Difference between revisions of "Part:BBa K4235002"
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<partinfo>BBa_K4235002 short</partinfo> | <partinfo>BBa_K4235002 short</partinfo> | ||
+ | ===Usage and Biology=== | ||
The plasmid pFastBac is used for the expression of 6x His-TEV-tagged proteins in insect cells through the Bac-to-Bac baculovirus expression system. The main components of this vector are an Ampicillin resistance marker gene and a mini-attTn7 transposon segment, which contains the multiple cloning site for expressing recombinant proteins driven by the polyhedrin promoter, a SV40 polyA signal and Gentamicin resistance gene expressed from the pC promoter. The mini-attTn7 segment is flanked by terminal inverted repeats called Tn7-L and Tn7-R which are recognized by the enzyme transposase. Transposases are responsible for introducing double-stranded breaks at these mini Tn7 elements and freeing the DNA sequence from the transfer vector. This DNA segment can then be inserted/transposed into another bacterial or baculovirus genome propagated in E coli DH10Bac cells, allowing for the production of recombinant Bacmid particles. | The plasmid pFastBac is used for the expression of 6x His-TEV-tagged proteins in insect cells through the Bac-to-Bac baculovirus expression system. The main components of this vector are an Ampicillin resistance marker gene and a mini-attTn7 transposon segment, which contains the multiple cloning site for expressing recombinant proteins driven by the polyhedrin promoter, a SV40 polyA signal and Gentamicin resistance gene expressed from the pC promoter. The mini-attTn7 segment is flanked by terminal inverted repeats called Tn7-L and Tn7-R which are recognized by the enzyme transposase. Transposases are responsible for introducing double-stranded breaks at these mini Tn7 elements and freeing the DNA sequence from the transfer vector. This DNA segment can then be inserted/transposed into another bacterial or baculovirus genome propagated in E coli DH10Bac cells, allowing for the production of recombinant Bacmid particles. | ||
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<partinfo>BBa_K4235002 parameters</partinfo> | <partinfo>BBa_K4235002 parameters</partinfo> | ||
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+ | ===Source=== | ||
+ | We received this plasmid as a generous donation from the Airola Lab at Stony Brook University. |
Revision as of 22:35, 10 September 2022
pFastBac-Htb_miniTn7
Usage and Biology
The plasmid pFastBac is used for the expression of 6x His-TEV-tagged proteins in insect cells through the Bac-to-Bac baculovirus expression system. The main components of this vector are an Ampicillin resistance marker gene and a mini-attTn7 transposon segment, which contains the multiple cloning site for expressing recombinant proteins driven by the polyhedrin promoter, a SV40 polyA signal and Gentamicin resistance gene expressed from the pC promoter. The mini-attTn7 segment is flanked by terminal inverted repeats called Tn7-L and Tn7-R which are recognized by the enzyme transposase. Transposases are responsible for introducing double-stranded breaks at these mini Tn7 elements and freeing the DNA sequence from the transfer vector. This DNA segment can then be inserted/transposed into another bacterial or baculovirus genome propagated in E coli DH10Bac cells, allowing for the production of recombinant Bacmid particles.
This plasmid was modified by the Airola lab at Stony Brook University to have twin strep tags and a 6x His-tag with a TEV site on the C-terminal of the MCS instead of N-terminal, as found in the original pFastBac HT-B.
For our project, we intended on cloning our insert BBa_K4235000 just upstream of the twin strep tag and 6x His-tags which are on the C-terminal of the MCS in YmBac-II.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4069
Illegal XbaI site found at 4116
Illegal SpeI site found at 4097
Illegal PstI site found at 4124 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4069
Illegal SpeI site found at 4097
Illegal PstI site found at 4124 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4069
Illegal BglII site found at 2547
Illegal BglII site found at 3017
Illegal BamHI site found at 4062
Illegal XhoI site found at 4131 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4069
Illegal XbaI site found at 4116
Illegal SpeI site found at 4097
Illegal PstI site found at 4124 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4069
Illegal XbaI site found at 4116
Illegal SpeI site found at 4097
Illegal PstI site found at 4124
Illegal NgoMIV site found at 127 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1304
Illegal BsaI site found at 3661
Illegal SapI.rc site found at 2386
Source
We received this plasmid as a generous donation from the Airola Lab at Stony Brook University.