1

Examen de mi-session

CHMI 3216 – Bioingénierie de l’A.D.N.

Mardi, le 30 octobre 2007

Professeur : Eric R. Gauthier, Ph.D.

Votre nom : ___________________________________

Consignes :

1. Examen d’une durée de 85 minutes. 2. 6 questions, sur 13 pages, pour un total de 70 points. Compte pour 20% de la note

finale.

3. Répondre à toutes les questions, selon les instructions fournies.

4. Inscrire vos réponses directement sur le questionnaire. Limitez-vous à l’espace fourni (n’utilisez pas le verso des pages).

5. Un document accompagnant le questionnaire vous fourni les informations

nécessaires pour répondre aux questions 2.1, 3.1, 3.2 et 6 (cartes de restriction, liste d’enzymes, séquences, etc).

6. Note : toutes les séquences d’ADN sont présentées avec le brin codant dans

l’orientation 5’ vers 3’ placée de gauche à droite.

7. L’utilisation des notes de cours et de livre est interdite.

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 1. Indiquez si les affirmations suivantes sont vraies ou fausses (10 points - 2 points par question):

Enoncé Vrai ou Faux

1.1. L’ADN ligase du phage T4 requiert le NAD+ comme cofacteur 1.2. Les enzymes de restriction de type II sont des homodimères 1.3. L’activité 3’ 5’ exonucléase des ADN polymérases ne

fonctionne pas sur de l’ADN double brin.

1.4. Lors du PCR, une augmentation de la concentration d’ADN polymérase diminue la fidélité de l’amplification.

1.5. Dans la préparation des banques d’ADNc, une des étapes consiste à séparer les ADNc selon la quantité de radioactivité incorporée lors de la transcription inverse.

Question 2. Répondez aux questions suivantes (12 points - 2 points par question):

Enoncé Réponse

2.1. Donnez la séquence d’une amorce permettant de séquencer un ADNc cloné dans le site EcoR I du vecteur pEGFP-N1 (carte présentée en annexe).

2.2. Ceci est le nom que l’on donne au phénomène selon lequel un enzyme de restriction coupera des séquences d’ADN similaires, main non-identique, à sa séquence cible lorsque la digestion enzymatique est faite sous des conditions non-optimales.

2.3. Ceci est le type de séquence devant être présente en amont (i.e. en 5’) d’un ADNc afin que ce dernier soit transcrit par une ARN polymérase

2.4. Les bactériophages utilisés dans la préparation des banques d’ADNc sont déficients dans le cycle……..

2.5. Dans la purification d’ARN total, quel est le rôle de la guanidium thiocyanate?

2.6. Ceci est le substrat chromogène que l’on ajoute au milieu de culture bactérien afin de détecter la présence d’activité β-galactosidase.

2

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 3. Donnez une réponse brève aux questions suivantes (18 points – 3 points par question): 3.1. Notre ami Stéphane (ben oui, encore lui…) tente de cloner un fragment d’ADN de 450 pb

dans le vecteur pEGFP-N1 (un schéma de ce vecteur est montré en annexe). Après avoir coupé son fragment d’ADN avec EcoR I et fait la ligation dans le site EcoR I de pEGFP-N1, il transforme des bactéries E. coli qu’il étale ensuite sur un milieu de culture contenant de l’ampicilline. Lors de la transformation de ses bactéries, Stéphane a la merveilleuse idée d’inclure plusieurs échantillons contrôle. Il obtient les résultats suivants :

ADN ajouté aux bactéries lors de la transformation

Nombre de colonies par pétri

Contrôle : H2O 0 Contrôle : pBluescript > 1000 Contrôle : pEGFP-N1 0 ADN/EcoRI + pEGFP-N1

0

Quel est la faille majeure dans le design expérimental qui empêche Stéphane d’obtenir des bactéries E. coli contenant l’ADN cloné dans pEGFP-N1? 3.2. Vous trouverez en annexe la séquence ADN encodant la protéine p53. Quelle paire

d’amorce parmi les suivantes pourra être utilisée afin d’amplifier le cadre de lecture ouvert (open reading frame - ORF) complet de p53?

a) amorce 5’ : atggaggagccgcagtcag amorce 3’: agggcctgactcagactga b) amorce 5’ : ctgactgcggctcctccat amorce 3’ : tcagtctgagtcaggccct c) amorce 5’ : tcctcggcgtcagtcatgg amorce 3’ : agggcctgactcagactga d) amorce 5’ : atggaggagccgcagtcag amorce 3’ : tcagtctgagtcaggccct

3

Test de mi-session – CHMI 3216F – 30 octobre 2007

3.3. Expliquez le principe de la méthode « touchdown PCR ». Quel est l’avantage principal de cette approche?

3.4. Expliquez pourquoi il est important d’exprimer les ADNc sous forme de protéine de fusion

avec la β-galactosidase lors du criblage d’une banque d’ADNc (clonée dans le vecteur λgt11) avec un anticorps?

4

Test de mi-session – CHMI 3216F – 30 octobre 2007

3.5. Notre ami Chris prépare une banque d’ADNc. Malheureusement pour lui, l’analyse

aléatoire (i.e. au hasard) de plages de lyse révèle que la très grande majorité des ADNc clonés dans le phage λ sont de petite taille (en dessous de 2 kpb). Donnez une cause probable de ce problème, et expliquez comment Chris pourrait résoudre cette difficulté.

