Editing PKA Alpha pLVX-Plus2

Clone PKA alpha into pLVX-Plus2<br> 

<br> 

subclone into pLVX-Plus2 from pMF8400<br>cut PKA-alpha in pMF8400 with Nde1 + BamH1 --&gt; 1978 bp<br>cut pLVX-Plus2 with Nde+BamH1&nbsp;

CMV promoter with intron<br>CATATG CCAAGT ACGCCCCCTA TTGACGTCAA<br> 601 TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG 

701 GTGATGCGGT TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA CGTCAATGGG AGTTTGTTTT 

801 GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT ATATAAGCAG 

901 AGCTCGTTTA GTGAACCGTC AGATCGCCTG GAGACGCCAT CCACGCTGTT TTGACCTCCA TAGAAGACAC CGGGACCGAT CCAGCCTCCA TCGGCTCGCA 

1001 TCTCTCCTTC ACGCGCCCGC CGCCCTACCT GAGGCCGCCA TCCACGCCGG TTGAGTCGCG TTCTGCCGCC TCCCGCCTGT GGTGCCTCCT GAACTGCGTC 

1101 CGCCGTCTAG GTAAGTTTAA AGCTCAGGTC GAGACCGGGC CTTTGTCCGG CGCTCCCTTG GAGCCTACCT AGACTCAGCC GGCTCTCCAC GCTTTGCCTG<br> <br> <br> 1201 ACCCTGCTTG CTCAACTCTA GTTAACGGTG GAGGGCAGTG TAGTCTGAGC AGTACTCGTT GCTGCCGCGC GCGCCACCAG ACATAATAGC TGACAGACTA<br> IL2 secretory signal<br> 1301 ACAGACTGTT CCTTTCCATG GGTCTTTTCT GCAGTCACCG TCGTCGACAC GTGTGATCAG ATAGG GCC ACC ATGTACAGG ATGCAACTCC TGTCTTGCAT<br> <br> 1401 TGCACTAAGT CTTGCACTTG TCAC<br>EcoR1<br>GAATTC <br>6His<br> cac cac cac cat cac cac cac ACC GGT <br> ggcaacg ccgccgccgc caagaagggc agcgagcagg<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp; &nbsp;&nbsp; EcoR1<br> 241 agagcgtgaa a gaattc tta gccaaagcca aagaagattt tcttaaaaaa tgggaaagtc<br> 301 ccgctcagaa cacagcccac ttggatcagt ttgaacgaat caagaccctc ggcacgggct<br> 361 ccttcgggcg ggtgatgctg gtgaaacaca aggagaccgg gaaccactat gccatgaaga<br> 421 tcctcgacaa acagaaggtg gtgaaactga aacagatcga acacaccctg aatgaaaagc<br> 481 gcatcctgca agctgtcaac tttccgttcc tcgtcaaact cgagttctcc ttcaaggaca<br> 541 actcaaactt atacatggtc atggagtacg tgcccggcgg ggagatgttc tcacacctac<br> 601 ggcggatcgg aaggttcagt gagccccatg cccgtttcta cgcggcccag atcgtcctga<br> 661 cctttgagta tctgcactcg ctggatctca tctacaggga cctgaagccg gagaatctgc<br> 721 tcattgacca gcagggctac attcaggtga cagacttcgg tttcgccaag cgcgtgaagg<br> 781 gccgcacttg gaccttgtgc ggcacccctg agtacctggc ccctgagatt atcctgagca<br> 841 aaggctacaa caaggccgtg gactggtggg ccctgggggt tcttatctat gaaatggccg<br> 901 ctggctaccc gcccttcttc gcagaccagc ccatccagat ctatgagaag atcgtctctg<br> 961 ggaaggtgcg cttcccttcc cacttcagct ctgacttgaa ggacctgctg cggaacctcc<br> 1021 tgcaggtaga tctcaccaag cgctttggga acctcaagaa tggggtcaac gatatcaaga<br> 1081 accacaagtg gtttgccaca actgactgga ttgccatcta ccagaggaag gtggaagctc<br> 1141 ccttcatacc aaagtttaaa ggccctgggg atacgagtaa ctttgacgac tatgaggaag<br> 1201 aagaaatccg ggtctccatc aatgagaagt gtggcaagga gttttctgag ttt<br>TCT AGA TGG AGC CAC CCG CAG TTC GAA AAG TAA <br> 

<br> 

BamH1 SfiI&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; EcoR1 PmeI SfiI Xho1 <br> 

GGATCC GGCCGCTGCGGCC GAATTCGTTTAAAC GGCCAAACAGGCC TCGAG A<br> 

<br> 

Digest with EcoR1 --&gt;1060bp fragment

[[Image:PLVXPlus2-PKA-alpha_2011-5-3_digestion.JPG]] 


--[[User:Paul.xu|Paul.xu]] 16:18, 3 May 2011 (EST)
[[Category:Cloning]]