What does a P2A sequence do? -

What does a P2A sequence do?

Application. In genetic engineering, the 2A peptides are used to cleave a longer peptide into two shorter peptides.

What is Bicistronic lentiviral vector?

Lenti-X bicistronic lentiviral vectors and expression systems allow you to deliver and stably express your gene of interest, along with a coexpressed bright fluorescent protein or an antibiotic selection marker, in virtually any mammalian cell type.

What is IRES in cloning?

IRES enables the coordinated co-expression of two genes with the same vector. For example, you can monitor the delivery of one gene by using a second gene with a fluorescent tag or express a protein of interest and simultaneously biotinylate it with the same vector.IRES enables the coordinated co-expressionco-expressionA gene co-expression network (GCN) is an undirected graph, where each node corresponds to a gene, and a pair of nodes is connected with an edge if there is a significant co-expression relationship between them.https://en.wikipedia.org › wiki › Gene_co-expression_networkGene co-expression network – Wikipedia of two genes with the same vector. For example, you can monitor the delivery of one gene by using a second gene with a fluorescent tag or express a protein of interest and simultaneously biotinylate it with the same vector.

What causes polycistronic mRNA?

Polycistronic mRNA is a mRNA that encodes several proteins and is characteristic of many bacterial and chloroplast mRNAs. Polycistronic mRNAs consist of a leader sequence which precedes the first gene. The gene is followed by an intercistronic region and then another gene.

What does it mean when a gene is truncated?

Definition. A genetic variant which results in a shorter version of the protein being produced.

What is selective mRNA translation?

This selective mRNA translation is an important layer of gene expression regulationgene expression regulationRegulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).https://en.wikipedia.org › Regulation_of_gene_expressionRegulation of gene expression – Wikipedia in erythropoiesis. Ribosome profiling is a recently developed high throughput sequencing technique for global mapping of translation initiation sites across the transcriptome.

What is truncated mRNA?

Truncated mRNAs, generated by premature termination of transcription or by nucleolytic cleavage and partial degradation of full-length mRNAs, are common in the eubacterial cell.

What is IRES plasmid?

IRES Elements Translation in eukaryotes usually begins at the 5′ cap so that only a single translation event occurs for each mRNA. However, some bicistronic vectors take advantage of an element called an Internal Ribosome Entry Site (IRES) to allow for initiation of translation from an internal region of the mRNA.Sept 9, 2014

What is an IRES and what is its purpose?

IRES are sequences that can recruit ribosomes and allow cap-independent translation, which can link two coding sequences in one bicistronic vector and allow the translation of both proteins in hESCs.IRES are sequences that can recruit ribosomes and allow cap-independent translationcap-independent translationThe RNAs of many positive sense RNA viruses contain a cap-independent translation element (CITE)3 that allows efficient translation in the absence of a 5′ cap structure (11, 12, 13). In animal viruses and some plant viruses, the CITE is an internal ribosome entry site (IRES) located upstream of the initiation codon.https://www.sciencedirect.com › science › article › piiStructure of a Viral Cap-independent Translation Element That Functions , which can link two coding sequences in one bicistronic vector and allow the translation of both proteins in hESCs.

What is T2A plasmid?

T2A is a self-cleaving 2A peptide (The average length is 18–22 amino acids) that could be applied in expression of more than one gene in cells. It is a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. [ 1]

What is IRES in biology?

An internal ribosome entry site, abbreviated IRES, is an RNA element that allows for translation initiation in a cap-independent manner, as part of the greater process of protein synthesis.An internal ribosome entry site, abbreviated IRES, is an RNA element that allows for translation initiation in a cap-independentcap-independentIn molecular biology, a cap-independent translation element (CITE or 3’CITE) is an RNA sequence found in the 3’UTR of many RNA plant viruses. Eukaryotic mRNAs contain a 5′ cap structure which is required for efficient binding of translation initiation factors.https://en.wikipedia.org › Cap-independent_translation_elementCap-independent translation element – Wikipedia manner, as part of the greater process of protein synthesis.

