YEplac181

In E. coli plasmid pBR322, insert yeast genes into either EcoRI or Cla I using PolIK and dNTPs to fill in overhangs and T4 DNA ligase for blunt-end ligation. Any restriction site regenerated by this process was destroyed following digestion with the appropriate restriction enzyme, using PolIK and dNTPs to fill in overhangs and T4 DNA ligase to ligate blunt ends. Once the constructions were verified, the yeast genes were moved into the plasmid vector pUC19. Excise the gene from pBR322 by HindIII digestion, followed by mung-bean exonuclease treatment to produce blunt ends. Construct YCplac vectors (ARS1-CEN4) by blunt-end ligation of the 1.4-kb EcoRI fragment (TRP1 & ARS1 genes) [3] into pBR322 EcoRI site (to make pT322) and subsequent blunt-end ligation of the functional 850-bp PvuII-HpaI CEN4 fragment [4] into pT322 ClaI (to make pTC1). Digest with AatII, and isolate the fragment containing yeast DNA from agarose gels and ligate to the large AatII-Nde I fragment of pUC19, which had the NdeI end filled in with PolIK (to make YCplac22). Make the other YCplac vectors by blunt-end ligation of the EcoRV fragment from pTC1, containing the ARS1 and CEN4 sequences, into ClaI pBR322 ClaI (to make pAC5). Insert the 1.1-kb HindIII-SmaI fragment (containing URA3) or the 1.6-kb HpaI-AccI fragment (containing LEU2) at pAC5 EcoRI as described above. Move these constructs into pUC19 as described above, to make YCplac33 and YCplac111, respectively. Construct the YEplac vector series (with a yeast 2 mu origin) by blunt-end ligation of the 1.5-kb SauIII fragment from YEp24, which contains the yeast 2 mu origin [5], into pBR322 ClaI (to make p2mu19). Remove p2mu19 XbaI site by filling in with PolIK after digestion and blunt-end religation of this site. Ligate the three yeast genes into p2mu19 EcoRI. The fragments used for the LEU2 and the URA3 constructs were the same as those used in the YCplac constructions. However, the YEplac112 vector (TRP1) contains the 850-bp EcoRI-BglII fragment of the TRP1 gene which does not contain ARS1 function. Construct the YIplac vectors by ligating each yeast gene fragment used in YEplac vector constructions into the pUC19 EcoO109 site using PolIK to fill the sticky ends. NCBI gi: 415331 Hosts: E.coli, Saccharomyces cerevisiae. Related vectors: pUC19, pBR322, yeast 2-micron plasmid, YCplac22, YCplac33, YCplac111, YEplac112, YEplac195, YIplac128, YIplac204, YIplac211. (Information source: VectorDB ( http://seq.yeastgenome.org/vectordb ).)

YEplac181质粒序列.txt

YEplac181 载体序列(点击下载)