3.6. Expliquez comment on devra procéder afin de marquer radioactivement l’extrémité 5’

d’une courte séquence d’ADN simple brin.

5

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 4. (5 points). Décrivez les différentes étapes requises lors de l’isolation de plasmides à partir de colonies bactériennes.

6

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 5 (15 points). Expliquez en détail les étapes de la préparation et du criblage (avec un ADNc de 400pb) d’une banque d’ADNc à partir de la lignée cellulaire HepG2 (cellules de foie humain).

7

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 5 (suite)

8

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 5 (suite)

9

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 6 (15 points). On vous donne un fragment d’ADN correspondant au cadre de lecture ouvert complet de eIF2α. Préparez une stratégie de clonage de cet ADN dans le MCS de pOPRSV/MCS. Indiquez comment vous utiliseriez des enzymes de restriction afin de confirmer l’orientation de ce fragment dans pOPRSV. NOTE 1: Votre réponse doit inclure tous les réactifs (enzymes, cofacteurs, substrats) que vous utiliserez pour cette expérience. NOTE 2: le cadre de lecture ouvert complet (et seulement le cadre de lecture ouvert) doit être inséré dans le vecteur de manière directionnelle, de telle sorte que le brin codant de eIF2α soit dans la bonne orientation pour être transcrit avec la polymérase T7. Vous trouverez en annexe la séquence de l’ADNc encodant la protéine eIF2α, ainsi que sa carte de restriction. Vous trouverez aussi la carte de restriction du vecteur pOPRSV1/MCS. NOTE 3. Malheureusement, votre copain Eric (je vous laisse deviner lequel…) a brisé votre seul et unique « thermocycler ». Donc, vous ne pouvez pas utiliser le PCR pour cette expérience.

10

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 6 (suite)

11

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 6 (suite)

12

Test de mi-session – CHMI 3216F – 30 octobre 2007

Question 6 (suite)

13

CHMI 3216 F

Bioingénierie de l’ADN

Annexe au Test de mi-session

30 octobre 2007

Question 2.1 et 3.1

Question 3.2 LOCUS NM_000546 2629 bp mRNA linear PRI 24-DEC-2006 DEFINITION Homo sapiens tumor protein p53 (Li-Fraumeni syndrome) (TP53), mRNA. ACCESSION NM_000546 VERSION NM_000546.2 GI:8400737 KEYWORDS . SOURCE Homo sapiens (human) ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo. REFERENCE 1 (bases 1 to 2629) AUTHORS Zeng,P.Y. and Berger,S.L. TITLE LKB1 is recruited to the p21/WAF1 promoter by p53 to mediate transcriptional activation JOURNAL Cancer Res. 66 (22), 10701-10708 (2006) PUBMED 17108107 REMARK GeneRIF: Chromatin immunoprecipitation analyses show that LKB1 is recruited directly to the p21/WAF1 promoter, as well as to other p53 activated promoters, in a p53-dependent fashion. REFERENCE 2 (bases 1 to 2629) AUTHORS Laurie,N.A., Donovan,S.L., Shih,C.S., Zhang,J., Mills,N., Fuller,C., Teunisse,A., Lam,S., Ramos,Y., Mohan,A., Johnson,D., Wilson,M., Rodriguez-Galindo,C., Quarto,M., Francoz,S., Mendrysa,S.M., Guy,R.K., Marine,J.C., Jochemsen,A.G. and Dyer,M.A. TITLE Inactivation of the p53 pathway in retinoblastoma JOURNAL Nature 444 (7115), 61-66 (2006) PUBMED 17080083 REMARK GeneRIF: data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought REFERENCE 3 (bases 1 to 2629) AUTHORS Cai,Q.L., Knight,J.S., Verma,S.C., Zald,P. and Robertson,E.S. TITLE EC5S ubiquitin complex is recruited by KSHV latent antigen LANA for degradation of the VHL and p53 tumor suppressors JOURNAL PLoS Pathog. 2 (10), E116 (2006) PUBMED 17069461 REMARK GeneRIF: In transfected cells and KSHV-infected B lymphoma cells, KSHV-encoded latency-associated nuclear antigen (LANA) expression stimulates degradation of tumor suppressors von Hippel-Lindau and p53. REFERENCE 4 (bases 1 to 2629) AUTHORS Feuerborn,A., Moritz,C., Von Bonin,F., Dobbelstein,M., Trumper,L., Sturzenhofecker,B. and Kube,D. TITLE Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma JOURNAL Leuk. Lymphoma 47 (9), 1932-1940 (2006) PUBMED 17065008

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=9606http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=17108107http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=17080083http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=17069461http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=17065008