Do you need stop codon before IRES?

You absolutely need an IRES (internal ribosome entry site) in between your two gene constructs because after the stop codon on the first cDNA is released from the ribosome. Note that usually the second gene after the IRES is translated less efficiently respect to the first.

What is a Bicistronic vector?

IRES-containing bicistronic vectors allow the simultaneous expression of two proteins separately, but from the same RNA transcript (Jackson et al. 1990; Jang et al. 1988).

How does P2A work?

P2A sequences sit in between your two genes of interest and cause ribosomal “skipping” during translation, which results in a missing peptide bond and effectively separates the two proteins. The major advantage of P2A is its size—just 19 amino acids.

What is P2A in cloning?

When expressing more than one gene in a cell line, often bicistronic expression vectors with an internal ribosomal entry site (IRES) are used. Instead of these large IRES sequences, a much shorter, self-cleaving 2A peptide (P2A) can be used. The efficient cleaving activity of this peptide was described in Kim et al.Mar 5, 2015

What is P2A and T2A?

Four members of 2A peptides family are frequently used in life science research. They are P2A, E2A, F2A, and T2A. F2A is derived from foot-and-mouth disease virus 18; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; T2A is derived from thosea asigna virus 2A.

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What is Bicistronic mRNA?

Abstract. Mammalian bicistronic mRNA is a recently discovered mammalian gene structure. Several reported cases of mammalian bicistronic mRNA indicated that genes of this structure play roles in some important biological processes.Sept 4, 2012

What is P2A biology?

Peptide 2A (P2A) sequences P2A sequences sit in between your two genes of interest and cause ribosomal “skipping” during translation, which results in a missing peptide bond and effectively separates the two proteins. The major advantage of P2A is its size—just 19 amino acids.

How do self cleaving peptides work?

These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. They help generating polyproteins by causing the ribosome to fail at making a peptide bond. The members of 2A peptides are named after the virus in which they have been first described.

Addgene: pPromoterless-P2A Sequences

Addgene: pPromoterless-P2A Sequences. GenBank File: Plasmid sequence and annotations. Use text editor or plasmid mapping software to view sequence. SnapGene File: Plasmid sequence and SnapGene enhanced annotations. Use with SnapGene software or the free Viewer to visualize additional data and align other sequences. Skip to main content.

High Cleavage Efficiency of a 2A Peptide Derived from

Oligonucleotides encoding P2A, T2A, E2A or F2A (refer to Fig. 1B for sequences) were purchased from Bioneer (Daejeon, Korea), annealed and then individually cloned into SphI/BglII sites of a pCS4+ plasmid (provided by Chang-Yeol Yeo). Oligonucleotides used are as follows.

Addgene: pC5Kan-P2A Sequences

Sequence provided by depositing laboratory may be theoretical/predicted or based on Sanger/NGS sequencing results. Discrepancies between sequencing results obtained by Addgene and the original sequence provided by the depositor may be present. Analyze Sequence. GenBank. SnapGene. File Help. > pC5Kan-P2A sequence 2685 bps

Optimized P2A for reporter gene insertion into Nipah virus

Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo.

Systematic comparison of 2A peptides – Scientific Reports

A highly conserved sequence GDVEXNPGP is shared by different 2As at the C-terminus (i670) varied in all possible positions with intervening P2A and T2A sequences

AmCyan-P2A-mCherry – Addgene

the nAmCyan and the mCherry genes are separated by a P2A sequence that results in 2 separate proteins. It also includes BsmBI restriction sites flanking the nAmCyan gene which result in BsiWI and BssHII overhangs for cloning in-frame with P2A-mCherry.

