BBa_M36850 1 AFP HeLa Optimized Type I Antifreeze Protein 2012-12-06T12:00:00Z 2016-01-28T12:19:58Z The amino acid sequence for this protein was published in the paper "A natural variant of type I antifreeze protein with four ice-binding repeats is a particularly potent antifreeze", by Heman Chao, et al. in Protein Science, Vol 5, Issue 6, pages 1150???1156 in June 1996. We optimized the nucleotides for expression in human cells. We are designing a HeLa cell actuator that produces Type I antifreeze protein. The antifreeze protein we chose is naturally found in winter flounder serum and is a Type I AFP, known as AFP9. This amino acid sequence was published in a paper by Chao in 1996. It is very short, consisting of only 52 amino acids, in which there are three repeats of the same 9-amino acid section. The amino acid content is very Alanine-rich. Our designed part is a patchwork of components all chosen to maximize the output of our antifreeze protein. The CMV promoter, a constitutive gene that is expressed at a constant level, was used in order to encourage the maximum transcription of our target gene in the HeLa cells. The antifreeze protein sequence starts with a spacer, made up primarily of G???s and C???s to increase the guanine and cytosine content in our sequence and provide room for the ribosome to bind. This is followed by DNA2.0 Gene Designer software???s template kozak sequence, which is strong and promotes ribosome binding well. The kozak is then followed by the start codon, ATG, and the optimized antifreeze protein sequence. The protein sequence is followed by the stop codon and a terminator sequence, SV40 poly A, preset in the Gene Designer template. SV40 is used for "high level expression". The antibiotic resistance marker is the default ampicillin. The AFP has not been shown to be toxic to cells, so in order to produce as much of the protein as possible, the origin of replication strength is high. false false _848_ 4206 15026 9 Not in stock false We used the OPTIMIZER software with standard human codons at a guided random mix to generate the DNA sequence for the amino acid, then further manually modified some of the codons to eliminate repeated sequences. false Karen Gomez, Ivy Nguyen, Yoo Hsiu Yeh annotation2213642 1 TAG range2213642 1 166 168 annotation2213643 1 Kozak range2213643 1 1 3 annotation2213641 1 ATG range2213641 1 4 6 annotation2213644 1 Antifreeze Protein range2213644 1 7 165 BBa_M36850_sequence 1 accatggatacagcttctgacgctgcagcggctgccgctgcaacagctgctaccgctgcagccgccgctgccgcaaccgccgtgaccgctgccaaggcagctgctctgacggcagctaatgcggccgctgctgcggctgcaacagcggccgcagcggctagaggatag igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z