pYPK4

pYPK4 is a version of pYPK1 that has a 2µ yeast ori instead of the CEN6/ARS and a HphMX4 marker instead of the bleomycin marker.



In [1]:

    
from pydna.all import *



In [2]:

    
githubuser="BjornFJohansson"
gistid = "c5424b7ebbf553c52053"
x=download_text('https://gist.githubusercontent.com/{user}/{gistid}/raw'.format(user=githubuser, gistid=gistid))
pAG32 =read(x)

pAG32 sequence



In [3]:

    
p150,p149,p325,p324,p678,p666 =parse_primers('''
>150_MX4fwd (25-mer) Primer in the Ashbya gossypi TEF promoter in forward direction. contaminated?
AAAATCTTGCTAGGATACAGTTCTC

>149_MX4rev (25-mer) Primer in the Ashbya gossypi TEF terminator in the reverse direction.
ACAAATGACAAGTTCTTGAAAACAA

>325_Hygfwd (27-mer)
GATGTAGGAGGGCGTGGATATGTCCTG

>324_Hygrev (27-mer)
TGCATCATCGAAATTGCCGTCAACCAA

>678_pYPK0_hygfwd: (77-mer)
ctcacgttaagggattttggtcatgagCACATACGATTTAGGTGACACTATAGAAC

>666_pYPK0_hygrev (70-mer)
catctttgacagcttatcatcgataagctCGACTCACTATAGGGAGACC''')



In [4]:

    
hygmarker1 =pcr( p678, p324, pAG32 )



In [5]:

    
hygmarker2 =pcr( p325, p666, pAG32 )



In [6]:

    
pYPK0=read("pYPK0.gb")



In [7]:

    
from Bio.Restriction import SbfI



In [8]:

    
vect = pYPK0.linearize(SbfI)



In [9]:

    
vect









    Out[9]:





Dseqrecord(-5766)



In [10]:

    
asm=Assembly((vect,hygmarker1,hygmarker2 ))
asm









    Out[10]:





Assembly
fragments..: 5766bp 1371bp 1262bp
limit(bp)..: 25
G.nodes....: 6
algorithm..: common_sub_strings



In [11]:

    
candidate = asm.assemble_circular()[0]



In [12]:

    
candidate.figure()









    Out[12]:





 -|pYKP0_lin|27
|            \/
|            /\
|            27|1371bp_PCR_prod|741
|                               \/
|                               /\
|                               741|1262bp_PCR_prod|29
|                                                   \/
|                                                   /\
|                                                   29-
|                                                      |
 ------------------------------------------------------



In [13]:

    
pYPK4=candidate.synced(pYPK0)



In [14]:

    
pYPK4.stamp()









    Out[14]:





cSEGUID_X0SJzGGAj7ulCEqpSsK4YXZW9Bw



In [15]:

    
pYPK4.locus = "pYPK4"



In [16]:

    
pYPK4.write("pYPK4.gb")









    




pYPK4.gb

DOWNLOAD



In [17]:

    
from pydna.all import *
reloaded=read("pYPK4.gb")
assert reloaded.cseguid() in reloaded.definition