filipa3_2018-01-23 (pMEC1137)

This vector expresses four genes and was assembled from four single gene expression cassettes:

Gene	Enzyme	Acronym	Cassette
ScTKL1	Transketolase	TKL	pYPK0_PDC1_ScTKL1_RPS19b
ScRPE1	xylitol dehydrogenase	RPE	pYPK0_RPS19b_ScRPE1_RPS19a
ScRKI1	Xylulokinase	RKI	pYPK0_RPS19a_ScRKI1_TPI1
CiGXF1	Transaldolase	GXF	pYPK0_TPI1_CiGXF1_ENO2

Yeast Pathway Kit Standard Primers

PDC1_ScTKL1_RPS19btp
            RPS19b_ScRPE1_RPS19atp
                          RPS19a_ScRKI1_TPI1tp
                                        TPI1_CiGXF1_ENO2tp

pYPK0-PDC1-ScTKL1-RPS19b-ScRPE1-RPS19a-ScRKI1-TPI1-CiGXF1-ScENO2



In [1]:

    
from pydna.all import *



In [2]:

    
p567,p577,p468,p467,p568,p578,p775,p778,p167,p166 = parse("yeast_pahtway_kit_standard_primers.txt")



In [3]:

    
pYPKp3 = read("pYPKp3.gb")



In [4]:

    
from Bio.Restriction import EcoRV



In [5]:

    
p1 =read("pYPK0_PDC1_ScTKL1_RPS19b.gb")
p2 =read("pYPK0_RPS19b_ScRPE1_RPS19a.gb")
p3 =read("pYPK0_RPS19a_ScRKI1_TPI1.gb")
p4 =read("pYPK0_TPI1_CiGXF1_ENO2.gb")



In [6]:

    
pcr( p775, p778, p3)









    Out[6]:




Amplicon(2047)



In [7]:

    
cas1  =pcr( p577, p778, p1)
cas2  =pcr( p775, p778, p2)
cas3  =pcr( p775, p778, p3)
cas4  =pcr( p775, p578, p4)



In [8]:

    
lin_vector = pYPKp3.linearize(EcoRV)



In [9]:

    
asm = Assembly( [lin_vector, cas1, cas2, cas3, cas4], limit = 61) # This limit is important!



In [10]:

    
asm









    Out[10]:





Assembly
fragments..: 5129bp 3904bp 2030bp 2047bp 3152bp
limit(bp)..: 61
G.nodes....: 12
algorithm..: common_sub_strings



In [11]:

    
candidate = asm.assemble_circular()[0]



In [12]:

    
candidate.figure()









    Out[12]:





 -|pYPKp3_lin|124
|             \/
|             /\
|             124|3904bp_PCR_prod|639
|                                 \/
|                                 /\
|                                 639|2030bp_PCR_prod|557
|                                                     \/
|                                                     /\
|                                                     557|2047bp_PCR_prod|596
|                                                                         \/
|                                                                         /\
|                                                                         596|3152bp_PCR_prod|242
|                                                                                             \/
|                                                                                             /\
|                                                                                             242-
|                                                                                                |
 ------------------------------------------------------------------------------------------------



In [13]:

    
asm.assemble_circular()[0].cseguid()









    Out[13]:





tgvhIH110nJjoBUSnn0xajU4gk4



In [14]: