The Myc-DDK-tagged pCMV-PAX9 expression vector (RC200380) was purchased from OriGene Technologies (Rockville, MD, USA) and named pAM6. P20L mutation was introduced by replacing the 0.3-kb EcoRI-BspEI fragment of pAM6 with a synthesized fragment containing the mutation (GeneArt Gene Synthesis Service, ThermoFisher Scientific). A240P mutation was introduced by PCR-based site-directed mutagenesis using a pair of primers. For the null mutant, a 0.3-kb BglII-BspEI fragment containing the first ATG was deleted from pAM6. These plasmids were named pAM7 (P20L), pAM8 (A240P), pAM9 (P20L/A240P) and pAM4 (null). A reporter plasmid was constructed by replacing the SV40 early promoter of the pGL4.13 luciferase reporter vector (Promega, Fitchburg, WI, USA) with a 2.3-kb BMP4 promoter fragment and named pAM5. Nucleotide sequences were verified after the construction.
Pgl4.13 luciferase reporter vector
Manufactured by Promega
Sourced in United States
The PGL4.13 luciferase reporter vector is a plasmid that contains the firefly luciferase gene as a reporter. It can be used to measure gene expression in a variety of cell types.
Automatically generated - may contain errors
2 protocols using pgl4.13 luciferase reporter vector
1
Generating PAX9 Mutant Expression Vectors
2
Constructing Luciferase Reporters for ABCB1 Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The upstream region (from bp −2091 to +701, where +1 indicates the transcription start site) and 3′ UTR (from bp +1 to +391, where +1 indicates the stop codon) of the human ABCB1 gene were amplified using PrimeSTAR MAX DNA polymerase (Takara Bio Inc). These sequences were subcloned into the pGL4.12 luciferase-reporter vector (Promega) using KpnI and NheI restriction sites and into the pGL4.13 luciferase-reporter vector (Promega) using XbaI and FseI restriction sites. The minigene sequence was amplified using PrimeSTAR MAX DNA polymerase (Takara Bio Inc). The sequence was subcloned into the pcDNA3.1 (Invitrogen; Life Technologies) vector using NheI and KpnI restriction sites. Sequences of primers are listed in Table 2 .
Primer sets for plasmid construction and minigene assay
| Gene | Primers |
|---|---|
| Human ABCB1 promoter | |
| Forward for −2091 bp | 5′-GCTGGTACCTCAACTTGCAAGGGGACCAG-3′ |
| Reverse for +703 bp | 5′-ATAGCTAGCCGACCTGAAGAGAAACCGCA-3′ |
| Human ABCB1 3′ UTR | |
| Forward for +1 bp | 5′-GCGCTCTAGAAACTCTGACTGTATGAGATG-3′ |
| Reverse for +391 bp | 5′-TAAGGCCGGCCAGTCACATGAAAGTTTAG-3′ |
| Human ABCB1 minigene | |
| Forward for exon 27 | 5′-CGTGCTAGCGCAAGCTGTTAGAACTTTACTTTCA-3′ |
| Reverse for exon 28 | 5′-CGCGGTACCACAGGCAGTTTGGACAAGATGA-3′ |
The numbers indicate the distance from the transcription site (+1) for the promoter vector or from the stop codon (+1) for the 3′ UTR vector.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!