REFERENCE 5 (bases 1 to 2629) AUTHORS Perra,M.T., Maxia,C., Corbu,A., Minerba,L., Demurtas,P., Colombari,R., Murtas,D., Bravo,S., Piras,F. and Sirigu,P. TITLE Oxidative stress in pterygium: relationship between p53 and 8-hydroxydeoxyguanosine JOURNAL (er) Mol. Vis. 12, 1136-1142 (2006) PUBMED 17093398 REMARK GeneRIF: Although pterygium has limited local invasion and ainability to metastasize, concomitant presence of altered p53 in 8-OHdG-immunoreactive cells could provide evidence of apparent genetic instability, which is in contrast to its benign clinical course. REFERENCE 6 (bases 1 to 2629) AUTHORS Seto,E., Usheva,A., Zambetti,G.P., Momand,J., Horikoshi,N., Weinmann,R., Levine,A.J. and Shenk,T. TITLE Wild-type p53 binds to the TATA-binding protein and represses transcription JOURNAL Proc. Natl. Acad. Sci. U.S.A. 89 (24), 12028-12032 (1992) PUBMED 1465435REFERENCE 7 (bases 1 to 2629) AUTHORS Baudier,J., Delphin,C., Grunwald,D., Khochbin,S. and Lawrence,J.J. TITLE Characterization of the tumor suppressor protein p53 as a protein kinase C substrate and a S100b-binding protein JOURNAL Proc. Natl. Acad. Sci. U.S.A. 89 (23), 11627-11631 (1992) PUBMED 1454855REFERENCE 8 (bases 1 to 2629) AUTHORS Crook,T., Wrede,D., Tidy,J.A., Mason,W.P., Evans,D.J. and Vousden,K.H. TITLE Clonal p53 mutation in primary cervical cancer: association with human-papillomavirus-negative tumours JOURNAL Lancet 339 (8801), 1070-1073 (1992) PUBMED 1349102REFERENCE 9 (bases 1 to 2629) AUTHORS Iavarone,A., Matthay,K.K., Steinkirchner,T.M. and Israel,M.A. TITLE Germ-line and somatic p53 gene mutations in multifocal osteogenic sarcoma JOURNAL Proc. Natl. Acad. Sci. U.S.A. 89 (9), 4207-4209 (1992) PUBMED 1349175REFERENCE 10 (bases 1 to 2629) AUTHORS Zhukov-Verezhnikov,N.N., Anisimov,P.I., Goncharova,N.S., Bochko,G.M. and Karaseva,Z.N. TITLE [Study of the heterogenetic antigens in vaccinal preparations of V. cholerae] JOURNAL Biull Eksp Biol Med 82 (8), 961-962 (1976) PUBMED 1088347COMMENT REVIEWED REFSEQ: This record has been curated by NCBI staff. The reference sequence was derived from M13121.1, M22881.1, U94788.1 and X02469.1. [WARNING] On Dec 29, 2006 this sequence was replaced by gi:120407067. On Jun 9, 2000 this sequence version replaced gi:4507636. Summary: Tumor protein p53, a nuclear protein, plays an essential role in the regulation of cell cycle, specifically in the

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=17093398http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=1465435http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=1454855http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=1349102http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=1349175http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=1088347http://www.ncbi.nlm.nih.gov/RefSeq/http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=M13121.1http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=M22881.1http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=U94788.1http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=X02469.1http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=120407067http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=4507636

transition from G0 to G1. It is found in very low levels in normal cells, however, in a variety of transformed cell lines, it is expressed in high amounts, and believed to contribute to transformation and malignancy. p53 is a DNA-binding protein containing DNA-binding, oligomerization and transcription activation domains. It is postulated to bind as a tetramer to a p53-binding site and activate expression of downstream genes that inhibit growth and/or invasion, and thus function as a tumor suppressor. Mutants of p53 that frequently occur in a number of different human cancers fail to bind the consensus DNA binding site, and hence cause the loss of tumor suppressor activity.

Alterations of the TP53 gene occur not only as somatic mutations in human malignancies, but also as germline mutations in some

cancer-prone families with Li-Fraumeni syndrome. Publication Note: This RefSeq record includes a subset of the publications that are available for this gene. Please see the Entrez Gene record to access additional publications. COMPLETENESS: full length. FEATURES Location/Qualifiers source 1..2629 /organism="Homo sapiens" /mol_type="mRNA" /db_xref="taxon:9606" /chromosome="17" /map="17p13.1" gene 1..2629 /gene="TP53" /note="tumor protein p53 (Li-Fraumeni syndrome); synonyms: p53, LFS1, TRP53" /db_xref="GeneID:7157" /db_xref="HGNC:11998" /db_xref="HPRD:01859" /db_xref="MIM:191170" CDS 252..1433 /gene="TP53" /GO_component="cytoplasm [PMID 7720704]; insoluble fraction [PMID 12915590]; mitochondrion [PMID 12667443]; nuclear matrix [PMID 11080164]; nucleolus [PMID 12080348]; nucleoplasm [PMID 11080164] [PMID 12915590]; nucleus [PMID 7720704]" /GO_function="ATP binding [PMID 8183576]; copper ion binding [PMID 7824276]; DNA strand annealing activity [PMID 8183576]; enzyme binding [PMID 10666337] [PMID 15577914]; metal ion binding; nuclease activity [PMID 11002423]; protein binding [PMID 15782130]; protein heterodimerization activity [PMID 10837489]; protein N-terminus binding [PMID 11861836]; transcription factor activity [PMID 7587074]; zinc ion binding [PMID 10065153]" /GO_process="apoptosis [PMID 7720704]; base-excision

