Target+-+Non-structural+protein+2B-+NS2B+(Dengue+Virus)+antibody


 * Target (protein/gene name):** Serine protease subunit NS2B

http://www.ncbi.nlm.nih.gov/protein/356624669
 * NCBI Gene # or RefSeq#:** 3U1I_C GI:356624669

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 * Protein ID (NP or XP #) or Wolbachia#:** Not found.

Viruses; ssRNA positive-strand viruses, no DNA stage; Flaviviridae; Flavivirus; Dengue virus group.
 * Organism (including strain):** Dengue Virus type 3 - Singapore/8120/1995


 * Etiologic Risk Group :**


 * Background/Disease Information :**

Dengue Fever & Dengue Hemorrhagic Fever
( Also known as :Hemorrhagic dengue; Dengue shock syndrome; Philippine hemorrhagic fever; Thai hemorrhagic fever; Singapore hemorrhagic fever ) ​ The four dengue viruses originated in monkeys and independently jumped to humans in Africa or Southeast Asia between 100 and 800 years ago. Dengue remained a relatively minor, geographically restricted disease until the middle of the 20th century. The 4 dengue viruses (DENV) are immunologically related, positive-strand RNA viruses of the genus //Flavivirus//, family Flaviviridae. These viruses cause both dengue fever (DF) and dengue hemorrhagic fever (DHF). Asymptomatic infection also occurs. Infection with 1 DENV produces lifelong immunity against reinfection with that DENV type but no long-term cross-protection against the other 3 DENV types (any cross-protection is ≤2 months). Early symptoms of dengue hemorrhagic fever are similar to those of dengue fever, but after several days the patient becomes irritable, restless, and sweaty. These symptoms are followed by a shock -like state. Bleeding may appear as tiny spots of blood on the skin (petechiae ) and larger patches of blood under the skin (ecchymoses ). Minor injuries may cause bleeding. Shock may cause death. If the patient survives, recovery begins after a one-day crisis period. No specific therapeutic agents exist for dengue virus infections. Encourage bed rest and maintenance of fluids to prevent dehydration while the patient is febrile. Control fever with acetaminophen. Headache, eye pain, joint pain, and muscle ache may require narcotics. Aspirin, aspirin-containing drugs, and other nonsteroidal anti-inflammatory drugs (such as ibuprofen) should be avoided because of their anticoagulant properties. Aspirin and other salicylates should be especially avoided in children because of the association with Reye syndrome.

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 * Essentiality of this protein:** NS2B is a subunit in the serine protease enzyme that is essential for the DENV virus functioning.


 * Complex of proteins?:** Forms a complex with the other subunit NS3p to form the NS2B(H)-NS3p complex.


 * Druggable Target:**

[] [|http://brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.91&Suchword=&organism[=Dengue+virus+3&show_tm=0]] [|http://www.brenda-enzymes.org/index.php4?page=taxonomy2/ncbi/tree/index.php4?f[id[value]=11069]]
 * *EC#: **3.4.21.91 ( Specific for Dengue Virus 3)
 * Link to BRENDA EC# page:**


 * --** Show screenshot of BRENDA enzyme mechanism schematic

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 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**

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 * -- link to Sigma (or other company) page for assay or assay reagents (substrates):**
 * -- paper that contains assay information:**
 * -- List cost and quantity of substrate reagents and supplier**
 * 52,00 EUR* **
 * Boc-Gly-Lys-Arg-AMC**

**Boc-GKR-AMC |Boc-Gly-Lys-Arg-MCA**


 * 3143-v 5 mg | 52.00 EUR**

t-Butyloxycarbonyl- glycyl- L-lysyl- L-arginine- 4-methylcoumaryl- 7-amide Synthetic Product A trace of impurity might be detectable by TLC when 100 µg is applied to the plate. The amount of contamination detectable by HPLC is less than 2 %.
 * **(M.W. 616.72)** || **C29H44N8O7** || **[109358-48-7]** ||

-- PDB # or closest PDB entry if using homology model: 3U1I_C Show pairwise alignment of your BLASTP search in NCBI against the PDB
 * Structure Available (PDB or Homology model)**

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 * Query sequence || >tr|Q8B5K4|Q8B5K4_9FLAV Nonstructural gene NS2B (Fragment) OS=Dengue virus 3 PE=4 SV=1 GVMAVGLVSILASSLLRNDVPMAGPLVAGGLLIACYVITGTSADLTVEKAADVAWEEEAE QTGVSHNLMITVDDDGTMRMKDDETENILTVLLRTALLIVSGIFPYSIPATLLVWHTWQK QT ||
 * Date of job execution || May 2, 2013 ||
 * Running time || 23.4 seconds ||
 * Program || blastp (BLASTP 2.2.28+) ||
 * Database || uniprotkb (Protein) generated for BLAST on Apr 30, 2013 ||
 * Sequences || 34,569,655 sequences consisting of 11,137,001,367 letters ||
 * Matrix || blosum62 ||
 * Threshold || 10 ||
 * Filtered || false ||
 * Gapped || true ||
 * Maximum number of hits reported || 250 ||

phthalazine-based compound, 7 ; pthalazine-based compound, 2; quanidine 5; quanidine 4; Bz-NKRR-H
 * Current Inhibitors:**

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 * Expression Information (has it been expressed in bacterial cells): Yes, in E-Coli**
 * Purification Method:**


 * Image of protein (PyMol with features delineated and shown separately):**


 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**

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 * Considered sequence fragment (NS2B) : (1344 to 1473)**

1321 1331 1341 1351 1361 1371 | | | | | |  1321 SWPLNEG VMAVGLVSIL ASSLLRNDVP MAGPLVAGGL 1380 1381 LIACYVITGT SADLTVEKAA DVTWEEEAEQ TGVSHNLMIT VDDDGTMRIK DDETENILTV 1440 1441 LLKTALLIVS GIFPYSIPAT LLVWHTWQKQ TQR


 * length of your protein in Amino Acids:**130 amino acids (from 1344 to 1473)
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam]website:** 14065.2

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 * Molar Extinction coefficient of your protein at 280 nm wavelength:**
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. Didn't work..