P2A Sequence : labrats

P2A Sequence. When you are trying to express two ORFs from the same construct, separated by a P2A, do you include a small stuffer between the first ORF and P2A sequence? It seems like many constructs have 6-27 extra nucleotides. Are they necessary? Got lab stories? Experiments gone wrong?

pLenti-Cas9-P2A-tGFP Sequence and Map – SnapGene

pLenti-Cas9-P2A-tGFP. 3rd-generation lentiviral vector for expressing Cas9 and TurboGFP under control of the CMV promoter. Sticky ends from different PasI sites may not be compatible. Sticky ends from different Bsu36I sites may not be compatible. BsrGI is typically used at 37°C, but is even more active at 60°C.

2A self-cleaving peptide-based multi – Scientific Reports

To date, 2A-like sequences in other viral mRNA molecules have been successfully identified, including the porcine teschovirus-1 2A (P2A), thosea asigna virus 2A (T2A), equine rhinitis A virus 2A

Plasmids 101: Multicistronic Vectors – Addgene

IRES was originally discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic cells. 1,2 Since then, a variety of IRES sequences have been discovered – many from viruses, but also some from cellular mRNAs. What they all have in common is the ability to spark translation initiation independent of the 5′ cap.

"Self-cleaving" 2A peptide from porcine teschovirus-1

P2A was a 66-base pair oligonucleotide from porcine teschovirus-1 . RFP was preceded by GRA8, a signal sequence, and followed by a His6 tag [ 7 ]. Restriction enzyme-mediated integration (REMI) was adapted for the transfection of sporozoites as previously described [ 16 , 17 ].

Addgene: Lenti-luciferase-P2A-Neo Sequences

Full Sequences from Depositor (1) Sequence provided by depositing laboratory may be theoretical/predicted or based on Sanger/NGS sequencing results. Discrepancies between sequencing results obtained by Addgene and the original sequence provided by the depositor may be present. > Lenti-luciferase-P2A-Neo sequence 8833 bps

IRES Or 2A In Polycistronic Expression – VectorBuilder

There are four commonly used 2A peptides, P2A, T2A, E2A and F2A, that are derived from four different viruses. Pros and cons of the two types of linkers. The key advantage of IRES over 2A is that it does not affect the protein sequence of either the upstream or the downstream ORF, which is not the case for 2A.

Express yourself: Gene Co-Expression in Bicistronic Constructs

pC5Kan-P2A – Addgene

P2A peptide coding sequence inserted in middle of MCS of pC5-Kan shuttle vector for easy transfer into C5-series Drosophila expression vectors. Vector type. shuttle vector for insect expression vectors Growth in Bacteria. Bacterial Resistance(s) Kanamycin Growth Temperature

Effects of different 2A peptides on transgene – PubMed

The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant

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2A peptides provide distinct solutions to driving stop

INTRODUCTION. A small number of nascent peptides are known to interact with the ribosomal exit tunnel and/or peptidyl-transferase centre (PTC) to regulate translation of downstream mRNA sequences (1,2).Most of these are encoded by short upstream ORFs (uORFs) in eukaryotes, or leader peptides in bacteria, and typically pause or stall ribosomes in response to small molecule effectors. ‘2A

Using a viral 2A peptide-based strategy to reconstruct the

Then, we replaced the second F2A linker with 2A-like sequences, from Thosea asigna (T2A) and Porcine teschovirus-1 (P2A), with the GSG sequence on the N-terminus. Studies have shown that these sequences can result in the more efficient cleavage of 2A-polyproteins than F2A ( Daniels et al., 2014 ; Szymczak-Workman et al., 2012 ; Souza-Moreira et

Efficient co-expression of multiple enzymes from a single

Two different 2A sequences, Porcine teschovirus-1 2A (P2A) and foot-and-mouth disease virus 2A (F2A), were evaluated. It was found that P2A sequence was more effective for co-expression of two antibiotic selection markers. Co-expression of three antibiotic resistance proteins was successful from a single promoter mediated by P2A sequence.

To use P2A system for multiproteins production in hESCs

The question is, whether I should remove the stop codon TGA of resistive gene and then add the P2A sequence and the ATG for second protein or I dont need to remove the stop codon of first protien.