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=9606http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=1&to=2629&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=full_report&list_uids=7157http://www.genenames.org/data/hgnc_data.php?hgnc_id=11998http://www.hprd.org/protein/01859http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=191170http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=252&to=1433&view=gbwithpartshttp://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005737http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=7720704http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005626http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005626http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=12915590http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005739http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=12667443http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0016363http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=11080164http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005730http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=12080348http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005654http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=11080164http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=12915590http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005634http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=7720704http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005524http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=8183576http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005507http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005507http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=7824276http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0000739http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=8183576http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0019899http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=10666337http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=15577914http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0046872http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0004518http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=11002423http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0005515http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=15782130http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0046982http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0046982http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=10837489http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0047485http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0047485http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=11861836http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0003700http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0003700http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=7587074http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0008270http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=10065153http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006915http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=7720704http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006284

repair [PMID 15116721]; caspase activation via cytochrome c [PMID 12667443]; cell aging [PMID 12080348]; cell cycle; cell cycle arrest [PMID 8675009]; cell differentiation [PMID 10065150]; cell proliferation [PMID 10065150]; DNA damage response, signal transduction by p53 class mediator resulting in induction of apoptosis [PMID 14654789]; negative regulation of cell growth [PMID 8986812]; negative regulation of helicase activity [PMID 7663514]; nucleotide-excision repair [PMID 7663514]; protein complex assembly [PMID 12915590]; protein tetramerization [PMID 15116721]; regulation of mitochondrial membrane permeability [PMID 15116721]; regulation of transcription, DNA-dependent [PMID 7587074]; response to DNA damage stimulus [PMID 8986812]; transcription" /note="p53 tumor suppressor" /codon_start=1 /product="tumor protein p53" /protein_id="NP_000537.2" /db_xref="GI:8400738" /db_xref="CCDS:CCDS11118.1" /db_xref="GeneID:7157" /db_xref="HGNC:11998" /db_xref="HPRD:01859" /db_xref="MIM:191170" /translation="MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLM LSPDDIEQWFTEDPGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWPLSSSVPSQKT YQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAM AIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVV PYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCA CPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRG RERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDS D" misc_feature 465..467 /gene="TP53" /note="A single base change (g or c at nt 466) gives rise to two variants with either R (more frequent) or P at aa position 72; this aa change is responsible for the observed difference in electrophoretic mobility between the two variants; Region: polymorphic variant" misc_feature 1185..1220 /gene="TP53" /note="Region: Nuclear localization signal" misc_feature 1194..1196 /gene="TP53"

http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006284http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=15116721http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0008635http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0008635http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0008635http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=12667443http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0007569http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=12080348http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0007049http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0007049http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0007050http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=8675009http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0030154http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=10065150http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0008283http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=10065150http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0042771http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0042771http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0042771http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0042771http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=14654789http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0030308http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=8986812http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0051097http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=7663514http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006289http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=7663514http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006461http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006461http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006461http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=12915590http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0051262http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=15116721http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0046902http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0046902http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=15116721http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006355http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006355http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006355http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=7587074http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006974http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006974http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&list_uids=8986812http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?view=details&depth=1&query=0006350http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NP_000537.2http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&DATA=CCDS11118.1http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=full_report&list_uids=7157http://www.genenames.org/data/hgnc_data.php?hgnc_id=11998http://www.hprd.org/protein/01859http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=191170http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400738&itemID=5&view=gpwithpartshttp://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400738&from=312&to=323&view=gpwithpartshttp://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400738&itemID=7&view=gpwithparts

