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The BAD DNA target sequences used were 5'-TGAAGGGACTTCCTCGCCCGT-3' and 5' GGCTTGGTCCCATCGGAAG-3'. HEK 293 cells were transfected with pLL3.7 vector containing either of these sequences or a scrambled sequence 5'-GGTACGGTCAGGCAGCTTCT-3' in combination with packaging vectors (VSVG, RSV-REV, and pMDL g/p RRE).
To prepare the translational reporter constructs, full length gtl-2 cDNA (4217 bp, without stop codon) was constructed by assembling PCR-amplified cDNA fragments and yk405b5 cDNA (a gift from Y. Kohara) into pPD95.79 vector containing either Pgtl-2 or Psupl-4.
After sequencing, the fragments were digested with the restriction enzymes HindIII and BamHI and subcloned into pcDNA3.1 vector or pcDNA3.1 vector containing either YFP or Rluc sequences.
We performed 3′UTR luciferase assay, and observed that luciferase activity was decreased after transfection of miR-183 precursor and a 3′UTR vector containing either Dkk-3 or SMAD4.
After overnight incubation, they were co-transfected with 50 nM miR-181b mimic or non-relevant control (NC), together with 0.5 μg reporter vector containing either wtMDM2 3'UTR or mutMDM23'UTR.
In addition to having both pfkA and pfkB deleted, RL257 contains the lacIq allele, which allows for inducible expression when coupled with an expression vector containing either the lac or tac promoter.
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In the multiplexed training case, each element of the transmitted vector contains either a pilot symbol or a data symbol, while in the superimposed case both pilot and data symbols are located in the same positions, typically distributed over the whole transmitted vector.
The exit vector contains either a univocal diagnosis (if the clinical data are sufficient) or a certain linear combination of vectors corresponding to several diseases.
To test whether overexpression or inhibition of C/EBPβ can influence the direction of reprogramming (i.e. hepatocyte vs ductal), we infected control, Dex and Dex/EGF-treated cells with the adenoviral vectors containing either LAP or LIP (Ad-CMV-LAP or Ad-CMV-LIP) and determined the expression of the hepatocyte marker TFN or the ductal marker PNA.
To obtain pre-adipocyte clones with long term down regulation of DNA-PKcs expression, the 3T3F442A cells were transfected using Amaxa electropation system (Nucleofector solution V; program: T030) with EBV-based vectors containing either control or short hairpin RNA coding sequences for murine DNA-PKcs as previously described [22].
This observation extends to expression vectors containing either TK or SV40 promoters, which were also potently transactivated by MAA in our experiments (data not shown).
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CEO of Professional Science Editing for Scientists @ prosciediting.com