Part:BBa_K1352003
Ice Nucleation Protein with YFP and His-tag
K1352003 has been created from an in-fusion of the brick K523013 created by Edinburgh 2011. This part contains a bglII/HIS/HindIII construct at the final codon of the YFP which allows for the insertion of extra peptides up to 40 kDa using a BglII and HindIII prefix and suffix.
K1352003 was created in two parts using primers created for in-fusion reactions. These parts were referred to in our experiments as the long and short fragments due to the difference in size (~2200 and ~150 bp respectively).
The short sequence primers consisted of a forward primer which was complimentary to the YFP stop codon and some of the following nucleotides, preceded in the 5’ to 3’ direction by the last 15 nucleotides of the flag sequence. The reverse codon contained a complimentary sequence to the end of K523013 and 15 nucleotides complimentary to the suffix of the backbone.
The long part was created using a primer which consisted of a complimentary sequence for the end of YFP and our whole insert. The sequences used can be seen as follows.
AAGCTTGTGATGGTGATGGTGATGAGATCTcttgtacagctcgtccatgc
As this sequence is written, this part consists of a HindIII cut site immediately followed by a HIS sequence which is in turn immediately followed by a BglII cut site. These are shown in capitals. The remainder of the sequence is a long tail complimentary to the end of K523013.
Below is the forward primer designed to anneal to the prefix region of a plasmid backbone.
CGCGGCCGCTTCTAGATTAATACGACTCACTATAGGG.
This sequence contains 15 nucleotides of the plasmid prefix sequence followed by a sequence complimentary to the start of the k523013 brick.
CACCATCACAAGCTTtaataatactagcaacatatcataacggagtg
This sequence is the complimentary sequence to the HIS tag sequence in the long fragment. The first 15 nucleotides are complimentary to the final (reverse direction) nucleotides of the HIS insert. The remainder is a long primer to bind to the YFP stop codons and the following 26 nucleotides of the K523013 biobrick.
AGCGGCCGCTACTAGTTATAAACGCAGAAAGGCCC
Similar to the vector sequence in the long fragment this contains 15 nucleotides that will compliment the suffix sequence of the backbone as well as containing a sequence that is complimentary to the sequence at the end of K523013.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1036
- 1000COMPATIBLE WITH RFC[1000]
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