/note="phosphorylation site" STS 354..552 /gene="TP53" /standard_name="GDB:177814" /db_xref="UniSTS:154969" STS 368..626 /gene="TP53" /standard_name="GDB:177797" /db_xref="UniSTS:154963" STS 729..803 /gene="TP53" /standard_name="PMC340938P3" /db_xref="UniSTS:273171" STS 1004..1202 /gene="TP53" /standard_name="PMC310707P2" /db_xref="UniSTS:272633" STS 1008..1082 /gene="TP53" /standard_name="GDB:190076" /db_xref="UniSTS:155620" STS 1503..1619 /gene="TP53" /standard_name="D17S1678" /db_xref="UniSTS:82485" STS 2503..2579 /gene="TP53" /standard_name="WI-20715" /db_xref="UniSTS:59997" polyA_signal 2612..2617 /gene="TP53" polyA_site 2629 /gene="TP53" ORIGIN 1 acttgtcatg gcgactgtcc agctttgtgc caggagcctc gcaggggttg atgggattgg 61 ggttttcccc tcccatgtgc tcaagactgg cgctaaaagt tttgagcttc tcaaaagtct 121 agagccaccg tccagggagc aggtagctgc tgggctccgg ggacactttg cgttcgggct 181 gggagcgtgc tttccacgac ggtgacacgc ttccctggat tggcagccag actgccttcc 241 gggtcactgc catggaggag ccgcagtcag atcctagcgt cgagccccct ctgagtcagg 301 aaacattttc agacctatgg aaactacttc ctgaaaacaa cgttctgtcc cccttgccgt 361 cccaagcaat ggatgatttg atgctgtccc cggacgatat tgaacaatgg ttcactgaag 421 acccaggtcc agatgaagct cccagaatgc cagaggctgc tccccgcgtg gcccctgcac 481 cagcagctcc tacaccggcg gcccctgcac cagccccctc ctggcccctg tcatcttctg 541 tcccttccca gaaaacctac cagggcagct acggtttccg tctgggcttc ttgcattctg 601 ggacagccaa gtctgtgact tgcacgtact cccctgccct caacaagatg ttttgccaac 661 tggccaagac ctgccctgtg cagctgtggg ttgattccac acccccgccc ggcacccgcg 721 tccgcgccat ggccatctac aagcagtcac agcacatgac ggaggttgtg aggcgctgcc 781 cccaccatga gcgctgctca gatagcgatg gtctggcccc tcctcagcat cttatccgag 841 tggaaggaaa tttgcgtgtg gagtatttgg atgacagaaa cacttttcga catagtgtgg 901 tggtgcccta tgagccgcct gaggttggct ctgactgtac caccatccac tacaactaca 961 tgtgtaacag ttcctgcatg ggcggcatga accggaggcc catcctcacc atcatcacac 1021 tggaagactc cagtggtaat ctactgggac ggaacagctt tgaggtgcgt gtttgtgcct 1081 gtcctgggag agaccggcgc acagaggaag agaatctccg caagaaaggg gagcctcacc 1141 acgagctgcc cccagggagc actaagcgag cactgcccaa caacaccagc tcctctcccc 1201 agccaaagaa gaaaccactg gatggagaat atttcaccct tcagatccgt gggcgtgagc 1261 gcttcgagat gttccgagag ctgaatgagg ccttggaact caaggatgcc caggctggga 1321 aggagccagg ggggagcagg gctcactcca gccacctgaa gtccaaaaag ggtcagtcta

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=354&to=552&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi?uid=154969http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=368&to=626&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi?uid=154963http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=729&to=803&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi?uid=273171http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=1004&to=1202&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi?uid=272633http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=1008&to=1082&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi?uid=155620http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=1503&to=1619&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi?uid=82485http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=2503&to=2579&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi?uid=59997http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&from=2612&to=2617&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=8400737&itemID=3&view=gbwithparts

1381 cctcccgcca taaaaaactc atgttcaaga cagaagggcc tgactcagac tgacattctc 1441 cacttcttgt tccccactga cagcctccca cccccatctc tccctcccct gccattttgg 1501 gttttgggtc tttgaaccct tgcttgcaat aggtgtgcgt cagaagcacc caggacttcc 1561 atttgctttg tcccggggct ccactgaaca agttggcctg cactggtgtt ttgttgtggg 1621 gaggaggatg gggagtagga cataccagct tagattttaa ggtttttact gtgagggatg 1681 tttgggagat gtaagaaatg ttcttgcagt taagggttag tttacaatca gccacattct 1741 aggtaggtag gggcccactt caccgtacta accagggaag ctgtccctca tgttgaattt 1801 tctctaactt caaggcccat atctgtgaaa tgctggcatt tgcacctacc tcacagagtg 1861 cattgtgagg gttaatgaaa taatgtacat ctggccttga aaccaccttt tattacatgg 1921 ggtctaaaac ttgaccccct tgagggtgcc tgttccctct ccctctccct gttggctggt 1981 gggttggtag tttctacagt tgggcagctg gttaggtaga gggagttgtc aagtcttgct 2041 ggcccagcca aaccctgtct gacaacctct tggtcgacct tagtacctaa aaggaaatct 2101 caccccatcc cacaccctgg aggatttcat ctcttgtata tgatgatctg gatccaccaa 2161 gacttgtttt atgctcaggg tcaatttctt ttttcttttt tttttttttt tttctttttc 2221 tttgagactg ggtctcgctt tgttgcccag gctggagtgg agtggcgtga tcttggctta 2281 ctgcagcctt tgcctccccg gctcgagcag tcctgcctca gcctccggag tagctgggac 2341 cacaggttca tgccaccatg gccagccaac ttttgcatgt tttgtagaga tggggtctca 2401 cagtgttgcc caggctggtc tcaaactcct gggctcaggc gatccacctg tctcagcctc 2461 ccagagtgct gggattacaa ttgtgagcca ccacgtggag ctggaagggt caacatcttt 2521 tacattctgc aagcacatct gcattttcac cccacccttc ccctccttct ccctttttat 2581 atcccatttt tatatcgatc tcttatttta caataaaact ttgctgcca