Does viral 2A peptide really outperform IRES sequence in

The question is, whether I should remove the stop codon TGA of resistive gene and then add the P2A sequence and the ATG for second protein or I dont need to remove the stop codon of first protien.

High Cleavage Efficiency of a 2A Peptide Derived – PLOS

To generate versatile cloning vectors harboring genes encoding viral 2A peptides, we inserted four different 2A sequences (P2A, T2A, E2A and F2A; Fig. 1B) into a pCS4+ plasmid, which has a simian cytomegalovirus (sCMV) promoter, SP6 and T7 RNA polymerase binding sites, multiple cloning sites (MCSs) and an SV40 polyadenylation site .

Linkers for Polycistronic Vectors – VectorBuilder

Sequence; T2A: Self-cleaving 2A peptide from Thosea asigna virus: Causes co-translational cleavage of the encoded polypeptide. Multiple proteins can be made from a polycistronic transcript containing multiple ORFs separated by 2A. P2A and T2A have higher cleavage efficiency compared to other 2As. PLoS One. 6:e18556 (2011)

Can a P2A-fluorophore sequence be used for making a

The gene of interest, which is located upstream of the P2A sequence, will be inserted into the ER, but I am not sure whether the fluorophore downstream of the P2A sequence will also be inserted

Problem with P2A self-cleavage peptide? – ResearchGate

The truly strange part is when I picked some clones to sequence, the results show that only part of the split GFP and part of the P2A sequence was present. The entire protein A sequence was

Spatiotemporal control of phosphatidic acid signaling with

CRY2-mCherry-PLD PMF or mCherry-CRY2-PLD PMF were amplified by PCR with insertion of a P2A sequence (primers: 5′-TTT AAA CTT AAG CTT GGT ACG CCA CCA TGA AGA TGG AC-3′ or 5′-TTT AAA CTT AAG CTT GGT ACG CCA CCA TGG TGA GCA AG-3′ and 5′-TAT AGC TCC ATT CAT GGT ACC GGG ACC TGG GTT TTC CTC GAC ATC GCC AGC CTG CTT GAG CAG ACT AAA GTT TGT

What if I add another cDNA sequence after the stop code

The question is, whether I should remove the stop codon TGA of resistive gene and then add the P2A sequence and the ATG for second protein or I dont need to remove the stop codon of first protien

pcDNA3.1(+)-P2A-eGFP-ORF3a plasmid from GenScript Biotech

pcDNA3.1 (+)-P2A-eGFP-ORF3a. The plasmid is used for SARS-CoV-2 ORF3a protein expression. The ORF3a protein is integrated with eGFP protein through P2A, which can be used for flow cytometry antibody screening or immunological research. Please select the target plasmid you will comment. *This material may be covered by one or more patents

Simian Immunodeficiency Virus (SIV)-Specific Chimeric

The P2A-sequence allows the cell to produce both proteins without re-initiation of translation due to a “stop and go” translational effect mediated by the ribosome, thereby resulting in similar levels of expression of the two proteins (70, 81). In contrast with the IRES-based CAR/CXCR5 construct, T cells transduced with the P2A-based

A Whole-Brain Cell-Type-Specific Sparse Neuron Labeling

To make the pAAV-hSyn-DIO-EGFP-P2A-EGFPf construct, EGFP and EGFPf sequences were PCR amplified from a pAAV-hSyn-EGFP-P2A-EGFPf-WPRE-HGHpA construct (Addgene, #74513). The loxP, loxP2272, and P2A sequences were added during the PCR amplification. The primer sequences were as follows: DIO-GFPf-F1: 5′-ATTGTAGCTGCTATT

Light-Activated ROS Production Induces Synaptic Autophagy

To allow for the independent expression of Syp-mCh and eGFP-LC3, a P2A cleavage site was placed between the two coding sequences (Fig. 1 A). P2A is a highly efficient “self-cleaving” small peptide derived from porcine teschovirus-1 leading to post-translational cleavage of the initially transcribed fusion protein ( Kim et al., 2011 ).