[] []  1 mnnqrkktgk psinmlkrvr nrvstgsqla krfsrgllng qgpmklvmaf iaflrflaip 61 ptagvlarwg tfkksgaikv lkgfkkeisn mlsiinkrkk tslclmmmlp atlafhltsr 121 dgeprmivgk nergksllfk tasginmctl iamdlgemcd dtvtykcpli aevepedidc 181 wcnltstwvt ygtcnqageh rrdkrsvala phvgmgldtr tqtwmsaega wrqvekvetw 241 alrhpgftil alflahyigt sltqkvvifi llmlvtpsmt mrcvgvgnrd fveglsgatw 301 vdvvlehggc vttmaknkpt ldielqktea tqlatlrklc iegkitnitt dsrcptqgea 361 ilpeeqdqny vckhtyvdrg wgngcglfgk gslvtcakfq clepiegkvv qyenlkytvi 421 itvhtgdqhq vgndtqgvtv eitpqastve avlpeygtlg lecsprtgld fnemilltmk 481 nkawmvhrqw fldlplpwts gatterptwn rkellvtfkn ahakkqevvv lgsqegamht 541 altgateiqn sggtsifagh lkcrlkmdkl elkgmsyamc lntfvlkkev setqhgtili 601 kveykgedap ckipfstedg qgkahngrli tanpvvtkke epvnieaepp fgesnivigi 661 gdkalkinwy kkgssigkmf eatargarrm ailgdtawdf gsvggvlnsl gkmvhqifgs 721 aytalfsgvs wimkigigvl ltwiglnskn tsmsfscivi giitlylgav vqadmgcvin 781 wkgkelkcgs gifvtnevht wteqykfqad spkrlataia gawengvcgi rsttrmenll 841 wkqianelny ilwenniklt vvvgdtigvl eqgkrtltpq pmelkyswkt wgkakivtae 901 tqnssfiidg pntpecpsas rawnvweved ygfgvfstni wlklrevytq scdhrlmsaa 961 ikderavhad mgywiesqkn gswklekasf ievktciwpk shtlwsngvl esdmiipksl 1021 agpisqhnhr pgyhtqtagp whlgkleldf nycegttvvi tencgtrgps lrtttvsgkl 1081 ihewccrsct lpplrymged gcwygmeirp inekeenmvk slvsagsgkv dnftmgvlcl 1141 ailfeevmrg kfgkkhmiag vlftfvllls gqitwrdmah tlimigsnas drmgmgvtyl 1201 aliatfkiqp flalgfflrk ltsrenlllg vglamattlq lpedieqman gialglmtlk 1261 litqfetyql wtalvsltcs ntiftltvaw rtatlilagv sllplcqsss mrktdwlpmt 1321 vaamgvpplp lfifslkdtp krrswplneg vmavglvsil assllrndvp magplvaggl 1381 liacyvitgt sadltvekaa dvtweeeaeq tgvshnlmit vdddgtmrik ddeteniltv 1441 llktallivs gifpysipat llvwhtwqkq tqrsgvlwdv psppetqkae leegvyrikq 1501 qgifgktqvg vgvqkegvfh tmwhvtrgav lthngkrlep nwasvkkdli sygggwrlsa 1561 qwqkgeevqv iavepgknpk nfqtmpgifq tttgeigaia ldfkpgtsgs piinregkvv 1621 glygngvvtk nggyvsgiaq tnaepdgptp eleeemfkkr nltimdlhpg sgktrkylpa 1681 ivreaikrrl rtlilaptrv vaaemeealk glpiryqtta tksehtgrei vdlmchatft 1741 mrllspvrvp nynliimdea hftdpasiaa rgyistrvgm geaaaifmta tppgtaeafp 1801 qsnapiqdee rdiperswns gnewitdfvg ktvwfvpsik agndianclr kngkkviqls 1861 rktfdteyqk trlndwdfvv ttdisemgan fkadrvidpr rclkpviltd gpervilagp 1921 mpvtaasaaq rrgrvgrnpq kendqyiftg qplnndedha hwteakmlld nintpegiip 1981 alfepereks aaidgeyrlk gesrktfvel mrrgdlpvwl ahkvasegik ytdrkwcfdg 2041 ernnqileen mdveiwtkeg erkklrprwl dartysdpla lkefkdfaag rksialdlvt 2101 eigrvpshla hrtrnaldnl vmlhtsehgg rayrhaveel petmetllll glmilltgga 2161 mlflisgkgi gktsiglicv ivssgmlwma dvplqwiasa ivleffmmvl lipepekqrt 2221 pqdnqlayvv igiltlaaiv aanemgllet tkrdlgmske pgvvsptsyl dvdlhpasaw 2281 tlyavattvi tpmlrhtien stanvslaai anqavvlmgl dkgwpiskmd lgvpllalgc 2341 ysqvnpltlt aavlllvthy aiigpglqak atreaqkrta agimknptvd gimtidldpv 2401 iydskfekql gqvmllvlca vqlllmrtsw alcealtlat gpittlwegs pgkfwnttia 2461 vsmanifrgs ylagaglafs imksvgtgrr gtgsqgetlg ekwkkklnql srkefdlykk 2521 sgitevdrte akeglkrget thhavsrgsa klqwfvernm vipegrvidl gcgrggwsyy 2581 caglkkvtev rgytkggpgh eepvpmstyg wnivklmsgk dvfylppekc dtllcdiges 2641 spsptveesr tirvlkmvep wlknnqfcik vlnpymptvi ehlerlqrky ggmlvrnpls 2701 rnsthemywi sngtgnivss vnmvsrllln rftmthrrpt iekdvdlgag trhvnaepet 2761 pnmdvigeri krikeehsst whyddenpyk twayhgsyev katgsassmi ngvvklltkp 2821 wdvvpmvtqm amtdttpfgq qrvfkekvdt rtprpmpgtr kvmeitaewl wrtlgrnkkp 2881 rlctreeftk kvrtnaamga vfteenqwds araavedeef wklvdrerel hklgkcgscv 2941 ynmmgkrekk lgefgkakgs raiwymwlga rylefealgf lnedhwfsre nsysgvegeg 3001 lhklgyilrd iskipggamy addtagwdtr iteddlhnee kitqqmdpeh kqlanaifkl 3061 tyqnkvvkvq rptpkgtvmd iisrkdqrgs gqvgtyglnt ftnmeaqlvr qmegegvlsk 3121 adlenphlle kkitqwletk gverlkrmai sgddcvvkpi ddrfanalla lndmgkvrkd 3181 ipqwqpskgw hdwqqvpfcs hhfhelimkd grklvvpcrp qdeligrari sqgagwslke 3241 taclgkayaq mwslmyfhrr dlrlasnaic savpvhwvpt srttwsihah hqwmttedml 3301 tvwnrvwied npwmedktpv ttwedvpylg kredqwcgsl igltsratwa qniltaiqqv 3361 rsligdeefl dympsmkrfr keeesegaiw
 * CDS Gene Sequence (paste as text only):**