Question 6 LOCUS BC016497 1492 bp mRNA linear ROD 29-JUN-2004 DEFINITION Mus musculus eukaryotic translation initiation factor 2, subunit 1 alpha, mRNA (cDNA clone MGC:25484 IMAGE:4501321), complete cds. ACCESSION BC016497 VERSION BC016497.1 GI:16741331 KEYWORDS MGC. SOURCE Mus musculus (house mouse) ORGANISM Mus musculus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Mus. REFERENCE 1 (bases 1 to 1492) AUTHORS Strausberg,R.L., Feingold,E.A., Grouse,L.H., Derge,J.G., Klausner,R.D., Collins,F.S., Wagner,L., Shenmen,C.M., Schuler,G.D., Altschul,S.F., Zeeberg,B., Buetow,K.H., Schaefer,C.F., Bhat,N.K., Hopkins,R.F., Jordan,H., Moore,T., Max,S.I., Wang,J., Hsieh,F., Diatchenko,L., Marusina,K., Farmer,A.A., Rubin,G.M., Hong,L., Stapleton,M., Soares,M.B., Bonaldo,M.F., Casavant,T.L., Scheetz,T.E., Brownstein,M.J., Usdin,T.B., Toshiyuki,S., Carninci,P., Prange,C., Raha,S.S., Loquellano,N.A., Peters,G.J., Abramson,R.D., Mullahy,S.J., Bosak,S.A., McEwan,P.J., McKernan,K.J., Malek,J.A., Gunaratne,P.H., Richards,S., Worley,K.C., Hale,S., Garcia,A.M., Gay,L.J., Hulyk,S.W., Villalon,D.K., Muzny,D.M., Sodergren,E.J., Lu,X., Gibbs,R.A., Fahey,J., Helton,E., Ketteman,M., Madan,A., Rodrigues,S., Sanchez,A., Whiting,M., Madan,A., Young,A.C., Shevchenko,Y., Bouffard,G.G., Blakesley,R.W., Touchman,J.W., Green,E.D., Dickson,M.C., Rodriguez,A.C., Grimwood,J., Schmutz,J., Myers,R.M., Butterfield,Y.S., Krzywinski,M.I., Skalska,U., Smailus,D.E., Schnerch,A., Schein,J.E., Jones,S.J. and Marra,M.A. TITLE Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences JOURNAL Proc. Natl. Acad. Sci. U.S.A. 99 (26), 16899-16903 (2002) PUBMED 12477932REFERENCE 2 (bases 1 to 1492) AUTHORS Strausberg,R. TITLE Direct Submission JOURNAL Submitted (31-OCT-2001) National Institutes of Health, Mammalian Gene Collection (MGC), Cancer Genomics Office, National Cancer Institute, 31 Center Drive, Room 11A03, Bethesda, MD 20892-2590, USA REMARK NIH-MGC Project URL: http://mgc.nci.nih.govCOMMENT Contact: MGC help desk Email: cgapbs-r@mail.nih.gov Tissue Procurement: The Cepko Laboratory cDNA Library Preparation: Life Technologies, Inc. cDNA Library Arrayed by: The I.M.A.G.E. Consortium (LLNL) DNA Sequencing by: Baylor College of Medicine Human Genome Sequencing Center Center code: BCM-HGSC

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=10090http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&query_hl=1&list_uids=12477932http://mgc.nci.nih.gov/

Web site: http://www.hgsc.bcm.tmc.edu/cdna/ Contact: amg@bcm.tmc.edu Gunaratne, P.H., Garcia, A.M., Lu, X., Hulyk, S.W., Loulseged, H., Kowis, C.R., Sneed, A.J., Martin, R.G., Muzny, D.M., Nanavati, A.N., Gibbs, R.A. Clone distribution: MGC clone distribution information can be found through the I.M.A.G.E. Consortium/LLNL at: http://image.llnl.gov Series: IRAK Plate: 31 Row: k Column: 7 This clone was selected for full length sequencing because it passed the following selection criteria: matched mRNA gi: 13385623. FEATURES Location/Qualifiers source 1..1492 /organism="Mus musculus" /mol_type="mRNA" /db_xref="taxon:10090" /clone="MGC:25484 IMAGE:4501321" /tissue_type="Eye, retina, mouse strain C57Bl\6" /clone_lib="NIH_MGC_94" /lab_host="DH10B" /note="Vector: pCMV-SPORT6" gene 1..1492 /gene="Eif2s1" /note="synonyms: 0910001O23Rik, eIF2alpha, 35kDa" /db_xref="GeneID:13665" /db_xref="MGI:95299" CDS 115..1062 /gene="Eif2s1" /codon_start=1 /product="eukaryotic translation initiation factor 2, subunit 1 alpha" /protein_id="AAH16497.1" /db_xref="GI:16741332" /db_xref="GeneID:13665" /db_xref="MGI:95299" /translation="MPGLSCRFYQHKFPEVEDVVMVNVRSIAEMGAYVSLLEYNNIEG MILLSELSRRRIRSINKLIRIGRNECVVVIRVDKEKGYIDLSKRRVSPEEAIKCEDKF TKSKTVYSILRHVAEVLEYTKDEQLESLFQRTAWVFDDKYKRPGYGAYDAFKHAVSDP SILDSLDLNEDEREVLINNINRRLTPQAVKIRADIEVACYGYEGIDAVKEALRAGLNC STETMPIKINLIAPPRYVMTTTTLERTEGLSVLNQAMAVIKEKIEEKRGVFNVQMEPK VVTDTDETELARQLERLERENAEVDGDDDAEEMEAKAED" misc_difference 1468..1492 /gene="Eif2s1" /note="polyA tail: 25 bases do not align to the mouse genome; Differences found between this sequence and the mouse C57BL/6J genome (build 35) are described in misc_difference features below" ORIGIN 1 gcgaagcgca cgctgaggag cttcggcgtc cggggttggt gttccgagtc tcctcgttgc

http://www.hgsc.bcm.tmc.edu/cdna/http://image.llnl.gov/http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=10090http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=16741331&itemID=2&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=retrieve&dopt=graphics&list_uids=13665http://www.informatics.jax.org/searches/accession_report.cgi?id=MGI:95299http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=16741331&itemID=1&view=gbwithpartshttp://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=AAH16497.1http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=retrieve&dopt=graphics&list_uids=13665http://www.informatics.jax.org/searches/accession_report.cgi?id=MGI:95299http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=16741331&itemID=4&view=gbwithparts