An Optogenetic Tool for Induced Protein – ScienceDirect

The P2A sequence contains an NPGP motif, which induces ribosomal skipping between G and P. Hence, free GFP is used as a reference marker for the overall synthesis level of the constructs. For expression in yeast, we used the strong ADH1 promoter. (B) AuLOV-cODC1 showed a low protein level in yeast cells kept in darkness or exposed to blue light.

A toolkit for Nannochloropsis oceanica CCMP1779 enables

Ribosomal skipping efficiency was less than 10% for F2A, and T2A sequences, while P2A had an efficiency of 10‐30% (Figure S4c). In order to increase ribosomal skipping efficiency, the N‐terminal F2A peptide‐encoding sequence was extended to 58 aa, and the P2A sequence to 30 aa, 45 aa and 60 aa.

A rapid and effective method for screening, sequencing and

We applied these principles to engineer a pCS2+MCS-P2A-sfGFP vector for generating mutation reporter constructs, which is based on the popular pCS2+ vector for mRNA expression and contains a multiple cloning site sequence with PacI and AscI rare-cutter restriction enzymes as well as alternative enzymes BstBI, EcoRI and SphI, which allow

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Generation of CRISPR/Cas9-mediated bicistronic knock-in

The donor plasmid pIns1/2A-cre contained the P2A sequence and nuclear translocation signal (NLS)-cre. The 2.0-kb 5′-arm (from 1.4 kb upstream of exon 1 to just before the stop codon of Ins1) and the 1.7-kb 3′-arm (from the stop codon to 1.1 kb downstream of exon 2) were cloned into this vector.

ARF-AID: A rapidly inducible protein degradation system

For C-terminal fusions, the repair construct consists of AID separated from the hygromycin resistance gene (HygR) by a porcine teschovirus-1 ribosomal skipping sequence (P2A) (Kim et al., 2011) (Figure 4). In the N-terminal repair construct, the order is reversed: HygR-P2A-AID (Figure 4). In both cases, the AID is separated from the protein of

Addgene: pCAG-RFP-p2a-postASAP

Plasmid pCAG-RFP-p2a-postASAP from Dr. Rafael Yuste’s lab contains the insert FingR.PSD95, ASAP2f and TagRFP and is published in Science. 2022 Jan 7;375(6576):82-86. doi: 10.1126/science.abg0501. Epub 2021 Nov 11. This plasmid is available through Addgene.

Mir-181-P2a

Mature sequence: Ocu-Mir-181-P2a_5p: mirBase accession: MIMAT0048278: Sequence: 0- AACAUUCAUUGCUGUCGGUGGGUU-24 Get sequence: Star sequence: Ocu-Mir-181-P2a_3p* mirBase accession: MIMAT0048279: Sequence: 40- CUCACUGAACAAUGAAUGCA-60 Get sequence

A New Mouse Line Reporting the Translation of – eNeuro

The GSG sequence is then followed by the teschovirus P2A sequence (Liu et al., 2017), the SV40 nuclear localization sequence (NLS; Ray et al., 2015) and a GFP sequence inserted between the last amino acid and the translation termination codon in exon 2 of the BDNF coding sequence. The presence of the P2A sequence should result in the

pcDNA3.1(+)-P2A-eGFP-ORF7b plasmid from GenScript Biotech

2019-nCov_pcDNA3.1 (+)-P2A-eGFP. The plasmid is used for 2019-nCoV (SARS-CoV-2) surface glycoprotein expression (Codon Optimized for Mouse expression system). The surface glycoprotein is integrated with eGFP protein through P2A, which can be used for flow cytometry antibody screening or immunological research.

Ribosomal skipping – ViralZone

Ribosomal “skipping” is an alternate mechanism of translation in which a specific viral peptide prevents the ribosome from covalently linking a new inserted aa, and let it continue translation. This result in apparent co-translational cleavage of the polyprotein. This process is induced by a “2A-like”, or CHYSEL (cis-acting hydrolase element) sequence.

