Difference between revisions of "Part:BBa K4235002"
(→Usage and Biology) |
|||
(6 intermediate revisions by the same user not shown) | |||
Line 2: | Line 2: | ||
__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K4235002 short</partinfo> | <partinfo>BBa_K4235002 short</partinfo> | ||
− | + | ||
+ | |||
+ | [[Image:PFastBac.jpg|700px|center|]] | ||
+ | |||
+ | |||
===Usage and Biology=== | ===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. | ||
Line 10: | Line 14: | ||
For our project, we intended on cloning our insert <partinfo>BBa_K4235000</partinfo> just upstream of the twin strep tag and 6x His-tags which are on the C-terminal of the MCS in YmBac-II. | For our project, we intended on cloning our insert <partinfo>BBa_K4235000</partinfo> just upstream of the twin strep tag and 6x His-tags which are on the C-terminal of the MCS in YmBac-II. | ||
+ | This vector contains the following parts: <br> | ||
+ | *Polyhedrin Promoter: <partinfo>BBa_K4235001</partinfo> <br> | ||
+ | *Gentamicin resistance cassette (pC+GmR): <partinfo>BBa_K4235024</partinfo> <br> | ||
+ | *Ampicillin resistance cassette (AmpR promoter+CDS): <partinfo>BBa_K4235025</partinfo> <br> | ||
+ | *SV40 polyA signal: <partinfo>BBa_K4235020</partinfo> | ||
+ | *miniatt-Tn7 transposon segment (polyhedrin+MCS+SV40polyA+GmR cassette): <partinfo>BBa_K4235010</partinfo> | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here |
Latest revision as of 02:07, 27 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.
This vector contains the following parts:
- Polyhedrin Promoter: BBa_K4235001
- Gentamicin resistance cassette (pC+GmR): BBa_K4235024
- Ampicillin resistance cassette (AmpR promoter+CDS): BBa_K4235025
- SV40 polyA signal: BBa_K4235020
- miniatt-Tn7 transposon segment (polyhedrin+MCS+SV40polyA+GmR cassette): BBa_K4235010
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.