61 cactaagcag cacggcgtgg gaccctactt cgggattcac acatccactt cagaatgccg 121 gggctaagtt gtagatttta tcaacacaaa tttcctgagg tggaagatgt agtgatggtg 181 aatgtaagat ccattgctga aatgggggcc tatgtcagct tgttggaata taataacatt 241 gaaggcatga ttcttcttag tgaattatcc agacgacgta tccgttctat aaacaaactg 301 atccgaattg gcagaaatga atgtgttgtt gtcattagag tggataaaga aaaaggatat 361 atagatttgt caaaaagaag agtttctcca gaggaagcaa tcaaatgtga ggacaaattc 421 acaaaatcca aaactgttta tagcattctt cgccatgttg ctgaggtatt agaatatacc 481 aaggatgagc agctggagag cctgttccag aggactgcct gggtcttcga tgacaagtac 541 aagagacctg gatacggtgc ctacgatgct tttaagcatg cagtctcaga cccatctatc 601 ttggatagtt tagatttgaa tgaagatgaa agagaagtac tcattaataa tatcaatagg 661 cgtttgaccc cacaagcggt caaaattcga gcagatattg aagtagcttg ctatggttat 721 gaaggcattg atgctgtaaa agaagccctg agggcaggtt tgaattgttc tacagaaacc 781 atgcccatca agattaatct aatagctcca cccaggtatg tgatgacaac aacgaccctg 841 gagaggacag aaggcctgtc tgtcctcaat caggctatgg cagttatcaa agagaagatc 901 gaggagaaga ggggcgtctt caatgttcag atggagccca aagtggtcac agatacagat 961 gagactgaac ttgcaaggca gctggaacgg ctggagagag aaaatgcaga agtggatgga 1021 gatgatgatg cagaagaaat ggaagccaaa gctgaagatt aacgttttgg caaacagtcc 1081 aatttaagga gtacgaagca gcccttcctg gctgtcaacc ctagacttga gagttttcca 1141 gtattgaaaa cttcaaagct gaatattttt atttccaagt atttaagtat tcaacaagcc 1201 agaatctaaa tgccctcctt catgtcagct gtttccacat agtggctcta acacctcaag 1261 catttttcaa gggagtggct tgatttgacc agagacaaat cttaaacagc agtcctaata 1321 ttgggcctac agtttccatt tctcatgtct ctggaatggc acccttatgg ttcagagatt 1381 taccagggac tccaaacaca aacaatccca atctttctat ataaaatgta ttcaagcaaa 1441 catcaaataa atttctggga tatttaaaaa aaaaaaaaaa aaaaaaaaaa aa

Question 6.

Name: eIF2a

Conformation: linear

Overhang: five_prime, three_prime, blunt

Minimum Site Length: 6 bases

Maximum Number of Cuts: 2

Included: all commercial, prototypes only

Noncutters: AarI, AatII, AccI, AflII, AflIII, AgeI, AlwNI, ApaI, ApaLI, AscI, AsuII, AvaI, AvrII, BaeI, BalI, BamHI, BcgI, BclI, BfiI, BglII, BsaAI, BsaBI, BsePI, BseSI, BseYI, BsgI, Bsp1407I, BspHI, BspLU11I, BsrBI, BstEII, BstXI, BtrI, BtsI, CfrI, Cfr10I, ClaI, DrdI, Eam1105I, EciI, Eco47III, EcoRI, EcoRV, Esp3I, FseI, FspAI, HaeII, HindIII, HpaI, KpnI, MfeI, MluI, NaeI, NarI, NcoI, NdeI, NheI, NotI, NruI, OliI, PI-PspI, PI-SceI, PacI, PfoI, PmaCI, PmeI, PshAI, PsiI, PsrI, PstI, PvuI, RsrII, SacI, SacII, SalI, SapI, SexAI, SfiI, SgfI, SgrAI, SmaI, SnaBI, SpeI, SrfI, Sse8387I, SwaI, TaqII, Tth111I, XbaI, XhoI

