•确定目标基因的形态：DNA or RNA
•查找目标基因
•比对目标基因同源性和特点
•选取目标基因序列
•选取目标基因的目标区段
1、mRNA 序列的查询；
以查大鼠cort为例；
先登录美国国立生物信息库网站https://www.ncbi.nlm.nih.gov/
在Nucleotide中，搜索rat cort
在NCBI上搜索到目的基因，找到该基因的mRNA
把以下内容存成txt文件：
Rattus norvegicus cortistatin (Cort), mRNA
NCBI Reference Sequence: NM_012835.1
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LOCUS NM_012835 438 bp mRNA linear ROD 22-JUN-2025
DEFINITION Rattus norvegicus cortistatin (Cort), mRNA.
ACCESSION NM_012835
VERSION NM_012835.1
KEYWORDS RefSeq; RefSeq Select.
SOURCE Rattus norvegicus (Norway rat)
ORGANISM Rattus norvegicus
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha;
Muroidea; Muridae; Murinae; Rattus.
REFERENCE 1 (bases 1 to 438)
AUTHORS Liu,Y., Lin,F., Fu,Y., Chen,W., Liu,W., Chi,J., Zhang,X. and Yin,X.
TITLE Cortistatin inhibits arterial calcification in rats via
GSK3beta/beta-catenin and protein kinase C signalling but not c-Jun
N-terminal kinase signalling
JOURNAL Acta Physiol (Oxf) 223 (3), e13055 (2018)
PUBMED 29436118
REMARK GeneRIF: inhibits aortic calcification and osteogenic
differentiation of vascular smooth muscle cells likely via the
GSK3beta/beta-catenin and PKC signalling pathways, but not JNK
signalling pathway
REFERENCE 2 (bases 1 to 438)
AUTHORS Yue,M., Xia,Y., Shi,C., Guan,C., Li,Y., Liu,R., Wei,Z. and Dai,Y.
TITLE Berberine ameliorates collagen-induced arthritis in rats by
suppressing Th17 cell responses via inducing cortistatin in the gut
JOURNAL FEBS J 284 (17), 2786-2801 (2017)
PUBMED 28636167
REMARK GeneRIF: Oral berberine exerted anti-arthritic effect through
inhibiting the Th17 cell response, which was closely associated
with the induction of cortistatin generation from gut through
augmenting autocrine/paracrine action between enteric nerve cells
and endocrine cells.
REFERENCE 3 (bases 1 to 438)
AUTHORS Liu,Y., Lin,F., Fu,Y., Chen,W., Liu,W., Chi,J., Zhang,X. and Yin,X.
TITLE Cortistatin inhibits calcification of vascular smooth muscle cells
by depressing osteoblastic differentiation and endoplasmic
reticulum stress
JOURNAL Amino Acids 48 (11), 2671-2681 (2016)
PUBMED 27480534
REMARK GeneRIF: CST directly inhibited beta-GP-induced calcification of
VSMCs in vitro, probably by suppressing ERS and phenotypic
transformation of VSMCs into osteoblastic cells.
REFERENCE 4 (bases 1 to 438)
AUTHORS Chen,X.M., Xia,J., Zhou,T., Yuan,Q., Zhang,W.F., Hu,C.P., Li,Y.J.
and Jiang,J.L.
TITLE Involvement of DDAH/ADMA pathway in the pathogenesis of rheumatoid
arthritis in rats
JOURNAL Int Immunopharmacol 16 (2), 322-331 (2013)
PUBMED 23619555
REMARK GeneRIF: The DDAH/asymmetric dimethylarginine pathway participates
in the pathogenesis of rheumatoid arthritis and may be mediated by
cortistatin.
REFERENCE 5 (bases 1 to 438)
AUTHORS Buga,A.M., Scholz,C.J., Kumar,S., Herndon,J.G., Alexandru,D.,
Cojocaru,G.R., Dandekar,T. and Popa-Wagner,A.
TITLE Identification of new therapeutic targets by genome-wide analysis
of gene expression in the ipsilateral cortex of aged rats after
stroke
JOURNAL PLoS One 7 (12), e50985 (2012)
PUBMED 23251410
REFERENCE 6 (bases 1 to 438)
AUTHORS Xidakis,C., Mastrodimou,N., Notas,G., Renieri,E., Kolios,G.,
Kouroumalis,E. and Thermos,K.
TITLE RT-PCR and immunocytochemistry studies support the presence of
somatostatin, cortistatin and somatostatin receptor subtypes in rat
Kupffer cells
JOURNAL Regul Pept 143 (1-3), 76-82 (2007)
PUBMED 17481746
REMARK GeneRIF: RT-PCR and immunocytochemistry studies support the
presence of cortistatin in rat Kupffer cells.
REFERENCE 7 (bases 1 to 438)
AUTHORS Robas,N., Mead,E. and Fidock,M.
TITLE MrgX2 is a high potency cortistatin receptor expressed in dorsal
root ganglion
JOURNAL J Biol Chem 278 (45), 44400-44404 (2003)
PUBMED 12915402
REFERENCE 8 (bases 1 to 438)
AUTHORS Deghenghi,R., Avallone,R., Torsello,A., Muccioli,G., Ghigo,E. and
Locatelli,V.
