Part:BBa_K1926011

The SNAP UNIT: SNAP-tag flanked by loxP

This part is the SNAP UNIT of cyclebow system including an sv40 nuclear location sequence (NLS), a covalent labeling of fusion proteins named SNAP-tag® and an sv40 terminator flanked by homodromous recognition site of recombinase CRE named loxP. The whole part can be cut off by CRE.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 419
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage

You may use this part to:

1) Localize proteins in vivo with BLOCK and dye from NEB company;

2) Dye after blocking, you are able to see whether an event have an influence on the localization and expression of the interested protein;

3) Fast cut off this part by transient transfecting recombinase gene into nucleus.

Source

The SNAP-tag® was brought from NEB company. NLS and sv40 terminator were got from commercial plasmid: pentry nls GFP/pcdna3.1.

Construction Design

The SNAP-tag® with tags and RTSs are constructed using PCR and oligonucleotide ligation by the following procedure. Our UNITs all contain XbaI and SpeI enzyme sites, so we can easily line up the UNITs using 3A assembly.

Figure 1: Three steps to construct our UNITs using PCR and oligonucleotide ligation.

Gel analysis after EcoR1 digestion

The length of our biobricks are confirmed by gel analysis after EcoR1 digestion. The biobrick BBa_R0040, which is the negative control in 2016’s interlab, was used as a control because it only have a 54 bp part.

Figure 2: AGE image of biobricks BBa_K1926001-BBa_K1926003. The samples were pre dyed with gelred