ATG AAC AAT CAA AGA AAA AAG ACA GGA AAG CCC TCA ATC AAT ATG CTC AAA AGA GTC AGG AAC AGG GTC AGC ACA GGA AGT CAA CTC GCT AAA AGA TTC TCT AAA GGA CTG TTG AAC GGA CAG GGA CCA ATG AAG TTG GTG ATG GCA TTC ATC GCC TTT CTC AGA TTC CTC GCA ATA CCA CCT ACA GCA GGG GTC TTA GCA AGA TGG GGA ACA TTT AAA AAG AGT GGA GCT ATA AAG GTC CTA AAA GGG TTT AAG AAA GAA ATA TCA AAT ATG CTG TCC ATA ATT AAT CAG AGA AAA AAG ACT AGT CTG TGT CTC ATG ATG ATA CTG CCA GCT GCA CTG GCT TTC CAC CTC ACC TCA AGA GAC GGA GAG CCA AGA ATG ATC GTG GGA AAA AAC GAG AGA GGA AAG TCC CTT CTA TTT AAG ACC GCC TCA GGA ATA AAC ATG TGT ACA CTC ATC GCA ATG GAT CTT GGA GAA ATG TGC GAT GAC ACA GTT ACA TAT AAG TGT CCA CAT ATC ACA GAG GTT GAA CCA GAG GAT ATC GAC TGT TGG TGT AAT CTC ACT TCA ACT TGG GTG ACT TAC GGA ACA TGC AAC CAG GCA GGG GAG CAC AGA AGG GAC AAA AGG AGC GTG GCT CTG GCA CCA CAC GTG GGC ATG GGA CTC GAT ACC AGG ACA CAG ACA TGG ATG TCT GCA GAG GGA GCT TGG AGA CAA GTT GAA AAG GTT GAA ACA TGG GCC TTG AGA CAT CCA GGA TTT ACC ATT TTG GCC CTT TTT CTC GCA CAT TAT ATA GGA ACA TCA CTT ACT CAA AAG GTG GTT ATC TTT ATA CTC TTA ATG TTG GTG ACA CCA TCA ATG ACA ATG AGG TGC GTT GGC GTG GGA AAC AGA GAC TTC GTG GAG GGA CTT AGC GGA GCA ACC TGG GTG GAT GTT GTC TTG GAA CAT GGC GGA TGC GTC ACA ACT ATG GCA AAA AAC AAA CCA ACC CTG GAT ATA GAA CTG CAA AAA ACT GAA GCA ACT CAG TTG GCT ACA CTA AGA AAA CTA TGC ATA GAA GGC AAA ATC ACT AAT ATT ACA ACC GAC TCC AGG TGT CCA ACC CAA GGA GAG GCC GTG TTA CCA GAG GAA CAG GAT CAA AAT TAT GTG TGT AAA CAC ACT TAT GTG GAT AGA GGC TGG GGA AAT GGA TGC GGG CTC TTC GGG AAG GGC TCT CTC GTC ACA TGC GCA AAG TTC CAA TGT TTG GAA CCA ATA GAA GGA AAG GTC GTG CAA TAC GAG AAC CTT AAA TAT ACA GTT ATA ATT ACA GTG CAC ACA GGA GAT CAA CAC CAG GTG GGC AAT GAG ACA CAA GGA GTG ACA GCC GAG ATA ACA CCC CAG GCT TCA ACA ACC GAA GCA ATA TTG CCC GAA TAC GGA ACT CTG GGA TTG GAG TGC AGC CCC AGA ACT GGA TTG GAT TTT AAT GAA ATG ATA TTG CTG ACA ATG AAA AAC AAG GCC TGG ATG GTC CAC AGA CAA TGG TTT TTC GAC CTC CCA CTG CCA TGG GCA AGC GGG GCT ACA ACT GAG ACC CCA ACT TGG AAT AGG AAA GAA CTC CTT GTT ACA TTC AAA AAT GCC CAC GCA AAA AAG CAA GAA GTT GTC GTG CTT GGA AGC CAG GAA GGA GCT ATG CAC ACA GCT CTC ACA GGG GCT ACA GAG ATA CAA AAC TCA GGA GGC ACT TCC ATT TTT GCA GGC CAC TTG AAG TGC AGA TTG AAA ATG GAT AAA CTG GAA TTA AAG GGA ATG TCA TAC GCC ATG TGT ACA AAT ACA TTC GTG CTG AAG AAA GAG GTT AGC GAA ACA CAG CAT GGA ACC ATC TTA ATT AAG GTG GAA TAT AAA GGA GAA GAC GCT CCT TGT AAA ATT CCA TTT TCC ACT GAA GAC GGA CAA GGA AAA GCA CAT AAC GGC AGA CTC ATC ACC GCA AAT CCC GTG GTT ACA AAA AAG GAA GAG CCT GTG AAC ATC GAA GCC GAG CCC CCA TTC GGG GAG AGC AAT ATC GTG ATC GGA ATA GGA GAC AAT GCA CTA AAA ATT AAT TGG TAT AAA AAG GGA TCA AGC ATC GGA AAG ATG TTC GAG GCT ACT