pYPK5

Cassette MX4 amplififcação:

primers 477 e 531
template pAG32.

Cassette 2u amplificação:

primers 534 e 535
template pYPK0

pYPK0 digerido com StuI.



In [1]:

    
from pydna.all import *



In [2]:

    
pYPK0 = read("pYPK0.gb")



In [3]:

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



In [4]:

    
pAG32.cseguid()









    Out[4]:





ttCV-qk-4UzU8BkVhaswwqdfGlg

pAG32 sequence



In [5]:

    
p477, p531, p535, p534=parse(''' 
>477_bleMX4r
agtggaacgaaaactcacgttaagggattttggtcatgaggagctcgttttcga

>531_pSU1_MX4f (67-mer)
taattctcatctttgacagcttatcatcgataagtccccgccgggtc

>535_pSU0_2u_Fwd (21-mer)
taactgtcagaccaagtttac

>534_pSU0_2u_rev (27-mer)
ttatcgatgataagctgtcaaagatga''')



In [6]:

    
hygmarker = pcr(p477, p531, pAG32)



In [7]:

    
two_micron =pcr(p534, p535, pYPK0)



In [8]:

    
from Bio.Restriction import StuI



In [9]:

    
vect,stuffer = pYPK0.cut(StuI)
vect, stuffer









    Out[9]:





(Dseqrecord(-4982), Dseqrecord(-780))



In [10]:

    
vect.name="pYPK0"



In [11]:

    
asm=Assembly( ( vect, hygmarker, two_micron ) )



In [12]:

    
asm









    Out[12]:





Assembly
fragments..: 4982bp 1721bp 1619bp
limit(bp)..: 25
G.nodes....: 8
algorithm..: common_sub_strings



In [13]:

    
a,b,c,d,e,f,g,*h = asm.assemble_circular()



In [14]:

    
a,b,c,d,e,f,g









    Out[14]:





(Contig(o6427),
 Contig(o6427),
 Contig(o6427),
 Contig(o6299),
 Contig(o6299),
 Contig(o6299),
 Contig(o6299))



In [15]:

    
f.figure()









    Out[15]:





 -|pYPK0|41
|        \/
|        /\
|        41|1721bp_PCR_pr_rc|33
|                            \/
|                            /\
|                            33|1619bp_PCR_prod|66
|                                               \/
|                                               /\
|                                               66-
|                                                  |
 --------------------------------------------------

The larger recombination product x (6427 bp) contains the pYPK0 vector two times in a row the product we want is the f sequence (6299bp) see below.

a:
 -|1619bp_PCR_prod|1505
|                  \/
|                  /\
|                  1505|pYPK0|66
|                             \/
|                             /\
|                             66|pYPK0|41
|                                      \/
|                                      /\
|                                      41|1721bp_PCR_pr_rc|33
|                                                          \/
|                                                          /\
|                                                          33-
|                                                             |
 -------------------------------------------------------------


f:
 -|1619bp_PCR_prod|66
|                  \/
|                  /\
|                  66|pYPK0|41
|                           \/
|                           /\
|                           41|1721bp_PCR_pr_rc|33
|                                               \/
|                                               /\
|                                               33-
|                                                  |
 --------------------------------------------------



In [16]:

    
pYPK5 = f.synced(pYPK0)



In [17]:

    
len(pYPK5)









    Out[17]:





6299



In [18]:

    
pYPK5.cseguid()









    Out[18]:





bYXaKaxDTMFN5_B-QWoDqATVAb0



In [19]:

    
pYPK5.locus = "pYPK5"
pYPK5.stamp()









    Out[19]:





cSEGUID_bYXaKaxDTMFN5_B-QWoDqATVAb0



In [20]:

    
pYPK5.write("pYPK5.gb")









    




pYPK5.gb



In [21]:

    
from pydna.all import *
r =read("pYPK5.gb")
assert r.cseguid() in r.definition