To generate NEIL1 and NEIL2 overexpression vectors, human NEIL1 and NEIL2 coding sequences excluding the stop codon were cloned into the SalI-XhoI restriction sites of pENTR1A no CCDB (w48-1) vector (Addgene plasmid #17398), which had been modified to contain EGFP in the XhoI-XbaI site (a kind gift from Dr. P. Herr). Following sequence verification, entry clones were shuttled using Gateway cloning LR clonase reaction (Thermo fisher scientific) into pLENTI-CMV Puro DEST vector (w118-1) (E. Campeau, Addgene plasmid #17452). Kanamycin was used to select putative positive clones. Destination vectors were verified by sequencing. U2OS cells were then transfected with destination constructs using jetPEI (Polyplus) and selected with 1 μg/mL puromycin for 10 days. To minimize variability in expression levels among the different positive clones, clonal expansion was carried out to generate a single clone of U2OS cells, constitutively expressing C-terminus GFP-tagged NEIL1 or GFP-tagged NEIL2.
Pentr1a no ccdb w48 1 vector
Manufactured by Addgene
The PENTR1A no CCDB (w48-1) vector is a plasmid that is commonly used in recombination cloning techniques. The vector contains the attL1 and attL2 recombination sites, which allow for the transfer of DNA sequences between compatible entry and destination vectors. The CCDB gene, which encodes a protein that is toxic to most E. coli strains, has been removed from this version of the vector.
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Lab products found in correlation
2 protocols using pentr1a no ccdb w48 1 vector
1
Generating NEIL1 and NEIL2 Overexpression Vectors
2
Cell Culture and Genetic Modification
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RenCa were maintained in RPMI-1640 (Gibco, BE12-702F) growth medium
supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma, F9665)
and 100 units/mL penicillin, 100 μg/mL streptomycin (Sigma,
6SLBJ7114U). wt-TG2 cDNA (coding 224Val) was subcloned into the pENTR1A
no CCDB (w48-1) vector (gift from Eric Campeau;81 (link) Addgene #17398) at the EcoRI position, and mutants were
generated based on QuikChange site-directed mutagenesis kit protocol
(Stratagene). The restriction digestion and sequencing confirmed constructs
were recombined into a pLenti CMV Blast DEST (706-1) destination vector,
which was a gift from Eric Campeau,35 (link) (Addgene
#17451) using LR Clonase and GATEWAY technology based on the manufacturer
protocol (Thermo #11791020).
supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma, F9665)
and 100 units/mL penicillin, 100 μg/mL streptomycin (Sigma,
6SLBJ7114U). wt-TG2 cDNA (coding 224Val) was subcloned into the pENTR1A
no CCDB (w48-1) vector (gift from Eric Campeau;81 (link) Addgene #17398) at the EcoRI position, and mutants were
generated based on QuikChange site-directed mutagenesis kit protocol
(Stratagene). The restriction digestion and sequencing confirmed constructs
were recombined into a pLenti CMV Blast DEST (706-1) destination vector,
which was a gift from Eric Campeau,35 (link) (Addgene
#17451) using LR Clonase and GATEWAY technology based on the manufacturer
protocol (Thermo #11791020).
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