克隆可以简单理解为复制品，例如假设通过提取mRNA，反转录后得到cDNA序列，然后将这段序列转入载体，再通过划线不断的繁殖，就会得到许多装有这段cDNA序列的克隆，实验室为了方便，在给得到的这些克隆起名时，一般会取cDNA序列的名，但实际上在这个克隆里面不仅包括了这个cDNA，还包括了载体的DNA。

    STS（sequence-tagged site）序列标记位点，是基因组上定位明确、作为界标并能通过PCR扩增被唯一操作的短的、单拷贝DNA序列，一般长度为200-500bp，一个 DNA序列要成为STS，首先序列必须已知，能用PCR方法检测，第二STS必须在基因组上具有唯一的定位点。通过STS可以判断在不同条件下测序得到的 DNA序列的准确性。

    EST（expressed sequence tag）表达序列标签，是从一个随机选择的cDNA克隆，进行5’端和3’端单一次测序挑选出来获得的短的cDNA序列。全基因组测序发现基因即昂贵又费 时，因为基因组中只有2%序列编码蛋白质，因此可以对真正编码蛋白质的mRNA构建cDNA文库，对cDNA进行测序，得到EST序列，从而发现新基因。

可以通过Ensembl查找基因或转录本序列：

http://www.ensembl.org/info/website/tutorials/sequence.html

但是，若采用手动提取转录本序列，则要注意strand的正负链：+/1代表forward strand；-/-1代表的是reverse strand。具体的意思：参考：https://www.biostars.org/p/3423/        也就是如下：

• DNA is double-stranded. By convention, for a reference chromosome, one whole strand is designated the "forward strand" and the other the "reverse strand". This designation is arbitrary. Sometimes the terms "plus strand" and "minus strand" are used instead.

• Visually (I'm not talking about the transcription machinery yet), you would typically read the sequence of a strand in the 5-3 direction. For the forward strand, this means reading left-to-right, and for the reverse strand it means right-to-left.

• A gene can live on a DNA strand in one of two orientations. The gene is said to have a coding strand(also known as its sense strand), and a template strand (also known as its antisense strand). For 50% of genes, its coding strand will correspond to the chromosome's forward strand, and for the other 50% it will correspond to the reverse strand.

• The mRNA (and protein) sequence of a gene corresponds to the DNA sequence as read (again, visually) from the gene's coding strand. So the mRNA sequence always corresponds to the 5-3 coding sequence of a gene.

• Now, the RNA polymerase machinery moves along the DNA in the 5-3 orientation of the coding strand (e.g. left-to-right for a forward strand gene). It reads the bases from the template strand (so it is reading in the 3-5 direction from the point-of-view of the template strand), and builds the mRNA as it goes. This means that the mRNA matches the coding sequence of the gene, not the template sequence. (Thisdiagram from Wikipedia illustrates).

• Annotations such as Ensembl and UCSC are concerned with the coding sequences of genes, so when they say a gene is on the forward strand, it means the gene's coding sequence is on the forward strand. To follow through again, that means that during transcription of this forward-strand gene, the gene's template sequence is read from the reverse strand, producing an mRNA that matches the sequence on the forward strand.

注意：strand Is either 1 for forward strand or -1 for reverse strand也就是说若strand为1，拼接后的序列通过碱基互补和T->U成为mRNA序列，若strand为-1，则需要将序列反向再经过T->U成为mRNA序列。