Target-+Apyrase+(Shigella+flexneri)

NOTE: Indra - we need to make a truncated version of your for the pNIC-Bsa4 (Version3 cloning) because there is a transmembrane region on the edge of the protein (see http://www.cbs.dtu.dk/services/TMHMM/) We can do this by changing the primers for Version 3 with the tails. We can still use the pUC19 clone that you have.

//Shigella flexneri// Apyrase Phosphotase 876677 3.6.1.5 1D2T
 * Organism:**
 * Target:**
 * NCBI Gene # or RefSeq#:**
 * EC#:**
 * PDB # or closest PDB entry is using homology model:**

//**Shigella flexneri**// is a species of Gram-negative bacteria in the genus //Shigell// that can cause diarrhea in humans. There are several different serogroups of //Shigella//; //S. flexneri// belongs to group //B//. //S. flexneri// infections can usually be treated with antibiotics although some strains have become resistant. Less severe cases are not usually treated because they become more resistant in the future. S. flexneri contians a virulence plasmid that codes for three virulence factors: a type-3 secretion system (T3SS), invasion plasmid antigen proteins (ipa proteins), and IcsA (used for cell-to-cell spread). Upon infection, //S. flexneri// injects the host cell cytoplasm with ipa proteins using the T3SS--a needle-and-syringe like apparatus common to many gram negative pathogens. These ipa proteins induce "membrane ruffling" by the host cell. Membrane ruffling creates membrane pockets which capture and engulf the bacteria. Once inside, S. flexneri utilizes host cell actin for propulsion to move directly from cell to cell using a cellular mechanism known as paracytophagy, similarly to the bacterial pathogen //Listeria monocytogenes//
 * Background/Disease:**

ATGAAAACCAAAAACTTCCTGCTGTTTTGCATTGCGACCAATATGATCTTCATCCCGTCTGCGAACGCCCTGAAAGCAGAAGGT TTCCTCACCCAGCAGACCTCTCCGGATTCTCTGTCTATCCTGCCGCCACCGCCAGCGGAGGATTCTGTTGTTTTCCTGGCGGAC AAAGCGCACTACGAATTTGGTCGTTCTCTCCGTGACGCGAACCGCGTTCGTCTGGCGTCTGAAGATGCGTACTACGAGAACTTC GGTCTCGCCTTCTCTGACGCGTACGGTATGGACATCTCTCGTGAAAACACCCCGATCCTGTACCAGCTGCTGACCCAGGTACTG CAGGACTCTCATGACTATGCTGTTCGCAACGCTAAAGAATACTACAAACGTGTTCGCCCGTTCGTTATCTACAAAGACGCGACCT GCACCCCGGATAAAGACGAAAAAATGGCGATCACCGGTTCTTACCCGTCCGGTCACGCGAGCTTCGGTTGGGCGGTTGCGCTG ATCCTGGCAGAAATCAACCCGCAGCGTAAAGCGGAAATCCTGCGTCGTGGTTACGAGTTCGGTGAATCTCGTGTTATCTGCGGC GCACACTGGCAGTCCGACGTTGAAGCGGGTCGTCTGATGGGTGCGTCTGTAGTTGCGGTGCTGCACAACACGCCGGAATTCAC CAAATCTCTGAGCGAAGCGAAGAAAGAATTCGAAGAACTGAACACTCCGACCAACGAACTGACCCCGTAA
 * Image of protein (PyMol or etc):**
 * Codon Optimized Sequence:**

