首先，限制性酶切位点就不用多说了吧，它就是DNA分子上被特异性的核苷酸序列（4-8bp），能被限制性内切酶识别，从而切割。
保护碱基：限制性内切酶识别特定的DNA序列，除此之外，酶蛋白还要占据识别位点两边的若干个碱基，这些碱基对内切酶稳定的结合到DNA双链并发挥切割DNA作用是有很大影响的，被称为保护碱基。【百度百科】
Kozak序列：KOZAK是一个女科学家，她研究过起始密码子AUG周边碱基定点突变后对转录和翻译所造成的影响，并总结出在真核生物中，起始密码子两端序列为：—— G/N-C/N-C/N-ANNAUGG——，如GCCACCAUGG、GCCAUGAUGG时，转录和翻译效率最高，特别是-3位的A对翻译效率非常重要。该序列被后人称为Kozak序列，并被应用于表达载体的构建中。
所以，引物设计上游引物就是在起始密码子前面5‘-保护碱基+酶切位点+Kozak+ATG+一小段目的基因序列
下游引物不用加Kozak序列，通常是5‘-保护碱基+酶切位点+终止密码子+（如果你有要表达的标签序列）+一小段目的基因序列
注意，我这里的介绍的引物设计针对目的基因要表达的，pcr出来后是要连接到表达载体上的，所以引物比较长，一般都有40多bp了，不过一样能够p出来。
好了，下面随便找一个基因为例来实战一下，比如我要将Stat5a从一个公司给的质粒上p出来，然后连接到一个表达载体上，采用直接双酶切目的基因和表达载体，然后连接的方式。在UCSC上找到Stat5a的CDs区。
从起始密码子ATG到终止密码子TGA复制到一个word里，
接下来用DNAMAN看这段CDs区里面存在哪些酶切位点，然后根据你表达载体多克隆位点存在哪些酶切位点，选择两个在表达载体多克隆位点有但是CDs区里面没有的两个酶切位点进行设计。不然内切酶把你的目的基因切断了就玩不下去了。。。。。
假设我以BamH1、Sal1分别作为上下游的酶切位点
好了，现在去百度找酶切位点保护碱基
百度文库很多这样的表，找到BamH1、Sal1，注意该表格后两列表示酶切2h、20h的酶切效率，当然是选最高的那个最好。
百度文库很多这样的表，找到BamH1、Sal1，注意该表格后两列表示酶切2h、20h的酶切效率，当然是选最高的那个最好。
上游：CGCGGATCCGCCACCATGGCGGGCTGGATTCAGGC
下游（特别注意要你设计出来的要反向互补）：GCGTCGACTCAGGACAGGGAGCTTCTA
好了，引物就这样设计好了，很简单有没有~~~
？？用DNAMAN怎么找酶切位点，下次再说~~~
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