You have a cDNA, part of the sequence of which is reproduced in Figure 3. You want to insert the portion encoding the highlighted region (WW3) into an expression vector, pAcGFP1 (Fig. 4) to produce a protein of fusion with the sequence of the fluorescent protein GFP. This protein can be detected by fluorescence microscopy.

FIGURE 3 - Region 1243 to 1559 of the cDNA encoding the ITCH protein (GenBank XM_024452005). The translation in amino acids is indicated above the sequence with the amino acid aligned with the zth nucleotide of each codon. The portion encoding the W W3 domain is shown by the black line above the sequence.

Figure 4 - Graphic representation of the vectors pAcGFP1-C1, C2 and C3. The three vectors are identical except for the nucleotides between position 1330 and the BglII site. The last two codons of the AcGFP sequence followed by the multicloning site of each of the vectors are detailed.

a) Using these primers in a PCR reaction, you will be able to create a new sequence containing the BglII and EcoRI restriction sites at each end of the WW3 region. What will be the sequence of the amplified product? BglII 5-5-GAGAAGATCTTTGATCCTCTTGGTCCATTG-3 EcoRI 5-GAGAGAATTCACTTCTGGGGTCTTCCC-3 b) Which of the vectors (C1, C2 or C3) will allow you to obtain a fusion protein between GFP and the WW3 domain of ITCH, using the enzymes BglII and EcoRI to produce sticky ends and recombine the DNAs? c) Write the sequence resulting from the recombination of the chosen vector and the amplified DNA in nucleotides and amino acids (one letter). Show only the beginning of the sequence, from position 1330 of the vector to position 1377 of the Itch cDNA sequence, inclusive. Separate the nucleotide sequence into triplets respecting the reading frame of the vector and align the amino acids with these triplets. Use a fixed-width font for easier alignment.

d) You must also combine the WW3 domain in a pro-karyotic expression vector to produce the protein in large quantities. The vector you have at your disposal for this is the vector pGEX-4T-1 (Fig. 5). Which of the two primers would you need to adapt and what would be the sequence of that primer? Note: there is more than one possible solution.

Figure 5 - Graphical representation of the expression vector pGEX-4T-1. This vector allows the GST sequence to be fused to the N-terminal side of the fusion protein. The sequence at the top of the image shows the end of the GST sequence followed by the vector multicloning site.

