The pYPkpw is a version of the pYPK0 vector that has a deletion from 45 bp upstream of the cut site of ZraI to 53 bp downstream of the EcoRV cut site which gives a total deletion of 179 bp.
The pYPKpw can be used as an alternative to the pYPK0 for the assembly of pathways from tp_gene_tp cassettes generated by pYPK0 specific primers flanking the cassette. The risk of potential misassembly is reduced since the pYPK0 derived flanking sequences are not present in the pYPKpw vector.
The pYPKpw also has an inactivated CRP gene which means that it can be propagated in CYA+ E. coli strains.
In :from pydna.all import *
In :pYPK0 =read("pYPK0.gb")
This sequence was removed
Inverse PCR was performed using the primers below.
In :pf, pr =parse(''' >866_pYPKpwF (39-mer) GCATGATATCttcacaggcggttttcgcacgtacccatg >865_pYPKpwR (39-mer) GCATGACGTCaccagacgctatgactcacccggacggca''', ds=False)
In :pcr_vector_backbone_prod =pcr(pr, pf, pYPK0)
In :candidate = pcr_vector_backbone_prod.looped()
In :pYPKpw = candidate.looped().synced(pYPK0)
The pYPKpw should have cseguid
WeyovdMmqwA4bc9EqEwUDmbo3Lg and length should be 5603 bp.
In :print(len(pYPKpw)) pYPKpw.cseguid()
In :from Bio.Restriction import EcoRV, FspAI, ZraI
The pYPKpw can be linearized with EcoRV, FspAI or ZraI. The result of the digestions below should be a linear fragment with the same length as the
These enzymes all cut between 486 and 506
Out:Dseq(-20) GACGTCATGCGCATGATATC CTGCAGTACGCGTACTATAG
ZraI GAC↓GTC --- FspAI RTGC↓GCAY ... EcoRV GAT↓ATC === --- ---.... ....=== === GAC↓GTCATGC↓GCATGAT↓ATC CTG↓CAGTACG↓CGTACTA↓TAG
In :from pydna.all import * reloaded=read("pYPKpw.gb") assert reloaded.cseguid() in reloaded.definition