entry vectors were recombined into the plant binary destination vector pMDC32 [ 3].
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