Simulated restriction enzyme digestion with the following enzymes:
Pattern created in the style of Paul Vanouse’s Latent Figure Protocol artworks:
EcoRI, HindIII, BamHI, KpnI, EcoRV, SacI, SalI
I have chosen STX18. STX18 regulates ER structure by mediating homotypic membrane fusion, maintaining ER tubular networks and dynamic clusters. Its degradation by the intramembrane protease SPP directly alters ER morphology, demonstrating its critical role in organelle architecture.
MAVDITLLFRASVKTVKTRNKALGVAVGGGVDGSRDELFRRSPRPKGDFSSRAREVISHI
GKLRDFLLEHRKDYINAYSHTMSEYGRMTDTERDQIDQDAQIFMRTCSEAIQQLRTEAHK
EIHSQQVKEHRTAVLDFIEDYLKRVCKLYSEQRAIRVKRVVDKKRLSKLEPEPNTKTRES
TSSEKVSQSPSKDSEENPATEERPEKILAETQPELGTWGDGKGEDELSPEEIQMFEQENQ
RLIGEMNSLFDEVRQIEGRVVEISRLQEIFTEKVLQQEAEIDSIHQLVVGATENIKEGNE
DIREAIKNNAGFRVWILFFLVMCSFSLLFLDWYDS
atggcggtggatattaccctgctgtttcgcgcgagcgtgaaaaccgtgaaaacccgcaac aaagcgctgggcgtggcggtgggcggcggcgtggatggcagccgcgatgaactgtttcgccgcagcccgcgcccgaaaggcgattttagcagccgcgcgcgcgaagtgattagccatattggcaaactgcgcgattttctgctggaacatcgcaaagattatattaacgcgtatagccat accatgagcgaatatggccgcatgaccgataccgaacgcgatcagattgatcaggatgcgcagatttttatgcgcacctgcagcgaagcgattcagcagctgcgcaccgaagcgcataaagaaattcatagccagcaggtgaaagaacatcgcaccgcggtgctggattttattgaagat tatctgaaacgcgtgtgcaaactgtatagcgaacagcgcgcgattcgcgtgaaacgcgtg gtggataaaaaacgcctgagcaaactggaaccggaaccgaacaccaaaacccgcgaaagcaccagcagcgaaaaagtgagccagagcccgagcaaagatagcgaagaaaacccggcgaccgaagaacgcccggaaaaaattctggcggaaacccagccggaactgggcacctggggcgatggcaaaggcgaagatgaactgagcccggaagaaattcagatgtttgaacaggaaaaccagcgcctgattggcgaaatgaacagcctgtttgatgaagtgcgccagattgaaggccgcgtggtggaaattagccgcctgcaggaaatttttaccgaaaaagtgctgcagcaggaagcggaaattgatagcattcatcagctggtggtgggcgcgaccgaaaacattaaagaaggcaacgaagatattcgcgaagcgattaaaaacaacgcgggctttcgcgtgtggattctgttttttctggtgatgtgcagctttagcctgctgtttctggattggtatgatagc
Codon optimization is important as it enhances protien expression levels, improves mRNA stability and Structure, ensures proper protien folding, avoids undesired motifs. Optimizing the codon leads to high yield outcomes. I have chosen to optimize STX18 to avoid problematic motifs such as removing restriction enzyme sites, ribosomal binding site blockers or mRNA instability elements.
Optimized from Twist BioScience: ATGGCCGTGGACATCACCCTGCTGTTCAGGGCCAGCGTGAAGACCGTGAAGACCAGGAACAAGGCCCTGGGCGTGGCCGTGGGCGGCGGCGTGGATGGCAGCAGGGATGAGCTGTTCAGGAGGAGCCCCAGGCCCAAGGGCGACTTCAGCAGCAGGGCCAGGGAGGTGATCAGCCACATCGGCAAGCTGAGGGACTTCCTGCTGGAGCACAGGAAGGACTACATCAACGCCTACAGCCACACCATGAGCGAGTACGGCAGGATGACCGACACCGAGAGGGACCAGATCGACCAGGACGCCCAGATCTTCATGAGGACCTGCAGCGAGGCCATCCAGCAGCTGAGGACCGAGGCCCACAAGGAGATCCACAGCCAGCAGGTGAAGGAGCACAGGACCGCCGTGCTGGACTTCATCGAGGACTACCTGAAGAGGGTGTGCAAGCTGTACAGCGAGCAGAGGGCCATCAGGGTGAAGAGGGTGGTGGACAAGAAGAGGCTGAGCAAGCTGGAGCCCGAGCCCAACACCAAGACCAGGGAGAGCACCAGCAGCGAGAAGGTGAGCCAGAGCCCCAGCAAGGACAGCGAGGAGAACCCCGCCACCGAGGAGAGGCCCGAGAAGATCCTGGCCGAGACCCAGCCCGAGCTGGGCACCTGGGGCGACGGCAAGGGCGAGGACGAGCTGAGCCCCGAGGAGATCCAGATGTTCGAGCAGGAGAACCAGAGGCTGATCGGCGAGATGAACAGCCTGTTCGACGAGGTGAGGCAGATCGAGGGCAGGGTGGTGGAGATCAGCAGGCTGCAGGAGATCTTCACCGAGAAGGTGCTGCAGCAGGAGGCCGAGATCGACAGCATTCACCAGCTGGTGGTGGGCGCCACCGAGAACATCAAGGAGGGCAACGAGGACATCAGGGAGGCCATCAAGAACAACGCCGGCTTCAGGGTGTGGATCCTGTTCTTCCTGGTGATGTGCAGCTTCAGCCTGCTGTTCCTGGACTGGTACGACAGC
What technologies could be used to produce this protein from your DNA? Describe in your words the DNA sequence can be transcribed and translated into your protein. You may describe either cell-dependent or cell-free methods, or both.
In vivo (cell-dependant)
DNA sequence design
Promoter (strong and inducible)
Codon Optimization (replace rare codons and enhance translation efficiency)
Tags ( His-tags or FLAG-tags for purification)
Terminatior (Transcription stop stop signal)
Production
Cloning
Transformation
Induction
Purification
In vitro (cell-free)
Design
T7 promoter
Ribosome-binding site
Linear DNA avoids plasmid preperation
Production
Lysate Preperation
Reaction Mix
Incubation
Purification