Name Sequence Site Length Overhang Frequency Cut Positions

HindII GTYRAC 6 blunt 1 1116

ScaI AGTACT 6 blunt 1 638

StuI AGGCCT 6 blunt 1 854

XmnI GAANNNNTTC 6 blunt 1 381

AclI AACGTT 6 five_prime 1 1062

BspMI ACCTGC 6 five_prime 1 745

Eco31I GGTCTC 6 five_prime 1 537

EcoNI CCTNNNNNAGG 6 five_prime 1 868

PpuMI RGGWCCY 7 five_prime 1 80

SanDI GGGWCCC 7 five_prime 1 80

StyI CCWWGG 6 five_prime 1 479

XhoII RGATCY 6 five_prime 1 187

http://rebase.neb.com/rebase/enz/HindII.htmlhttp://rebase.neb.com/rebase/enz/ScaI.htmlhttp://rebase.neb.com/rebase/enz/StuI.htmlhttp://rebase.neb.com/rebase/enz/XmnI.htmlhttp://rebase.neb.com/rebase/enz/AclI.htmlhttp://rebase.neb.com/rebase/enz/BspMI.htmlhttp://rebase.neb.com/rebase/enz/Eco31I.htmlhttp://rebase.neb.com/rebase/enz/EcoNI.htmlhttp://rebase.neb.com/rebase/enz/PpuMI.htmlhttp://rebase.neb.com/rebase/enz/SanDI.htmlhttp://rebase.neb.com/rebase/enz/StyI.htmlhttp://rebase.neb.com/rebase/enz/XhoII.html

Name Sequence Site Length Overhang Frequency Cut Positions

BglI GCCNNNNNGGC 6 three_prime 1 751

BsrDI GCAATG 6 three_prime 1 191

MmeI TCCRAC 6 three_prime 1 203

NspI RCATGY 6 three_prime 1 580

PflMI CCANNNNNTGG 6 three_prime 1 1241

SduI GDGCHC 6 three_prime 1 938

SphI GCATGC 6 three_prime 1 580

XcmI CCANNNNNNNNNTGG 6 three_prime 1 198

MslI CAYNNNNRTG 6 blunt 2 337, 1364

SspI AATATT 6 blunt 2 1164, 1319

AcyI GRCGYC 6 five_prime 2 26, 914

BbvCI CCTCAGC 7 five_prime 2 13, 461

Bpu10I CCTNAGC 6 five_prime 2 13, 461

DraII RGGNCCY 6 five_prime 2 80, 206

Ksp632I CTCTTC 6 five_prime 2 372, 901

SmlI CTYRAG 6 five_prime 2 1126, 1255

TatI WGTACW 6 five_prime 2 536, 636

VspI ATTAAT 6 five_prime 2 644, 794

AloI GAACNNNNNNTCC 7 three_prime 2 487, 519

BciVI GTATCC 6 three_prime 2 289, 544

BplI GAGNNNNNCTC 6 three_prime 2 623, 655

BsaXI ACNNNNNCTCC 6 three_prime 2 487, 517

BsmI GAATGC 6 three_prime 2 119, 443

DraIII CACNNNGTG 6 three_prime 2 76, 1241

Eco57I CTGAAG 6 three_prime 2 93, 1073

FalI AAGNNNNNCTT 6 three_prime 2 1087, 1119

Hin4I GAYNNNNNVTC 6 three_prime 2 263, 295

PpiI GAACNNNNNCTC 7 three_prime 2 487, 519

http://rebase.neb.com/rebase/enz/BglI.htmlhttp://rebase.neb.com/rebase/enz/BsrDI.htmlhttp://rebase.neb.com/rebase/enz/MmeI.htmlhttp://rebase.neb.com/rebase/enz/NspI.htmlhttp://rebase.neb.com/rebase/enz/PflMI.htmlhttp://rebase.neb.com/rebase/enz/SduI.htmlhttp://rebase.neb.com/rebase/enz/SphI.htmlhttp://rebase.neb.com/rebase/enz/XcmI.htmlhttp://rebase.neb.com/rebase/enz/MslI.htmlhttp://rebase.neb.com/rebase/enz/SspI.htmlhttp://rebase.neb.com/rebase/enz/AcyI.htmlhttp://rebase.neb.com/rebase/enz/BbvCI.htmlhttp://rebase.neb.com/rebase/enz/Bpu10I.htmlhttp://rebase.neb.com/rebase/enz/DraII.htmlhttp://rebase.neb.com/rebase/enz/Ksp632I.htmlhttp://rebase.neb.com/rebase/enz/SmlI.htmlhttp://rebase.neb.com/rebase/enz/TatI.htmlhttp://rebase.neb.com/rebase/enz/VspI.htmlhttp://rebase.neb.com/rebase/enz/AloI.htmlhttp://rebase.neb.com/rebase/enz/BciVI.htmlhttp://rebase.neb.com/rebase/enz/BplI.htmlhttp://rebase.neb.com/rebase/enz/BsaXI.htmlhttp://rebase.neb.com/rebase/enz/BsmI.htmlhttp://rebase.neb.com/rebase/enz/DraIII.htmlhttp://rebase.neb.com/rebase/enz/Eco57I.htmlhttp://rebase.neb.com/rebase/enz/FalI.htmlhttp://rebase.neb.com/rebase/enz/Hin4I.htmlhttp://rebase.neb.com/rebase/enz/PpiI.html

Sites de restriction pour pOPRSV/MCS : Enzyme Nombre de nucléotides additionnels Site de restriction requis en 5’ pour couper cette séquence Cla I : 2 EcoR V : 1 Kpn I : 2 Not I : 8 Sma I : 3 Spe I : 2 Xba I : 1 Xho I : 1