Subcloning issue - (Mar/14/2016 )

Hi all,

I need!! I have fragment

GCTCAGCACTACTCTGTTGCCTGGTCTTCCTGGCTGGGGTGGCAGCCAGCCGAGATGCGAGCACCCTGTCTGACAGCAGCTGTATCCACTTGCCAACCAGCCTGCCCCACATGCTGCGGGAGCTCCGAGCTGCCTTCGGCGAGGCGAAGACTTTCTTTCAAATGAAGGACCAACTGCACAGCTTACTGTTGACCCAGTCTCTGCTGGATGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCGGAAATGATCCAGTTTTACCTGGAAGAGGTGATGCCACAGGCTGAGAACCACGGGCCTGACATCAAGGAGCACGTGAACTCACTGGGGGAGAAGCTGAAGACCCTGCGGCTGCGGCTGCGGCGCTGTCATCGCTTTCTGCCCTGCGAAAACAAGAGCAAGGCGGTGGAGAAGGTGAAGAGAGTCTTCAGTGAGCTCCAAGAGAGGG

Inserted into pGEM T Easy vector. I need to subclone this fragment into an expression vector o obtain a protein. I have tried, in silico, to insert this fragment into vectors pET32b+ and pET22b+, however, the ORF keeps shifting where I can get the His tag aligned at the end. Can someone please help??

I tried various RE and restriction sites without success. 

I would really appreciate the help

Thanks,

PCR the fragment with

Yes, PCR cloning would be advantageous here.  If the his tag is already in the vector, you could also add/subtract bases from the respective end of your insert by including them in the primer, and thus bring the epitope tag into frame.