BspTI (AflII) (10 U/µL)

Protocol tips

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	To generate a pCMV-MCS-T2A-hHO1 plasmid, a pcDNA3.1-iCasper-T2A-HO1 vector (Addgene #64278) (To et al., 2015) was treated with BspTI and SgsI restriction endonucleases (Thermo Scientific) to cut out iCasper gene and then MCS (multiple cloning site) was inserted by ligation of MCS_HO1_sense and MCS_HO1_asense oligonucleotides there.

Protocol tips
To generate a pCMV-MCS-T2A-hHO1 plasmid, a pcDNA3.1-iCasper-T2A-HO1 vector (Addgene #64278) (To et al., 2015) was treated with BspTI and SgsI restriction endonucleases (Thermo Scientific) to cut out iCasper gene and then MCS (multiple cloning site) was inserted by ligation of MCS_HO1_sense and MCS_HO1_asense oligonucleotides there.

Publication protocol

"To generate a pCMV-MCS-T2A-hHO1 plasmid, a pcDNA3.1-iCasper-T2A-HO1 vector (Addgene #64278) (To et al., 2015) was
treated with BspTI and SgsI restriction endonucleases (Thermo Scientific) to cut out iCasper gene and then MCS (multiple cloning
site) was inserted by ligation of MCS_HO1_sense and MCS_HO1_asense oligonucleotides there. To generate a pEGFP-T2A-HO1
plasmid, an EGFP gene was amplified with EGFP_HindIII_fw and EGFP_XhoI_rv oligonucleotides, treated with HindIII and XhoI restriction endonucleases (Thermo Scientific) and inserted into the pCMV-MCS-T2A-HO1 plasmid digested with the same enzymes.
To generate a pIRES2-HO1 plasmid, an AgeI restriction site was removed from HO1 gene in the pcDNA3.1-iCasper-T2A-HO1 vector
by quick-change mutagenesis using HO1_AgeI_del_fw and HO1_AgeI_del_rv oligonucleotides and PrimeSTAR DNA Polymerase
(Takara Bio). Before transformation into E.coli TOP10 (Invitrogen), the PCR product was treated with DpnI restriction endonuclease.
Resulted HO1 gene without AgeI recognition site was amplified with HO1_AgeI_fw and HO1_NotI_rv oligonucleotides, treated with
BshTI and NotI restriction endonucleases (Thermo Scientific) and inserted into the pIRES2-EGFP plasmid digested with the same
enzymes to cut out an EGFP gene. EGFP-T2A-HO1 DNA fragment was amplified from pEGFP-T2A-HO1 with EGFP_SacI_fw and
HO1_XmaI_rv oligonucleotides, digested with SacI and Cfr9I restriction endonucleases (Thermo Scientific) and inserted into a
pEGFP-N1 plasmid (Clontech) treated with the same enzymes. Then, a SpeI restriction site was added before a HO1 gene by
quick-change mutagenesis using HO1_SpeI_add_fw and HO1_SpeI_add_rv oligonucleotides and PrimeSTAR DNA Polymerase
(Takara Bio). The miRFP670 and the miRFP703 genes (Shcherbakova et al., 2016) was PCR-amplified with miRFP_SpeI_fw and
miRFP_XmaI_rv oligonucleotides. The mIFP gene was PCR-amplified with mIFP_SpeI_fw and mIFP_XmaI_rv oligonucleotides
from a pmIFP-N1 plasmid (Addgene #54620) (Yu et al., 2015). The smURFP gene was PCR-amplified with smURFP_SpeI_fw and
smURFP_XmaI_rv oligonucleotides from a pcDNA3-smURFP-IRES-HO1 plasmid (Addgene #80345) (Rodriguez et al., 2016).
Then, the genes of all NIR FPs were treated with BcuI and Cfr9I restriction endonucleases (Thermo Scientific) and inserted into
the pEGFP-T2A-HO1 plasmid (with SpeI recognition site added previously) digested with the same enzymes. An EGFP gene was
cut out with BshTI and NotI restriction endonucleases, the plasmids were treated with T4 DNA Polymerase (Thermo Scientific)
and self-ligated with T4 DNA Ligase (Thermo Scientific). In this way, the pEGFP-T2A-NIR FP plasmids were generated where NIR FP
is miRFP670, miRFP703, mIFP or smURFP. To generate a pEGFP-T2A-NIR FP-IRES2-HO1 plasmid, the pIRES2-HO1 vector was
treated with Cfr9I and NotI restriction endonucleases (Thermo Scientific) and then the IRES2-HO1 fragment was inserted into the
pEGFP-T2A-NIR FP plasmids digested with the same enzymes. To obtain pEGFP-T2A-NIR FP-IRES2-HO1 plasmids with inactive
HO1, a two-step quick-change mutagenesis was performed to introduce the H25A and D140F substitutions."

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