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Over time, planks are removed from O′ and used to replace corresponding planks of O.
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These alleles together with the previously identified GUT1CBS 6412-13allelele were used to replace the corresponding alleles in the strain CEN.PK113-1A CEN.PK113-1A
The identified SNPs and InDels in this step were used to replace the corresponding nucleotides in the reference MSU7 cDNA sequences to construct the pseudo-reference sequence.
This fragment is used to replace the corresponding attP1 fragment of pDONR-221.
To generate attP4 and attP5 from attP2, the same strategy leads to a 721bp fragment flanked by EcoR1 and EcoRV, which are used to replace the corresponding attP2 fragment of pDONR-221.
Then, the amplified PCR fragment was digested with ApaI and SalI restriction enzymes and was used to replace the corresponding region in HxBru-Vif+, and the resultant provirus was called HxBru-Vif−.
Similarly, an AvrII -SacI fragment from pTM238 was used to replace the corresponding fragment from pTM242, to yield pTM253.
The HindIII -AvrII fragment from pTM238 was used to replace the corresponding fragment from pTM238, yielding pTM252.
An NgoMI -KpnI fragment was used to replace the corresponding fragment in pTM034 [ 20] to yield pTM192 and its sequence was confirmed (Fig. 3).
An NcoI- SacI fragment was used to replace the corresponding fragment in pTM034 to yield pTM194 and its sequence was confirmed.
A BglII/ PshAI fragment containing the tag was then liberated from this plasmid and used to replace the corresponding fragment in PCRA10kb to generate PCRA-10kb-tag.
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