TMEM16F mediates bystander TCR-CD3 membrane dissociation

The Ii sequence was a gift from J. Thibodeau (University of Montreal, Canada) and was subcloned into a pHAGE vector by overlap PCR. The WT and Mut TMEM16F sequences were a gift from S. Nagata (Kyoto University, Japan). The TMEM16F sequences were subcloned into a pHAGE vector containing a P2A sequence, followed by the mAmetrine fluorescent protein.

2a肽 – 维基百科，自由的百科全书

这种肽都有一段的序列模体（英语： sequence motif ），经常会在最后甘氨酸（G）和脯氨酸（P）连接处导致核糖体无法连接，从而造成”剪切”的效果。这种肽在很多科病毒中都有分布。目前一共有4種常用的2A肽：T2A、P2A、E2A、F2A。

PSF-CMV-COOH-P2A-2 – FMDV P2A PLASMID plasmid vector for

PSF-CMV-COOH-P2A-2 – FMDV P2A PLASMID plasmid vector for molecular cloning; Synonyms: expression vector,molecular cloning vector,vector,snapfast vector,plasmid vector,cloning vector,plasmid; find Sigma-Aldrich-OGS64 MSDS, related peer-reviewed papers, technical documents, similar products & more at Sigma-Aldrich

PSF-CMV-COOH-P2A-2 – FMDV P2A PLASMID plasmid vector for

PSF-CMV-COOH-P2A-2 – FMDV P2A PLASMID plasmid vector for molecular cloning; Synonyms: 発現ベクター,プラスミドベクター,ベクター,クローニングベクター,分子クローニングベクター,プラスミド; find Sigma-Aldrich-OGS64 MSDS, related peer-reviewed papers, technical documents, similar products & more at Sigma-Aldrich

pLenti-EnCMV-TLR4 -P2A-mCherry-SV40-Puro Plasmid

pLenti-EnCMV-TLR4 -P2A-mCherry-SV40-Puro Plasmid. PVT42564 2ug. pLenti-EnCMV-TLR4 -P2A-mCherry-SV40-Puro Plasmid Desciption. Promoter:CMV. Replicon:pUC. Competent cells:Stbl3. Culture temperature:37 degrees. Host：Mammalian cells,Lentivirus. Plasmid use(s):Protein expression. Fragment type：ORF. Fragment species：Mouse. Prokaryotic

pLenti-C-mGFP Lentiviral Gene Expression Vector – PS100071

Special offer: 20% off on this product. Use code “Shuttle20”. This Lenti vector is a third generation of lentiviral vector. All OriGene’s TrueORF clone can be shuttled into this vector via PrecisionShuttling, a simple cut-and-ligation process. The ORF cloned in this vector can be expressed as a tagged protein with a c-terminal monomeric GFP tag.

Compositions and Methods for Genetically-encoded High

compositions and methods for genetically-encoded high voltage-activated calcium channel blockers using engineered ubiquitin ligases

(PDF) CTCF Regulates Ataxin-7 Expression through Promotion

C) This pattern was reversed in SCA7-CTCF-I-mut mice that display robust activation of the P2A promoter, as significant enrichment of the H3K9/14Ac mark was present in the ataxin-7 sense P2A region and initial transcript region (p < 0.05, NIH-PA Author Manuscript ANOVA).

US Patent Application for MODIFIED IMMUNE CELLS CO

A population of immune cells comprising modified immune cells co-expressing a chimeric antigen receptor and an IL-6 signaling antagonist (e.g., an anti-IL6 or anti-IL-6R antibody) and optionally an IL-1 signaling antagonist. Also provided herein are methods of producing such immune cell populations comprising the modified immune cells and methods of using such in cell therapy (e.g., to treat

Two-gene Vectors for Generating Car-t Cells and Uses

TWO-GENE VECTORS FOR GENERATING CAR-T CELLS AND USES THEREOF is an invention by Rizal Ismail, Singapore SINGAPORE. This patent application was filed with the USPTO on Thursday,

(PDF) Transcriptional activation induced by snail 1 during

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