In several experiments with libraries made in different vectors, we have found that correct inserts are not in frame with the vector sequences (4, 5).
When K−1 is a prime power, we obtain a tight frame with K2+1 vectors and when K is a prime power, with K2+K−1 vectors.
The NcoI site puts the Wilms3 cDNA in frame with the vector-included his-tag.
This synthetic gene was inserted into (i) pET-28a, in-frame with the vector-encoded 6x-His tag and stop codon at its carboxyl end, to express r-Δgp160 antigen, and (ii) pTrxBAP in frame with the vector-encoded Trx (thioredoxin -6x-His thioredoxin -6x-Histhioredoxin -6x-Hiss, to express tag-BAPrx-Bat-Δgp160 antheen.
Native P38α was amplified using primers incorporating a flanking 5' NdeI site such that after subcloning, the 5' ATG would fall in-frame with the vector derived N-terminal tags.
Both selected targeting sequences do not contain an ATG initiation codon that is out-of-frame with the vector URA3, which avoids the translational incompetence of URA3 mRNA and resistance to 5-FOA (see Methods).
The bacterial clones obtained were PCR screened using vector and receptor specific primers and colonies that contained the coding regions in frame with the promoter vector pCMV were selected and plasmid DNA was extracted using the DNA Midi-Prep kit (Roche, Germany).
Firstly, we represent the position and velocity of target t in the x-y coordinate frame with the state vector x t = [ x t x ̇ t y t y ̇ t ] T, (2).
The interaction between full-length API5 and NP was validated in yeast two-hybrid system, by cloning full-length human cDNA of API5 in frame with activation domain vector pYESTrp2, followed by co-transformation with pHybLex/Zeo-NP in L40 strain of yeast cells.
The PCR product after digestion was subcloned in frame with mCherry into the vector pcDNA3.
The PCR product was then subcloned in frame with GFP into pARL vector (Kind gift of the Dr C. Sanchez Heidelberg, Germany) [ 47] digested with XhoI and KpnI.