TITLE Growth hormone-inhibiting activity of cortistatin in the rat
JOURNAL J Endocrinol Invest 24 (11), RC31-RC33 (2001)
PUBMED 11817718
REFERENCE 9 (bases 1 to 438)
AUTHORS Fukusumi,S., Kitada,C., Takekawa,S., Kizawa,H., Sakamoto,J.,
Miyamoto,M., Hinuma,S., Kitano,K. and Fujino,M.
TITLE Identification and characterization of a novel human
cortistatin-like peptide
JOURNAL Biochem Biophys Res Commun 232 (1), 157-163 (1997)
PUBMED 9125122
REFERENCE 10 (bases 1 to 438)
AUTHORS de Lecea,L., Criado,J.R., Prospero-Garcia,O., Gautvik,K.M.,
Schweitzer,P., Danielson,P.E., Dunlop,C.L., Siggins,G.R.,
Henriksen,S.J. and Sutcliffe,J.G.
TITLE A cortical neuropeptide with neuronal depressant and
sleep-modulating properties
JOURNAL Nature 381 (6579), 242-245 (1996)
PUBMED 8622767
COMMENT VALIDATED REFSEQ: This record has undergone validation or
preliminary review. The reference sequence was derived from
U51919.1.
Publication Note: This RefSeq record includes a subset of the
publications that are available for this gene. Please see the Gene
record to access additional publications.
##Evidence-Data-START##
Transcript exon combination :: U51919.1, BI281700.1 [ECO:0000332]
RNAseq introns :: single sample supports all introns
SAMEA5760396 [ECO:0000348]
##Evidence-Data-END##
##RefSeq-Attributes-START##
RefSeq Select criteria :: based on conservation, expression
##RefSeq-Attributes-END##
FEATURES Location/Qualifiers
source 1..438
/organism="Rattus norvegicus"
/mol_type="mRNA"
/strain="Sprague-Dawley"
/db_xref="taxon:10116"
/chromosome="5"
/map="5q36"
gene 1..438
/gene="Cort"
/note="cortistatin"
/db_xref="GeneID:25305"
/db_xref="RGD:2383"
exon 1..155
/gene="Cort"
/inference="alignment:Splign:2.1.0"
CDS 30..368
/gene="Cort"
/note="Preprocortistatin"
/codon_start=1
/product="cortistatin preproprotein"
/protein_id="NP_036967.1"
/db_xref="GeneID:25305"
/db_xref="RGD:2383"
/translation="MGGCSTRGKRPSALSLLLLLLLSGIAASALPLESGPTGQDSVQD
ATGGRRTGLLTFLAWWHEWASQDSSSTAFEGGTPELSKRQERPPLQQPPHRDKKPCKN
FFWKTFSSCK"
sig_peptide 30..110
/gene="Cort"
/note="/evidence=ECO:0000255; propagated from
UniProtKB/Swiss-Prot (Q62949.1)"
misc_feature 225..332
/gene="Cort"
/note="propagated from UniProtKB/Swiss-Prot (Q62949.1);
Region: Disordered. /evidence=ECO:0000256|SAM:MobiDB-lite"
mat_peptide 279..365
/gene="Cort"
/product="Cortistatin-29. /evidence=ECO:0000255.
/id=PRO_0000033160"
/note="propagated from UniProtKB/Swiss-Prot (Q62949.1)"
mat_peptide 324..365
/gene="Cort"
/product="cortistatin-14"
exon 156..438
/gene="Cort"
/inference="alignment:Splign:2.1.0"
ORIGIN
1 aaagcacaga cttcaggtct ccaaggagga tgggtggctg cagcacaaga ggcaagcggc
61 cgtcagccct cagtctgctg ctgctgctgc tgctctcggg gatcgcagcc tctgccctcc
121 ccctggagag cggtcccacc ggccaggaca gtgtgcagga tgccacaggc gggaggagga
181 ccggccttct gactttcctt gcctggtggc atgagtgggc ttcccaagac agctccagca
241 ccgctttcga agggggtacc ccggagctgt ctaagcggca ggaaagacca cccctccagc
301 agcccccaca ccgggataaa aagccctgca agaacttctt ctggaaaacc ttctcctcgt
361 gcaagtagcc cgagcctgac cggagcctga ccggccaccc tgtgaatgca gccgtggcct
421 gaataaagag tgtcaagt
//
其中上面后origin后面的序列，是我们用来设计的序列。
要点：
1、种属判断不能有错：
——Homo sapiens 代表人
——Rattus norvegicus 代表大鼠
——Mus musculus 代表小鼠
2、有别名，要判断是或者不是；
3、有受体，配体，名称中正好含这个关键词的，要判断是或者不是；
4、多个转录本时需要查找同源序列（即核酸序列相同区域）；
还有其他内容，可以向BioTNT 技术部咨询：
1、启动子序列的查询；
2、microRNA 序列的查询；
3、SNP 序列的查询；