GAA AGA GGA GCC AGA AGG ATG GCC ATT TTG GGA GAT ACA GCA TGG GAC TTC GGG TCC GTG GGA GGC GTC TTG AAT TCA CTG GGA AAA ATG GTG CAC CAA ATA TTT GGA TCA GCA TAT ACT GCA TTG TTC TCA GGA GTC TCA TGG GTG ATG AAG ATA GGA ATA GGA GTC CTC TTG ACA TGG ATT GGA CTG AAC TCA AAA AAT ACA AGT ATG AGT TTC TCC TGT ATA GCT ATT GGA ATA ATC ACC TTG TAC CTG GGA GCT GTT GTG CAG GCA GAC ATG GGA TGC GTG ATT AAC TGG AAA GGC AAA GAA CTG AAG TGC GGA TCT GGC ATC TTT GTC ACA AAC GAG GTC CAT ACA TGG ACA GAA CAG TAC AAG TTT CAA GCA GAT TCA CCA AAA AGA CTC GCT ACT GCT ATT GCT GGA GCA TGG GAG AAC GGA GTC TGT GGA ATA AGA AGC ACC ACT AGA ATG GAA AAT TTG TTA TGG AAG CAA ATA GCA AAT GAA CTG AAT TAT ATA TTG TGG GAA AAT AAC ATA AAG TTA ACA GTT GTG GTT GGA GAC ACA CTA GGA GTC CTA GAA CAA GGA AAA AGG ACA CTG ACA CCT CAA CCA ATG GAG TTA AAA TAC TCT TGG AAA ACA TGG GGC AAG GCT AAG ATA GTG ACC GCA GAG ACA CAG AAT TCA AGC TTC ATT ATA GAC GGC CCA AAC ACT CCA GAA TGT CCA TCA GCC TCA AGA GCT TGG AAT GTC TGG GAA GTC GAA GAT TAC GGG TTC GGG GTC TTT ACC ACA AAT ATC TGG CTG AAG CTG AGA GAA GTG TAC ACA CAG TTG TGC GAC CAT AGA TTA ATG TCC GCA GCT GTT AAG GAC GAG AGA GCA GTT CAC GCC GAC ATG GGA TAT TGG ATT GAA TCA CAA AAA AAC GGC TCT TGG AAA CTA GAG AAA GCA TCA CTC ATC GAG GTG AAA ACT TGC ACA TGG CCA AAA TCA CAC ACA TTG TGG ACA AAT GGA GTG TTA GAG TCA GAT ATG ATA ATT CCA AAG TCC CTG GCT GGG CCA ATT AGT CAA CAT AAC TAT AGA CCA GGA TAT CAC ACA CAG ACA GCT GGG CCA TGG CAC TTG GGA AAA CTT GAG TTG GAC TTT AAT TAT TGT GAA GGG ACC ACT GTG GTT ATT ACA GAG AGC TGC GGA ACA AGA GGA CCC TCC CTA AGA ACC ACA ACT GTT TCT GGG AAA TTA ATA CAC GAA TGG TGT TGC AGA TCA TGT ACA TTG CCC CCA TTG AGG TAT ATG GGG GAG GAT GGA TGT TGG TAT GGA ATG GAG ATA AGA CCA ATA TCT GAA AAG GAG GAA AAC ATG GTT AAA TCC CTT GTG AGC GCT GGA TCA GGG AAA GTT GAC AAC TTC ACA ATG GGG GTG TTA TGC TTG GCT ATT CTT TTT GAG GAA GTG CTA AGA GGA AAG TTC GGA AAA AAG CAT ATG ATT GCC GGA GTG TTC TTT ACA TTT GTC CTA TTA CTA TCA GGG CAA ATA ACA TGG AGA GAC ATG GCA CAT ACA CTA ATA ATG ATT GGA TCA AAC GCC TCA GAC AGA ATG GGG ATG GGG GTT ACA TAC CTG GCA CTG ATT GCA ACA TTT AAA ATT CAA CCA TTC TTG GCC TTA GGG TTC TTT CTC AGA AAA TTG ACA TCA AGG GAA AAC CTA TTA CTG GGA GTC GGC CTG GCA ATG GCT ACA ACC CTA CAA CTC CCA GAA GAC ATC GAA CAG ATG GCC AAC GGG GTG GCA CTT GGC CTG ATG GCT CTT AAA CTT ATA ACA CAA TTC GAA ACA TAT CAG CTT TGG ACA GCT CTG GTT TCA CTT ACA TGT AGT AAT ACA ATC TTC ACA CTT ACA GTG GCT TGG AGA ACA GCC ACA TTG ATT CTA GCA GGA GTT AGC CTT TTG CCC GTC TGC CAA TCA TCT AGC ATG AGA AAG ACT GAT TGG TTG CCT ATG ACA GTG GCT GCC ATG GGA GTG CCT CCA CTG CCT CTT TTT ATT TTC TCA TTA AAA GAC ACA TTG AAA AGA AGG TCA TGG CCA TTG AAC GAG GGG GTT ATG GCA GTC GGA CTC GTG AGT ATC CTA GCA TCA TCT TTA CTT AGA