Plasmid

Part:BBa_K4235002

Designed by: Maulik Masaliya   Group: iGEM22_Stony_Brook   (2022-09-10)
Revision as of 02:07, 27 September 2022 by Maulik masaliya (Talk | contribs) (Usage and Biology)

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pFastBac-Htb_miniTn7


PFastBac.jpg


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:

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4069
    Illegal SpeI site found at 4097
    Illegal PstI site found at 4124
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE 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
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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.

[edit]
Categories
//dna/transposon
//plasmid
//plasmid/chromosomalintegration
Parameters
None