ATGAAAACCAAAAACTTTCTTCTTTTTTGTATTGCTACAAATATGATTTTTATCCCCTCAGCAAATGCTC TGAAG GCAGAAGGTTTTCTCACTCAACAAACTTCACCAGACAGTTTGTCAATACTTCCGCCGCCTCCGGC AGAGGATTCAGTAGTATTTCTGGCTGACAAAGCTCATTATGAATTCGGCCGCTCGCTCCGGGATGCTAAT CGTGTACGTCTCGCTAGCGAAGATGCATACTACGAGAATTTTGGTCTTGCATTTTCAGATGCTTATGGCA TGGATATTTCAAGGGAAAATACCCCAATCTTATATCAGTTGTTAACACAAGTACTACAGGATAGCCATGA TTACGCCGTGCGTAACGCCAAAGAATATTATAAAAGAGTTCGTCCATTCGTTATTTATAAAGACGCAACC TGTACACCTGATAAAGATGAGAAAATGGCTATCACTGGCTCTTATCCCTCTGGTCATGCATCCTTTGGTT GGGCAGTAGCACTGATACTTGCGGAGATTAATCCTCAACGTAAAGCGGAAATACTTCGACGTGGATATGA GTTTGGAGAAAGTCGGGTCATCTGCGGTGCGCATTGGCAAAGCGATGTAGAGGCTGGGCGTTTAATGGGA GCATCGGTTGTTGCAGTACTTCATAATACACCTGAATTTACCAAAAGCCTTAGCGAAGCCAAAAAAGAGTTTGAAGAATTAAATACTCCTACCAATGAACTGACCCCATAA
 * CDS Gene Sequence Non-Codon Optimized:**

Updated CDS Gene Sequence after being Cut with only 681 bp Non-Codon Optimized:
TACTTCCAATCCATG GCAGAAGGTTTTCTCACTCAACAAACTTCACCAGACAGTTTGTCAATACTTCCGCCGCCTCCGGCAGA GGATTCAGTAGTATTTCTGGCTGACAAAGCTCATTATGAATTCGGCCGCTCGCTCCGGGATGCTAATCGTGTACGTCTCGCTA GCGAAGATGCATACTACGAGAATTTTGGTCTTGCATTTTCAGATGCTTATGGCATGGATATTTCAAGGGAAAATACCCCAATCT TATATCAGTTGTTAACACAAGTACTACAGGATAGCCATGATTACGCCGTGCGTAACGCCAAAGAATATTATAAAAGAGTTCGTC CATTCGTTATTTATAAAGACGCAACCTGTACACCTGATAAAGATGAGAAAATGGCTATCACTGGCTCTTATCCCTCTGGTCATGC ATCCTTTGGTTGGGCAGTAGCACTGATACTTGCGGAGATTAATCCTCAACGTAAAGCGGAAATACTTCGACGTGGATATGAGTT TGGAGAAAGTCGGGTCATCTGCGGTGCGCATTGGCAAAGCGATGTAGAGGCTGGGCGTTTAATGGGAGCATCGGTTGTTGCA GTACTTCATAATACACCTGAATTTACCAAAAGCCTTAGCGAAGCCAAAAAAGAGTTTGAAGAATTAAATACTCCTACCAATGAACTGACCCCATAA

MKTKNFLLFCIATNMIFIPSANALKAEGFLTQQTSPDSLSILPPPPAEDSVVFLADKAHY EFGRSLRDANRVRLASEDAYYENFGLAFSDAYGMDISRENTPILYQLLTQVLQDSHDYAV RNAKEYYKRVRPFVIYKDATCTPDKDEKMAITGSYPSGHASFGWAVALILAEINPQRKAE ILRRGYEFGESRVICGAHWQSDVEAGRLMGASVVAVLHNTPEFTKSLSEAKKEFEELNTP TNELTP*
 * Codon Optimized Amino Acid Sequence:**

>gi|13448943|ref|NP_085159.1| apyrase [Shigella flexneri 5a] MKTKNFLLFCIATNMIFIPSANALKAEGFLTQQTSPDSLSILPPPPAEDSVVFLADKAHYEFGRSLRDAN RVRLASEDAYYENFGLAFSDAYGMDISRENTPILYQLLTQVLQDSHDYAVRNAKEYYKRVRPFVIYKDAT CTPDKDEKMAITGSYPSGHASFGWAVALILAEINPQRKAEILRRGYEFGESRVICGAHWQSDVEAGRLMG ASVVAVLHNTPEFTKSLSEAKKEFEELNTPTNELTP ~52% homology to a Escherichia blattae acid phosphatase 1D2T on PDB
 * Amino Acid Sequence:**