AAT GAC GTC CCA ATG GCA GGA CCA TTG GTG GCC GGG GGA CTG CTT ATT GCC TGC TAC GTC ATA ACA GGA ACT TCA GCT GAT CTG ACT GTT GAA AAG GCA CCA GAT GTG ACT TGG GAA GAG GAA GCC GAG CAA ACA GGA GTC AGC CAT AAC CTA ATG ATA ACT GTT GAC GAT GAC GGC ACA ATG AGA ATA AAG GAT GAC GAA ACA GAA AAC ATA CTG ACA GTG TTG CTG AAA ACA GCA TTG TTA ATC GTT TCT GGC ATA TTC CCC TAT AGC ATT CCC GCC ACC TTG TTA GTT TGG CAC ACA TGG CAA AAA CAA ACC CAA AGA TCT GGA GTG CTG TGG GAC GTG CCT TCA CCT CCC GAA ACA CAG AAA GCT GAG CTA GAA GAG GGA GTC TAC AGA ATC AAA CAA CAG GGG ATA TTT GGG AAA ACA CAA GTC GGC GTG GGA GTG CAA AAG GAA GGC GTG TTT CAC ACA ATG TGG CAC GTC ACA AGA GGG GCT GTC TTG ACA CAT AAC GGA AAG AGA CTG GAG CCT AAT TGG GCT AGT GTC AAG AAA GAT CTC ATC AGC TAT GGA GGC GGG TGG AGG TTG TCA GCA CAG TGG CAG AAG GGA GAG GAA GTG CAA GTG ATA GCC GTG GAA CCC GGC AAA AAT CCA AAA AAC TTT CAA ACC ACA CCC GGG ACC TTC CAA ACA ACT ACA GGA GAA ATT GGG GCA ATA GCC TTA GAC TTC AAA CCA GGG ACT TCT GGA TCA CCC ATT ATC AAT AGG GAG GGA AAG GTG GTT GGA CTG TAC GGC AAC GGA GTG GTT ACA AAG AAC GGA GGG TAC GTG AGT GGA ATT GCA CAG ACA AAT GCT GAA CCC GAT GGA CCA ACA CCA GAA TTG GAG GAA GAG ATG TTT AAA AAG AGA AAT TTG ACA ATA ATG GAT CTT CAC CCC GGA TCA GGA AAG ACA AGG AAA TAC CTA CCA GCT ATA GTG AGA GAA GCT ATA AAA AGG AGA TTG AGG ACC TTG ATA CTA GCA CCA ACA AGA GTG GTC GCT GCA GAG ATG GAG GAA GCA CTC AAA GGA CTC CCT ATC AGG TAC CAG ACA ACT GCC ACC AAA AGC GAA CAT ACT GGG AGA GAA ATC GTC GAC TTG ATG TGC CAT GCC ACA TTC ACA ATG AGA CTC CTA AGC CCC GTG AGG GTG CCA AAC TAC AAC TTG ATT ATC ATG GAT GAG GCA CAC TTT ACT GAC CCT GCC TCT ATT GCA GCT AGA GGG TAT ATA AGC ACA AGA GTG GGG ATG GGG GAA GCA GCC GCA ATA TTT ATG ACA GCC ACA CCA CCT GGA ACT GCT GAT GCA TTC CCT CAG TCA AAC GCC CCA ATA CAG GAT GAG GAA AGA GAT ATC CCA GAA AGA TCA TGG AAC TCA GGA AAT GAA TGG ATT ACA GAC TTC GCT GGC AAG ACA GTG TGG TTT GTG CCA TCA ATT AAG GCT GGC AAT GAC ATC GCC AAC TGT CTT AGA AAG AAC GGA AAA AAG GTG ATA CAG CTA TCA AGG AAA ACC TTC GAT ACA GAA TAC CAA AAG ACA AAG CTG AAC GAT TGG GAC TTT GTT GTG ACA ACC GAC ATA TCT GAA ATG GGA GCC AAC TTC AAG GCA GAC AGA GTG ATA GAT CCA AGG AGA TGC TTG AAA CCT GTG ATA TTA ACA GAC GGA CCT GAG AGA GTT ATC CTA GCT GGC CCA ATG CCC GTG ACC GCC GCA TCA GCC GCT CAG AGA AGG GGA AGG GTG GGA AGA AAC CCA CAA AAA GAG AAT GAT CAA TAC ATC TTT ACC GGA CAA CCT CTG AAC AAT GAC GAG GAC CAC GCT CAT TGG ACA GAA GCA AAA ATG TTG CTC GAC AAC ATT AAT ACT CCT GAG GGC ATT ATC CCC GCC CTC TTT GAA CCT GAG AGA GAA AAG AGT GCA GCT ATC GAC GGA GAG TAC AGG TTA AAA GGA GAG TCA AGA AAA ACC TTC GTT GAA CTC ATG AGG AGA GGC GAC TTA CCA GTC TGG TTG GCA CAC AAA GTG GCC AGT GAA GGG ATA AAG TAC ACA GAC AGA AAA TGG TGT TTC GAT GGA CAA AGA AAC AAT CAG ATA CTC GAA