公开(公告)号：EP3128006A1

公开(公告)日：2017-02-08

申请号：EP15179986.3

申请日：2015-08-06

申请人： Consejo Superior de Investigaciones Cientificas (CSIC) ,  EMBL (European Molecular Biology Laboratory)

发明人： VEGA FERNÁNDEZ, María Cristina ,  FERNÁNDEZ PÉREZ, Francisco José ,  BERGER, Imre

IPC分类号： C12N15/00

CPC分类号： C12N15/815

摘要： The invention provides an isolated multiple integration element (MIE) comprising, in the specified order: a) a homing endonuclease site (HE); b) a promoter selected from the group consisting of AOX1, FLD1, FMD1, GAP and TEF1; c) a BstBI restriction site; d) a sequence encoding a secretion signal; e) a Ncol restriction site; f) a selection marker gene; g) a Pmel restriction site; h) a DNA sequence encoding a detection and/or purification polypeptide; i) a terminator selected from the group consisting of AOX1, FLD1, FMD1, GAP and TEF1; and j) a BstXI restriction site, wherein the HE and BstXI sites are selected such that HE and BstXI result in compatible cohesive ends when cut by the homing endonuclease and the BstXI restriction enzyme, respectively, and the ligation product of HE and BstXI cohesive ends can neither be cleaved by the homing endonuclease nor the BstXI restriction enzyme.

摘要翻译： 本发明提供一种分离的多重整合元件（MIE），其以指定的顺序包括：a）归巢内切核酸酶位点（HE）; b）选自AOX1，FLD1，FMD1，GAP和TEF1的启动子; c）BstBI限制性位点; d）编码分泌信号的序列; e）Nco限制性位点; f）选择标记基因; g）Pmel限制性位点; h）编码检测和/或纯化多肽的DNA序列; i）选自AOX1，FLD1，FMD1，GAP和TEF1的终止子; 和j）BstXI限制性位点，其中选择HE和BstXI位点，使得HE和BstXI分别在归巢内切核酸酶和BstXI限制酶切割时分别产生相容的粘性末端，并且HE和BstXI粘性末端的连接产物 不能被归巢内切核酸酶和BstXI限制酶切割。