Updated Amino Acid Sequence after being Cut:
MYFQSMAEGFLTQQTSPDSLSILPPPPAEDSVVFLADKAHYEFGRSLRDANRVRLASEDAY YENFGLAFSDAYGMDISRENTPILYQLLTQVLQDSHDYAVRNAKEYYKRVRPFVIYKDAT CTPDKDEKMAITGSYPSGHASFGWAVALILAEINPQRKAEILRRGYEFGESRVICGAHWQ SDVEAGRLMGASVVAVLHNTPEFTKSLSEAKKEFEELNTPTNELTP*

//Forward Primer:// 5' TACTTCCAATCCATG AAAACCAAAAACTTCCT 3' 32 bp GC Content: 34.4% 0mM Mg: 59.2 C 1.5mM Mg: 67.2 C 2mM Mg: 67.8 C 4mM Mg: 68.9 C 6mM Mg: 69.5 C
 * Primer design results for pNIC-Bsa4 cloning:**

Reverse Primer: 5' TATCCACCTTTACTG '3 35 bp GC Content: 48.6% 0mM Mg: 64.8 C 1.5mM Mg: 71.8 C 2mM Mg: 72.2 C 4mM Mg: 73.1 C 6mM Mg: 73.5 C

Actual Primers ordered in July:

 * ||  ||   ||   || front of gene |||||||| ATGAAAACCAAAAACTTCCTGCTG ||   ||   ||   ||   || GC% || Tm ||
 * Upstream ||  |||| remove an ATG || together |||||||||| TACTTCCAATCCATGAAAACCAAAAACTTCCTG || Length || 33 || sfAPY_for || 36.4 || 59.3 ||
 * |||||| TACTTCCAATCCATG || For Primer ||||||||||  || 32 ||   ||   ||   ||   ||
 * Downstream: ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * |||||| TATCCACCTTTACTG || end of gene |||||||| ACCAACGAACTGACCCCGTAA ||  ||   ||   ||   ||   ||   ||
 * ||  ||   ||   || rev comp |||||||| TTACGGGGTCAGTTCGTTGGT ||   ||   ||   ||   ||   ||   ||
 * ||  ||   ||   || together |||||||||| TATCCACCTTTACTGTTACGGGGTCAGTTCGTT || Length || 33 || sfAPY_rev || 45.5 || 63 ||
 * ||  ||   ||   || Rev Primer ||||||||||   || 35 ||   ||   ||   ||   ||

Updated Primers ordered in September:

 * ||  ||   ||   || front of gene |||||||| GCAGAAGGTTTTCTCACTCAACAA ||   ||   ||   ||   || GC% || Tm ||
 * Upstream ||  |||| remove an ATG || together |||||||||| TACTTCCAATCCATGGCAGAAGGTTTTCTCACT || Length || 33 || sfAPY_for2 || 36.4 || 59.3 ||
 * |||||| TACTTCCAATCCATG || For Primer ||||||||||  || 32 ||   ||   ||   ||   ||
 * Downstream: ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * |||||| TATCCACCTTTACTG || end of gene |||||||| ACCAACGAACTGACCCCGTAA ||  ||   ||   ||   ||   ||   ||
 * ||  ||   ||   || rev comp |||||||| TTACGGGGTCAGTTCGTTGGT ||   ||   ||   ||   ||   ||   ||
 * ||  ||   ||   || together |||||||||| TATCCACCTTTACTGTTACGGGGTCAGTTCGTT || Length || 33 || sfAPY_rev2 || 45.5 || 63 ||
 * ||  ||   ||   || Rev Primer ||||||||||   || 35 ||   ||   ||   ||   ||

Virtual Screening:
RMS = 4.806 (206 to 206 atoms)