GAG AAC ATG GAC GTG GAA ATC TGG ACA AAA GAG GGA GAA AAG AAA AAG CTA AGA CCA AGA TGG CTT GAC GCT AGA ACA TAC TCA GAC CCT CTC GCC CTT AAA GAA TTT AAA GAT TTT GCA GCT GGA AGA AAA TCC ATA GCA CTC GAT CTG GTC ACA GAG ATA GGG AGG GTC CCA TCA CAT TTG GCA CAT AGG ACC AGA AAC GCT CTT GAT AAT CTG GTG ATG CTA CAC ACC TCA GAA GAT GGA GGG AGG GCA TAC AGG CAC GCC GTT GAG GAA TTA CCC GAA ACA ATG GAA ACC CTC TTA CTG CTA GGA TTA ATG ATA CTA CTG ACA GGC GGA GCA ATG TTG TTT TTG ATT TCA GGG AAA GGA ATA GGA AAA ACA AGC ATA GGG CTT ATA TGT GTG ATA GCC TCA TCT GGA ATG CTT TGG ATG GCA GAG GTG CCA CTA CAA TGG ATA GCC TCA GCA ATC GTG CTT GAA TTT TTC ATG ATG GTG CTG TTG ATA CCT GAA CCA GAA AAG CAA AGA ACA CCA CAA GAC AAC CAA TTA GCA TAC GTT GTC ATC GGA ATA TTG ACA TTA GCC GCA ACT ATA GCC GCA AAC GAG ATG GGG CTA CTG GAG ACA ACT AAA AGA GAT CTT GGG ATG TCC AAA GAA CCA GGG GTT GTC TCC CCT ACA TCT TAC CTA GAT GTG GAC CTA CAT CCT GCA TCT GCA TGG ACA CTC TAT GCC GTT GCA ACC ACA GTG ATT ACC CCA ATG TTA AGG CAT ACC ATC GAA AAC TCT ACA GCC AAC GTG TCC CTA GCT GCA ATC GCA AAC CAG GCC GTT GTG CTG ATG GGA TTA GAT AAG GGC TGG CCA ATT AGT AAA ATG GAT CTC GGA GTC CCC TTG CTA GCC CTG GGA TGC TAT TCT CAG GTT AAT CCA TTA ACA TTA ACA GCC GCT GTT CTC CTA CTT ATA ACC CAC TAC GCA ATC ATA GGA CCA GGC CTG CAA GCC AAG GCT ACA AGA GAA GCT CAA AAG AGA ACA GCA GCT GGA ATT ATG AAA AAC CCA ACA GTG GAC GGA ATC ATG ACA ATA GAT TTG GAT TCA GTC ATT TTC GAT AGT AAA TTT GAG AAA CAG CTG GGA CAA GTG ATG TTA CTC GTG CTT TGC GCT GTT CAA CTT TTG CTA ATG AGA ACA TCC TGG GCT TTG TGC GAG GCA TTA ACA TTG GCA ACT GGA CCC ATA ACT ACA CTG TGG GAA GGA TCC CCT GGG AAG TTC TGG AAC ACA ACC ATA GCA GTG TCA ATG GCA AAC ATT TTT AGG GGA TCC TAC TTG GCA GGA GCT GGA CTG GCC TTC TCC ATT ATG AAA TCC GTT GGA ACC GGC AAA AGA GGA ACC GGA TCA CAA GGA GAA ACA TTA GGA GAA AAG TGG AAA AAG AAA TTG AAC CAA CTC TCA AGA AAA GAA TTC GAT TTG TAT AAA AAG TCA GGA ATT ACA GAA GTG GAC AGA ACA GAG GCA AAG GAA GGC CTC AAG AGA GGA GAA ACA ACT CAC CAT GCT GTC TCA AGG GGA TCA GCA AAG TTA CAA TGG TTT GTC GAA AGA AAC ATG GTG GTT CCA GAA GGC AGA GTG ATT GAC TTG GGG TGT GGC AGA GGA GGG TGG TCA TAT TAC TGC GCA GGG CTG AAA AAG GTG ACA GAG GTG AGA GGA TAT ACA AAA GGA GGG CCA GGA CAT GAA GAG CCA GTG CCA ATG TCC ACA TAC GGA TGG AAC ATA GTC AAG TTA ATG AGT GGA AAA GAC GTC TTC TAC CTA CCA CCC GAG AAA TGT GAC ACC CTA CTT TGT GAT ATT GGC GAA TCA TCT CCA TCT CCA ACA GTG GAA GAG AGC AGA ACA ATT AGA GTG CTT AAA ATG GTG GAG CCA TGG CTA AAA AAT AAC CAA TTT TGC ATT AAA GTC CTT AAT CCA TAC ATG CCA ACA GTG ATC GAA CAC CTG GAG AGG TTG CAG AGG AAA CAT GGA GGC ATG CTT GTG AGA AAC CCA CTG TCA AGA AAC AGC ACT CAT GAA ATG TAC TGG ATT TCA AAC GGA ACA GGA AAC ATT GTT TCA AGC GTT AAC ATG GTG TCC AGA CTG TTA CTT AAT AGA