Done using IDT tool as Twist Bioscience link provided was not working, optimised for expression in S. cerevisiae, removed type II restriction enzymes BsaI, BsmBI and BpsI.
The sequence entered of OsRGLP1 having start codon and a stop codon:
ATG GCT TCT GAT CCA TCT CCA TTA CAA GAT TTC TGT GTT GCT GAC ATG CAT TCT
CCA GTT TTG GTC AAC GGT TTT GCT TGT TTG AAC CCA AAG GAT GTC AAT GCT GAT
CAT TTC TTC AAG GCT GCT ATG TTG GAC ACT CCA AGA AAG ACC AAC AAG GTT GGT
TCC AAT GTC ACT TTG ATC AAC GTT ATG CAA ATT CCA GGT TTG AAC ACT TTG GGT
ATC TCC ATT GCC AGA ATT GAT TAT GCT CCA TTG GGT CAA AAC CCA CCA CAC ACA
CAC CCA AGA GCC ACT GAA ATT TTG ACT GTT TTG GAA GGT ACT TTG TAT GTT GGT
TTT GTC ACT TCC AAC CCA GAC AAC AAG TTC TTC TCC AAG GTT TTG AAC AAG GGT
GAT GTC TTT GTC TTC CCA GTT GGT TTG ATT CAC TTC CAA TTC AAC CCA AAC CCA
TAC AAG CCA GCT GTT GCT ATT GCT GCT TTA TCT TCT CAA AAC CCA GGT GCC ATC
ACC ATT GCC AAT GCT GTT TTT GGT TCC AAG CCA CCA ATT TCT GAT GAT GTT TTG
GCC AAG GCT TTC CAA GTT GAA AAG GGT ACC ATT GAT TGG TTA CAA GCT CAA TTC
TGG GAA AAC AAC CAC TAC TAA
These are the parameters used on the IDT codon optimisation tool; restriction sites to avoid were entered. However, when checking the sequence on Benchling for the absence of these enzymes, BsmBI and BsaI are not present, but one restriction site of BbsI does exist. The codon optimisation might have been more accurate if Twist or GenWiz software had been used.
3.4. You have a sequence! Now what?
The gene sequence can be recombinantly expressed in S. cerevisiae by using a protocol adapted from Gietz, R. D et al., 2007.
Part 4: Prepare a Twist DNA Synthesis Order
Build Your DNA Insert Sequence
Added 5’ and 3’ sequences for Type II restriction digestion and have annotated the sequence 5’ end - CDS - 3’ end. Can be subsequently used for ordering through Twist Bioscience.
Used vectors of YTK kit and used the combinatorial assembly feature of Benchling to design the final vector: pYtk009 vector (pTDH3) promoter, pYTK051 vector (tENO1) terminator.
