You are interested in cloning a gene that codes for an enzyme that produces a blue pigment. You have chosen to use the plasmid pUC19 for cloning (shown below). Drdl 91 Acll 2297 Xmnl 2294 Bcgl 2215 Scal 2177 Pvul 2066 Avall 2059 BsrDI 1935 Acll 1924 Fspl 1919- Avall 1837 NmeAIII 1822 Bell 1813- Bpml 1784 BsrFI 1779 BciVI 2542 Aatll-Zral 2617 Sspl 2501 Bsal 1766 BsrDI 1753 Bmrl 1744 Ahdl 1694 BsmBI 51 BsmBI 2683 Eco01091 2674 PUC19 2,686 bp BoeAl 1292 ori BsTAPI 179 Ndel 183 Kasl - Narl - Sfol 235 Bell 245 Fspl 256 lacZo AlwNI 1217 Pvul 276 Pvull 306 Bmrl 364 BseYI 1110 BceAl 387 MCS BeiVI 1015 Til 781 AflIII - Pcil 806 Drdl 908 Apol-EcoRI 396 Banll-Sacl - Eco53KI 402 Acc651- Kpnl 408 Aval - BsoBI - Smal TspMI - Xmal 412 Pvull 628 Tfil 641 BsaXI 659 BspQI-Sapl 683 BamHI 417 Xbal 423 Accl - Hincll-Sall 429 BspMI -BfuAI 433 Sbfl 434 Pstl 435 Sphl 441 Hind 447 The plasmid codes for three elements, lacZ, ApR and ori. What are these three things? Which restriction enzyme cut sites would you use to clone your blue pigment gene into pUC19 and why? You digest the plasmid and your blue pigment gene with the restriction enzymes chosen in step b. Explain how the two fragments will stick together, and explain how you will stich the phosphate backbone back up.

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