TTT ACA ATG ACA CAC AGA AGG CCC ACA ATT GAA AAA GAC GTG GAC CTA GGA GCC GGA ACC AGA CAC GTG AAC GCA GAG CCC GAA ACA CCT AAT ATG GAT GTG ATA GGG GAG AGG ATA AAA AGA ATC AAA GAA GAG CAC AAC TCA ACA TGG CAT TAT GAC GAT GAA AAC CCT TAT AAA ACT TGG GCC TAC CAC GGA AGT TAT GAA GTT AAG GCA ACA GGC AGT GCC TCT TCC ATG ATT AAC GGA GTG GTT AAA TTG CTC ACT AAA CCT TGG GAC GTG GTT CCA ATG GTT ACA CAA ATG GCT ATG ACA GAC ACA ACC CCC TTT GGA CAA CAG AGG GTC TTC AAG GAA AAG GTC GAC ACT AGG ACC CCC AGA CCA ATG CCA GGA ACA AGG AAA GCA ATG GAA ATA ACA GCA GAA TGG TTG TGG AGA ACC CTG GGA AGA AAT AAA AGA CCA AGG TTA TGC ACC AGA GAG GAA TTC ACA AAG AAA GTG AGA ACA AAC GCA GCT ATG GGA GCA GTG TTC ACA GAA GAG AAC CAA TGG GAC TCA GCA AAA GCA GCC GTG GAA GAT GAA GAG TTT TGG AAA CTC GTG GAT AGA GAA AGA GAA CTT CAT AAA CTG GGA AAA TGT GGC TCA TGT GTG TAC AAT ATG ATG GGA AAG AGA GAG AAG AAA CTC GGA GAA TTC GGC AAG GCT AAA GGG TCA AGG GCC ATT TGG TAT ATG TGG TTA GGG GCC AGA TAC TTG GAA TTC GAG GCT CTA GGA TTT CTT AAC GAA GAC CAC TGG TTC AGT AGG GAG AAC TCA TAC TCA GGA GTG GAA GGA GAG GGA CTG CAT AAA CTC GGA TAC ATT CTT AGA GAC ATA AGT AAG ATA CCA GGA GGG GCC ATG TAC GCA GAC GAT ACT GCA GGG TGG GAT ACA AGA ATT ACA GAA GAC GAT CTG CAT AAC GAG GAA AAA ATA ATC CAA CAG ATG GAC CCC GAA CAC AGA CAA CTT GCT AAC GCC ATT TTT AAA CTA ACT TAC CAA AAC AAG GTG GTC AAA GTC CAA AGA CCA ACC CCT ACA GGA ACC GTT ATG GAC ATA ATT TCC AGA AAG GAT CAG AGA GGC TCT GGA CAA TTG GGA ACA TAC GGC TTG AAT ACC TTT ACA AAC ATG GAA GCA CAA CTT GTG AGA CAA ATG GAA GGG GAA GGG GTG CTG ACA AAA GCT GAC TTG GAA AAC CCA CAT TTA CTA GAG AAG AAA ATC ACA CAA TGG TTG GAA ACA AAG GGA GTG GAG AGA CTG AAG AGA ATG GCT ATA TCA GGC GAT GAC TGT GTG GTT AAG CCT ATA GAC GAT AGG TTT GCC AAT GCA CTG CTC GCC CTG AAT GAC ATG GGA AAG GTG AGA AAG GAT ATT CCA CAA TGG CAA CCA TCA AAA GGG TGG CAC GAT TGG CAA CAG GTG CCA TTT TGT AGC CAC CAT TTC CAT GAG CTG ATA ATG AAA GAC GGC AGA AAA TTA GTG GTC CCT TGT AGA CCA CAA GAT GAG CTC ATA GGG AGA GCC AGA ATT AGC CAG GGA GCC GGC TGG TCT CTG AGA GAG ACT GCA TGT CTG GGA AAA GCC TAC GCA CAA ATG TGG TCA CTG ATG TAT TTC CAC AGA AGG GAC CTC AGA TTA GCA TCC AAT GCA ATA TGC AGC GCA GTG CCA GTG CAC TGG GTG CCA ACA TCA AGA ACA ACT TGG TCC ATA CAC GCC CAC CAT CAA TGG ATG ACA ACA GAG GAC ATG CTG ACA GTG TGG AAT AGA GTG TGG ATT GAA GAG AAT CCA TGG ATG GAA GAT AAA ACA CCA GTC ACA ACA TGG GAA AAC GTG CCA TAC TTG GGG AAA AGA GAG GAT CAA TGG TGC GGA TCA CTC ATA GGC TTG ACA TCC AGA GCA ACA TGG GCA CAA AAC ATC CCA ACA GCC ATA CAG CAA GTG AGA TCA CTT ATC GGG AAT GAG GAA TTT CTC GAC TAC ATG CCC AGC ATG AAG AGA TTC AGA AAG GAG GAA GAG TCT GAG GGG GCC ATT TGG TAA
 * GC% Content for gene: 46.7 %**
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**


