SalI (10 U/µL)

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	This T2A-NeoR fragment was digested with XhoI (Thermo Scientific), HSVtk fragment was digested with SalI (Thermo Scientific), fragments were ligated and the fragment of predicted length was gel purified again.

Protocol tips
This T2A-NeoR fragment was digested with XhoI (Thermo Scientific), HSVtk fragment was digested with SalI (Thermo Scientific), fragments were ligated and the fragment of predicted length was gel purified again.

Publication protocol

"Selection cassette design (HSV thymidine kinase, T2A peptide, NeoR in one frame) was performed with Ugene7
. HSVtk andNeoR sequences were PCR amplified with Phusion polymerase (Thermo Scientific) from pBS246-neo/Tk (a gift from E.P.,
construction of this plasmid is described elsewhere8
) with primers HSVtk_F, HSVtk_R, and G418_F, G418_R, correspondingly. T2A peptide coding sequence, corresponding to amino
acid sequence GSGEGRGSLLTCGDVEENPGP, was synthesized by hybridization of oligonucleotides T2A(+) and T2A(-) in
annealing buffer and consequent treatment of hybridized duplex
with T4 polymerase (Thermo Scientific) and gel purified. The
NeoR fragment was digested with XbaI (Thermo Scientific), the
T2A fragment was digested with NheI (Thermo Scientific), then
these fragments were ligated and the fragment of expected size
(881bp) was gel purified. This T2A-NeoR fragment was digested
with XhoI (Thermo Scientific), HSVtk fragment was digested with
SalI (Thermo Scientific), fragments were ligated and the fragment of predicted length was gel purified again. This HSVtk-T2ANeoR fragment was double digested with EcoRI (Thermo Scientific) and BshTI (Thermo Scientific) and ligated with pX459-b1
double-digested with EcoRI and BshTI. This step gave the plasmid with full-length selection cassette. Below we refer to this
plasmid as pHSVtk-T2A-NeoR. Then the pHSVtk-T2A-NeoR
plasmid was double-digested with XbaI and Acc65I (Thermo
Scientific), gel purified and ligated with hybridized AdaptUp(+)-
AdaptUp(-) duplex. A successful insert was verified by digestion
of newly introduced restriction sites: SalI and BamHI. We refer
to this plasmid as pAdaptUp-HSVtk-T2A-NeoR. This plasmid
was digested with NotI (Thermo Scientific), treated with FastAP
(Thermo Scientific), gel purified and ligated with AdaptDown(+)-
AdaptDown(-) duplex to introduce a 2xBpiI site that generates
half-sites for BclI and XhoI after cleavage. The resulting plasmid
was transformed into E. coli Top-10 cells. We refer to this plasmid
as pAdaptUp-HSVtk-T2A-NeoR-AdaptDown. To obtain LoxPflanked sequences, we used a pBK-CMV-derived plasmid with a
modified multiple cloning site containing restriction sites for BclI,
NheI, and XhoI. This plasmid was transformed into E. coli JM110
to eradicate Dam methylation, double digested with BclI and NheI,
dephosphorylated, gel purified and ligated with LoxP(+)- LoxP(-)
duplex, treated with T4 PNK. This plasmid was transformed into
E. coli Top10 cells and referred to as pLoxP. To obtain homology regions that flank Cas9 cleavage site we PCR amplified them
from Caki1 gDNA, using primer pairs Bgl1Up_F and Bgl1Up_R
for an upstream fragment and Bgl1 Down_F and Bgl1Down_R
for a downstream fragment. These PCR fragments were double digested with NheI and XhoI and ligated with pLoxP, double
digested by the same sites and dephosphorylated. Ligation products
were transformed into E. coli JM110, and purified plasmids were
digested with BclI and XhoI to yield upstream and downstream
fragments of DNA bearing LoxP site on its end. The upstream
fragment was ligated with pAdaptUp-HSVtk-T2A-NeoRAdaptDown double digested with SalI and BamHI to give pUp1LHSVTK-T2A-NeoR-AdaptDown. The downstream fragment was
ligated with pUp1L-HSVTK-T2A-NeoR-AdaptDown treated
with BpiI and dephosphorylated. We refer to this plasmid as
pUp1L-HSVTK-T2A-NeoR-L1Down To obtain LoxPflanked sequences, we used a pBK-CMV-derived plasmid with a
modified multiple cloning site containing restriction sites for BclI,
NheI, and XhoI. This plasmid was transformed into E. coli JM110
to eradicate Dam methylation, double digested with BclI and NheI,
dephosphorylated, gel purified and ligated with LoxP(+)- LoxP(-)
duplex, treated with T4 PNK. This plasmid was transformed into
E. coli Top10 cells and referred to as pLoxP. To obtain homology regions that flank Cas9 cleavage site we PCR amplified them
from Caki1 gDNA, using primer pairs Bgl1Up_F and Bgl1Up_R
for an upstream fragment and Bgl1 Down_F and Bgl1Down_R
for a downstream fragment. These PCR fragments were double digested with NheI and XhoI and ligated with pLoxP, double
digested by the same sites and dephosphorylated. Ligation products
were transformed into E. coli JM110, and purified plasmids were
digested with BclI and XhoI to yield upstream and downstream
fragments of DNA bearing LoxP site on its end. The upstream
fragment was ligated with pAdaptUp-HSVtk-T2A-NeoRAdaptDown double digested with SalI and BamHI to give pUp1LHSVTK-T2A-NeoR-AdaptDown. The downstream fragment was
ligated with pUp1L-HSVTK-T2A-NeoR-AdaptDown treated
with BpiI and dephosphorylated. We refer to this plasmid as
pUp1L-HSVTK-T2A-NeoR-L1Down."

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