DNA

Part:BBa_K2605002

Designed by: Kaitlin Schaaf   Group: iGEM18_Calgary   (2018-10-09)


Multiple cloning site with flanking FRT sites

Multiple cloning site (MCS) flanked by FRT sites. FRT2 is upstream of the MCS and FRT5 is downstream of the MCS. The entire MCS is flanked by SapI restriction sites so that the entire cloning site and FRT sites can be transferred to different plasmids. AflII restriction sites flank the MCS interior of the FRT sites so that only the MCS can be transferred to different plasmids.


Usage and Biology

This part was deisgned as the first iteration of a novel toolkit for targeted gene integration and maintenance of expression in eukaryotic chassis. The collection contains chromatin-modifying elements that limit silencing of the integrated gene and minimize neighborhood effects. These elements, as well as the mCherry-BGH reporter gene with our improved CMV promoter, have restriction sites for directional cloning into the MCS. The FRT sites are leveraged for recombinase-mediated casette exchange (RMCE), more of which can be read on iGEM Calgary 2018 wiki and parts page.

Below is a schematic of how this MCS can be used with chromatin-modifying elements and a reporter gene. The restriction sites for bi-directional cloning of each part is shown. Parts BBa_K2605004 (insulator), BBa_K2605009 (CMV + mCherry reporter), and BBa_K2605003(UCOE) are represented in this image.

Characterization

The MCS (pSB1c3-BBa_K2605002) was digested with each restriction enzyme that contains a site within the part. SapI and AflII alone were used because these are flanking the MCS, therefore a single digest produces an insert band. The remaining enzymes (FseI, MfeI, BmtI, BamHI, NsiI, and SalI) were used in a double digest with NcoI, which contains a single site within the pSB1C3 backbone. Therefore, those double digests also produced an insert band. As shown in figure 1 below, each digest produced a pSB1c3 backbone band and an insert band, indicating that each restriction site within the MCS is functional.

Figure 1. Digest confirmations of pSB1C3-BBa_K2605002. The plasmid was single digested with SapI or AflII and double digested with NcoI and FseI/MfeI/BmtI/BamHI/NsiI/SalI then run on a 1.5% agarose gel at 100 V for 35 minutes. The molecular ladder (L) is visible on the far left and the expected band sizes, obtained from Benchling Virtual Digest, are visible in the diagram below. Undigested plasmid (U) was used as a control.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 191
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 207
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 125
  • 1000
    COMPATIBLE WITH RFC[1000]


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