W QL Q R SQL QGAM QQF NQRFI YG TGGCAGCTACAGCGTAGTCAGCTTCAAGGAGCAATGCAGCAGTTTAACCAGAGATTCATTTATGG 1243 WW3 NQ D L F A T S Q S K E F D P L G P L P P GAATCAAGATTTATTTGCTACATCACAAAGTAAAGAATTTGATCCTCTTGGTCCATTGCCACCTG 1308 WW3 GWE KRT DS N G R VYF V NH N T R I T GATGGGAGAAGAGA ACAGACAGCAATGGCAGAGTATATTTCGTCAACCACAACACACGAATTACA 1373 WW3 QW EDP R S QG QLN E K P L P E G W E M CAATGGGAAGACCCCAGAAGTCAAGGTCAATTAA ATGAAAAGCCCTTACCTGAAGGTTGGGAAAT 1438 RF TV D G I PYF V D H N R R TT GAGATTCACAGTGGATGGAATTCCATATTTTGTGGACCACAATAGAAGAACTACCAC 1503 Nhel (592) Agel (601) PCMV IE PUC ori ACGFP1 /HSV TK poly A Pvu ll (1147) PACGFP1 4.7 kb SV40 poly A Kan MCS (1330-1417) Neo SV40 ori P f1 Pori SV40 e Pvu ll (2862) Pvu ll (2254) PACGFP1-C1 1330 AcGFP1 1340 1350 1360 1370 1380 1390 TAC AAG TCC GGA CTC AGA TCT CGA GCT CAA GCT TCG AAT TCT GCA GTC GAC GGT ACC GCG GGC CCG GGA TCC ACC BspE Bg/ll Xhol Hind III EcoRI Pst Sall Sac KpnI Accl Asp7181 Sac II Xmal Smal 1400 STOPS GGA TCT AGA TAA CTG ATC A Xbal" Bc/I Apal ACGFP1 1350 1370 1380 STOPS PACGFP1-C2 1330 1340 1360 1390 1400 TAC AAG TCC GGC CGG ACT CAG ATC TCG AGC TCA AGC TTC GAA TTC TGC AGT CGA CGG TAC CGC GGG CCC GGG ATC CAC CGG ATC TAG ATA ACT GAT CA Eag! Bg/Il Xhol Sac Hind III EcoRIPst Sall KpnI Apal BamHI Xbal Bcll Accl Asp7181 Xmal Sac II Smal PACGFP1-C3 1330 1340 1350 1360 1370 1380 1390 STOPS TAC AAG TAC TCA GAT CTC GAG CTC AAG CTT CGA ATT CTG CAG TCG ACG GTA CCG CGG GCC CGG GAT CCA CCG GAT CTA GAT AAC TGA TCA Scal Bg/11 Xhol Hind III Sac EcoRI Pst Sall KpnI Apal BamHI Xbal Bcll Accl Asp7181 Xmal Sac II Smal AcGFP1. PGEX-4T-1 (27-4580-01) Thrombin Leu Val Pro Arg Gly Serl Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp CTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG, CGG CCG CAT CGT GAC TGA BamHI EcoRI Smal Sali Xho Not Stop codons Th1111 Aatii Bal glutathione S-transferase Ptac BspMI. pSj100Bam7Stop7 Amp Pst! PGEX -4900 bp Nar AlwNI EcoR V lac, 9 p4.5 BssH II Apa BstE 11 PBR322 ori Mlu! W QL Q R SQL QGAM QQF NQRFI YG TGGCAGCTACAGCGTAGTCAGCTTCAAGGAGCAATGCAGCAGTTTAACCAGAGATTCATTTATGG 1243 WW3 NQ D L F A T S Q S K E F D P L G P L P P GAATCAAGATTTATTTGCTACATCACAAAGTAAAGAATTTGATCCTCTTGGTCCATTGCCACCTG 1308 WW3 GWE KRT DS N G R VYF V NH N T R I T GATGGGAGAAGAGA ACAGACAGCAATGGCAGAGTATATTTCGTCAACCACAACACACGAATTACA 1373 WW3 QW EDP R S QG QLN E K P L P E G W E M CAATGGGAAGACCCCAGAAGTCAAGGTCAATTAA ATGAAAAGCCCTTACCTGAAGGTTGGGAAAT 1438 RF TV D G I PYF V D H N R R TT GAGATTCACAGTGGATGGAATTCCATATTTTGTGGACCACAATAGAAGAACTACCAC 1503 Nhel (592) Agel (601) PCMV IE PUC ori ACGFP1 /HSV TK poly A Pvu ll (1147) PACGFP1 4.7 kb SV40 poly A Kan MCS (1330-1417) Neo SV40 ori P f1 Pori SV40 e Pvu ll (2862) Pvu ll (2254) PACGFP1-C1 1330 AcGFP1 1340 1350 1360 1370 1380 1390 TAC AAG TCC GGA CTC AGA TCT CGA GCT CAA GCT TCG AAT TCT GCA GTC GAC GGT ACC GCG GGC CCG GGA TCC ACC BspE Bg/ll Xhol Hind III EcoRI Pst Sall Sac KpnI Accl Asp7181 Sac II Xmal Smal 1400 STOPS GGA TCT AGA TAA CTG ATC A Xbal" Bc/I Apal ACGFP1 1350 1370 1380 STOPS PACGFP1-C2 1330 1340 1360 1390 1400 TAC AAG TCC GGC CGG ACT CAG ATC TCG AGC TCA AGC TTC GAA TTC TGC AGT CGA CGG TAC CGC GGG CCC GGG ATC CAC CGG ATC TAG ATA ACT GAT CA Eag! Bg/Il Xhol Sac Hind III EcoRIPst Sall KpnI Apal BamHI Xbal Bcll Accl Asp7181 Xmal Sac II Smal PACGFP1-C3 1330 1340 1350 1360 1370 1380 1390 STOPS TAC AAG TAC TCA GAT CTC GAG CTC AAG CTT CGA ATT CTG CAG TCG ACG GTA CCG CGG GCC CGG GAT CCA CCG GAT CTA GAT AAC TGA TCA Scal Bg/11 Xhol Hind III Sac EcoRI Pst Sall KpnI Apal BamHI Xbal Bcll Accl Asp7181 Xmal Sac II Smal AcGFP1. PGEX-4T-1 (27-4580-01) Thrombin Leu Val Pro Arg Gly Serl Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp CTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG, CGG CCG CAT CGT GAC TGA BamHI EcoRI Smal Sali Xho Not Stop codons Th1111 Aatii Bal glutathione S-transferase Ptac BspMI. pSj100Bam7Stop7 Amp Pst! PGEX -4900 bp Nar AlwNI EcoR V lac, 9 p4.5 BssH II Apa BstE 11 PBR322 ori Mlu