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<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">Hi Tim, <div><br class="webkit-block-placeholder"></div><div>Though I am not so sure, lots of references seem to hint pPS904 was originally known as pKG5 in this article: Kahana, J., Schnapp, B.J.,Silver, P.A. (1995) PNAS 92:9707-9711. In its materials section, the author wrote the following:</div><div><br class="webkit-block-placeholder"></div><div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 11.5px/normal Courier; "><span style="font: 11.0px Courier">Plasmid </span>pKG5 <span style="font: 11.0px Courier">was </span>constructed <span style="font: 11.0px Courier">as <span class="Apple-style-span" style="font-size: 12px; "><span style="font: 11.5px Courier">follows: </span>wild-type <span style="font: 11.5px Courier">GFP </span><span style="font: 10.5px Courier">was </span>PCR-amplified <span style="font: 11.5px Courier">from </span>plasmid <span style="font: 11.0px Courier">TU<span class="Apple-style-span" style="font-size: 12px; "><span style="font: 11.0px Courier">65 </span><span style="font: 11.5px Courier">(5) using </span><span style="font: 11.0px Courier">the 5' </span><span style="font: 11.5px Courier">Xho </span><span style="font: 11.0px Courier">I-linked </span><span style="font: 11.5px Courier">primer </span>CCGCTCGAGCTATGAGTAAAGGAGAAGA and <span style="font: 12.0px Courier">the </span>3' 17 <span style="font: 12.0px Courier">primer. </span>The PCR <span class="Apple-style-span" style="font-size: 11px; "><span style="font: 11.5px Courier">product </span>was cut with <span style="font: 11.5px Courier">Xho </span>I and Pst I <span style="font: 12.0px Courier">(New </span><span style="font: 11.5px Courier">England </span><span style="font: 12.0px Courier">Biolabs).</span></span></span></span></span></span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 12px/normal Courier; "><span style="font: 11.0px Courier">The NUF2 open </span>reading <span style="font: 11.5px Courier">frame </span><span style="font: 10.5px Courier">was </span>amplified <span style="font: 11.5px Courier">from </span>plasmid <span class="Apple-style-span" style="font-size: 11px; "><span style="font: 11.5px Courier">pPS511 </span><span style="font: 12.0px Courier">(4) using </span><span style="font: 11.5px Courier">the BamHI-linked </span>5' <span style="font: 12.0px Courier">primer GCGGATC</span>CATGAGTAGGAATCAAGATGTC <span class="Apple-style-span" style="font-size: 12px; "><span style="font: 11.5px Courier">and the </span>in-frame, Xho<span style="font: 11.0px Courier">I-linked 3' </span>primer CCCTCGAGCTTGCATATATTCGAG<span style="font: 11.0px Courier">CAT<span class="Apple-style-span" style="font-size: 12px; ">CCCTCGAGCTTGCATATATTCGAG<span style="font: 11.0px Courier">CAT.</span></span></span></span></span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 11px/normal Courier; ">The PCR <span style="font: 11.5px Courier">product </span>was cut with BamHI and Xho I. The <span class="Apple-style-span" style="font-size: 12px; "><span style="font: 11.0px Courier">two </span>fragments <span style="font: 11.0px Courier">were </span>ligated together <span style="font: 11.0px Courier">into </span>plasmid pPS293 (a</span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 11.5px/normal Courier; "><span style="font: 12.5px Courier">2-gm </span>URA3 vector <span style="font: 11.0px Courier">with </span>the GALl <span style="font: 11.0px Courier">promoter </span>sequence) <span style="font: 11.0px Courier">cut <span class="Apple-style-span" style="font-size: 12px; ">with BamHI <span style="font: 11.0px Courier">and Pst I.</span></span></span></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 11.5px/normal Courier; "><font class="Apple-style-span" size="3"><span class="Apple-style-span" style="font-size: 11px;"><br class="webkit-block-placeholder"></span></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 11.5px/normal Courier; "><font class="Apple-style-span" size="3"><span class="Apple-style-span" style="font-size: 11px;"><span class="Apple-style-span" style="font-family: Helvetica; font-size: 14px; ">So the backbone of pPS904 is just double digested pPS293, whereas the insert is concatenation of GFP and NUF2.</span></span></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 11.5px/normal Courier; "><font class="Apple-style-span" face="Helvetica" size="4"><span class="Apple-style-span" style="font-size: 14px;"><br class="webkit-block-placeholder"></span></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 11.5px/normal Courier; "><font class="Apple-style-span" face="Helvetica" size="4"><span class="Apple-style-span" style="font-size: 14px;">Cheers,</span></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 11.5px/normal Courier; "><font class="Apple-style-span" face="Helvetica" size="4"><span class="Apple-style-span" style="font-size: 14px;"><br class="webkit-block-placeholder"></span></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 11.5px/normal Courier; "><font class="Apple-style-span" face="Helvetica" size="4"><span class="Apple-style-span" style="font-size: 14px;">Wei</span></font></div></div><div><div apple-content-edited="true"> <span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 14px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0; "><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; "><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; "><div><div>__________________________________</div><div><br class="webkit-block-placeholder"></div><div>Wei Chen</div><div><br class="webkit-block-placeholder"></div><div>Laboratory of Yeast Genetics, 6131</div><div>Department of Microbiology</div><div>College of Life Sciences,</div><div>Wuhan University</div><div>Wuhan, 430072</div><div>PR. China</div><div><br></div></div></span></div></span></div></span> </div><br></div></body></html>
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