pYPKpw

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.

This sequence was replaced by a short segment containing a ZraI, FspAI and EcoRV site in the given order, which are all unique in the vector.

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 [1]:

    
from pydna.all import *



In [2]:

    
pYPK0 =read("pYPK0.gb")

This sequence was removed



In [3]:

    
print(str(pYPK0[485:664].seq))









    



TTGTTTTGCCAGATTCAGCAGAGTCTGTGCAATGCGGCCGCTGACGTCGAGGAACGCCAGGTTGCCCACTTTCTCACTAGTGACCTGCAGCCGGCGCGCCATCTGTGCAGACAAACGCATCAGGATATCCGGATTTACCTGAATCAATTGGCGAAATTTTTTGTACGAAATTTCAGCCA

Inverse PCR was performed using the primers below.



In [4]:

    
pf, pr =parse('''  >866_pYPKpwF (39-mer)
                          GCATGATATCttcacaggcggttttcgcacgtacccatg

                          >865_pYPKpwR (39-mer)
                          GCATGACGTCaccagacgctatgactcacccggacggca''', ds=False)



In [5]:

    
pcr_vector_backbone_prod =pcr(pr, pf, pYPK0)



In [6]:

    
candidate = pcr_vector_backbone_prod.looped()



In [7]:

    
pYPKpw = candidate.looped().synced(pYPK0)

The pYPKpw should have cseguid WeyovdMmqwA4bc9EqEwUDmbo3Lg and length should be 5603 bp.



In [8]:

    
print(len(pYPKpw))
pYPKpw.cseguid()









    



5603






    Out[8]:





WeyovdMmqwA4bc9EqEwUDmbo3Lg



In [9]:

    
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



In [10]:

    
pYPKpw.linearize(ZraI)









    Out[10]:





Dseqrecord(-5603)



In [11]:

    
pYPKpw.linearize(FspAI)









    Out[11]:





Dseqrecord(-5603)



In [12]:

    
pYPKpw.linearize(EcoRV)









    Out[12]:





Dseqrecord(-5603)

These enzymes all cut between 486 and 506



In [13]:

    
pYPKpw[486:506].seq









    Out[13]:





Dseq(-20)
GACGTCATGCGCATGATATC
CTGCAGTACGCGTACTATAG

ZraI   GAC↓GTC      ---
FspAI  RTGC↓GCAY    ...
EcoRV  GAT↓ATC      ===


--- ---.... ....=== ===
GAC↓GTCATGC↓GCATGAT↓ATC
CTG↓CAGTACG↓CGTACTA↓TAG



In [14]:

    
pYPKpw.stamp()









    Out[14]:





cSEGUID_WeyovdMmqwA4bc9EqEwUDmbo3Lg



In [15]:

    
pYPKpw.locus="pYPKpw"



In [16]:

    
pYPKpw.write("pYPKpw.gb")









    




pYPKpw.gb

Download

pYPKpw



In [17]:

    
from pydna.all import *
reloaded=read("pYPKpw.gb")
assert reloaded.cseguid() in reloaded.definition