 * GC% Content for gene (codon optimized): 60.06%**

Do Not Need this info for Spring (but still copy these lines to your Target page for now) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):**
 * ( link to DNA Works output text file - **that should be saved in your Google Docs folder after you did the primer design protocol)


 * Primer design results for 'tail' primers (this is just 2 sequences):**



AssayMethodsUsingPep t i d y l - AMC(MCA)Subs t rates(3) (Example f o r C a s p a s e - 7 u s in g a n A u t o - F l u o r es ce n c e S p e c t r op h o t o m e t e r f o r M u l t i p l a t e )

Reagen t s 1. S u b s t r a t e s t o c k s o l u ti o n:C o n t e n t o f v ia l(C o de317 1 -v A c - D E VD - AM C), i n DMS O a t10 m M 2. AM C s t o c k s o l u ti o n:C o n t e n t o f v ia l(C o de30 9 9- v AM C), i n DMS O a t1 m M 3. B u f f e r :I C E s ta n d a r d b uf f e r(100 m M H E P E S - KO H ,pH7 . 5, 1 0 % s u c r o s e( w / v ), 0 . 1 %C H AP S( w / v ),10 m M D T T, 0 . 1 m g / m l ov al b u m i n ) 4. E n z y m e s o l u ti o n: r ec. C a s p a s e -7 i n b u f f e r

Procedure a) In i t i a lr at e a ss a yf o r30 mi n a tλ e x=390n m, λ e m=460 n m <span style="display: block; height: 1px; left: -40px; margin: 0.45pt 0in 0in 20.25pt; overflow: hidden; position: absolute; text-indent: -0.15in; top: 1376.5px; width: 1px;"><span style="font-family: 'Arial','sans-serif'; font-size: 9pt; letter-spacing: -0.05pt;">b) E n d-p o i n t(30 m i n ) a s s a y a t5d i ff e r e n t s u b s t r at e c o n ce n t r a ti o n s a tλ e x= 3 55nm o r380 n m ,λ e m=4 6 0nm

<span style="display: block; height: 1px; left: -40px; line-height: 95%; margin: 0in 238.8pt 0in 25.8pt; overflow: hidden; position: absolute; text-indent: -9.15pt; top: 1376.5px; width: 1px;">1. S e ta f l u o r e s ce n c e s p ec t r o p o t o m e t e r a t λ e x=380 nm( o r380n m ,355n m ) a n dλ e m= 4 60nm R e lati v e f l u o r e s ce n c e i sd e t e r m i n e d w i t h 1 0 -6 M o f AM C a t e a c h c o n d i t i o n <span style="display: block; height: 1px; left: -40px; margin: 0.1pt 234.7pt 0in 28.2pt; overflow: hidden; position: absolute; text-indent: -9.85pt; top: 1376.5px; width: 1px;">2. S u b s t r at e : D il u t e t h e s t o c k s o l u ti o n w i t h t h e b uf f e r (x10, x2 0 ,x4 0, x 8 0, x160) <span style="display: block; height: 1px; left: -40px; margin-left: 28.2pt; overflow: hidden; position: absolute; text-indent: -9.85pt; top: 1376.5px; width: 1px;">3.C a s p a s e -7: D i ss o l ve i n b u f f e r <span style="display: block; height: 1px; left: -40px; margin: 0in 248.7pt 0in 28.2pt; overflow: hidden; position: absolute; text-indent: -9.85pt; top: 1376.5px; width: 1px;">4. P i p e tt e160 µ l o f b uff e r a n d20 M l o f s ub s t r a t e s o l u ti o n s( 1, ½ , ¼ , 1 / 8, 1 / 16 m M ) i n e a c h w e l l <span style="display: block; height: 1px; left: -40px; margin: 0in 247.65pt 0in 28.2pt; overflow: hidden; position: absolute; text-indent: -9.85pt; top: 1376.5px; width: 1px;">5. I n c u b at e i n t h e f l u o r e s ce n c e s p ec t r o p h o t o m e t e r fo r3-4 mi n( fo r t e m p e r a t u r e e qu i li b r a t i o n ) <span style="display: block; height: 1px; left: -40px; line-height: 11.3pt; margin: 0.3pt 286.35pt 0in 28.2pt; overflow: hidden; position: absolute; text-indent: -9.85pt; top: 1376.5px; width: 1px;">6. T a ke t h e m u l t i p l at e o ut a n d a dd20 µ l o f e n z y m e s o l u ti o n i n e a c h w e l l <span style="display: block; height: 1px; left: -40px; line-height: 11.4pt; margin-left: 28.2pt; overflow: hidden; position: absolute; text-indent: -9.85pt; top: 1376.5px; width: 1px;">7. M o u n t t h ep l at e i n t h e f l u o r e s ce n c e s p ec t r o m e t e r <span style="display: block; height: 1px; left: -40px; margin: 0.5pt 0in 0in 18.35pt; overflow: hidden; position: absolute; top: 1376.5px; width: 1px;">8 C al c u l at e t h e a m ou n t o f r e l e a s e d AM C

<span style="display: block; height: 1px; left: -40px; overflow: hidden; position: absolute; top: 1376.5px; width: 1px;"><span style="height: 317px; left: 0px; margin-left: 431px; margin-top: 11px; mso-ignore: vglayout; position: absolute; width: 239px; z-index: 251658237;"> R e sul t s

<span style="display: block; height: 1px; left: -40px; margin-left: 31.05pt; overflow: hidden; position: absolute; top: 1376.5px; width: 1px;">

<span style="display: block; height: 1px; left: -40px; margin-right: 2.45pt; overflow: hidden; position: absolute; text-align: center; top: 1376.5px; width: 1px;">PeptaNova G mb H [|www][|.][|peptanova.de]info@peptanova.de