Part:BBa_K4761107
pTarget
pTarget plasmid vector used for expressing sgRNA. This plasmid is the original pTarget plasmids we use to construct other pTargets by using overlapping PCR.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2064
Illegal XbaI site found at 2070
Illegal SpeI site found at 1955
Illegal PstI site found at 2082 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2064
Illegal NheI site found at 1932
Illegal SpeI site found at 1955
Illegal PstI site found at 2082 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2064
Illegal BglII site found at 2094
Illegal BamHI site found at 1920 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2064
Illegal XbaI site found at 2070
Illegal SpeI site found at 1955
Illegal PstI site found at 2082 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2064
Illegal XbaI site found at 2070
Illegal SpeI site found at 1955
Illegal PstI site found at 2082
Illegal NgoMIV site found at 771 - 1000COMPATIBLE WITH RFC[1000]
Characterization
This plasmid, known as the Original pTarget in the Wet Lab members in 2023 HiZJU-China, is used as the template for creating other pTargets(BBa_K4761420 to BBa_K4761428) for the CRISPR-Cas9 dual plasmid system in our Gene Knockout Module.
By using pTargets made from this original pTarget plasmid, we have successfully knocked out many desired genes in DH5α chassis, as pheA, pykA, pykF, etc., which may compete tyrosine with our salidroside production pathway.
These pTargets are easy to be cured by incubation overnight at 30℃ after the knockout was successfully carried out.
Primer Design Guide for Building New pTargets
All the pTargets we use is made from this plasmid using overlapping PCR. The primer consists of two parts: the sgRNA sequence(BBa_K4761020 to BBa_K4761028) in the middle, and the homologous sequence on its left and right sides at an appropriate Tm, as shown in Fig.2.
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