pMEC1135

Also referred to as pYPK0-C4. This vector is identical to pMEC1136 but for a point mutation in XYL1 N272D. The systematic name of this vector is :

pYPK0-TEF1-SsXR_N272D-TDH3-SsXDH-PGI1-XK-FBA1-ScTAL1-PDC1

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

Gene	Enzyme	Acronym	Cassette
SsXYL1_N272D	D-xylose reductase	XR	pYPK0_TEF1_PsXYL1_N272D_TDH3
SsXYL2	xylitol dehydrogenase	XDH	pYPK0_TDH3_PsXYL2_PGI1
ScXKS1	Xylulokinase	XK	pYPK0_PGI1_ScXKS1_FBA1
ScTAL1	Transaldolase	tal1p	pYPK0_FBA1_ScTAL1_PDC1

The vector pMEC1135 is identical to this vector, but has a point mutation in XYL1.

Yeast Pathway Kit Standard Primers



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]:

    
pYPK0 =read("pYPK0.gb")



In [4]:

    
pYPK0.cseguid()









    Out[4]:





qSJ95NM815X60yBy4l6iI69a_Dw



In [5]:

    
from Bio.Restriction import ZraI, AjiI, EcoRV



In [6]:

    
p417,p626 =parse('''   >417_ScTEF1tpf (30-mer)
                              TTAAATAACAATGCATACTTTGTACGTTCA
                              >626_ScTEF1tpr_PacI (35-mer)
                              taattaaTTTGTAATTAAAACTTAGATTAGATTGC''', ds=False)

p415,p623 =parse('''   >415_ScTDH3tpf (29-mer)
                              TTAAATAATAAAAAACACGCTTTTTCAGT                                            
                              >623_ScTDH3tpr_PacI (33-mer)
                              taattaaTTTGTTTGTTTATGTGTGTTTATTCG''', ds=False)

p549,p622 =parse('''   >549_ScPGI1tpf (27-mer)
                              ttaaatAATTCAGTTTTCTGACTGAGT                                            
                              >622_ScPGI1tpr_PacI (28-mer)
                              taattaaTTTTAGGCTGGTATCTTGATT''', ds=False)

p409,p624 =parse('''   >409_ScFBA1tpf (37-mer)
                              TTAAATAATAACAATACTGACAGTACTAAATAATTGC                                            
                              >624_ScFBA1tpr_PacI (29-mer)
                              taattaaTTTGAATATGTATTACTTGGTT''', ds=False)



In [7]:

    
p1 =read("pYPK0_TEF1_PsXYL1_N272D_TDH3.gb")
p2 =read("pYPK0_TDH3_PsXYL2_PGI1.gb")
p3 =read("pYPK0_PGI1_ScXKS1_FBA1.gb")
p4 =read("pYPK0_FBA1_ScTAL1_PDC1.gb")



In [8]:

    
cas1  =pcr( p167, p623, p1)
cas2  =pcr( p415, p622, p2)
cas3  =pcr( p549, p624, p3)
cas4  =pcr( p409, p166, p4)



In [9]:

    
pYPK0_E_Z, stuffer = pYPK0.cut((EcoRV, ZraI))



In [10]:

    
asm =Assembly( [pYPK0_E_Z, cas1, cas2, cas3, cas4] , limit = 61)



In [11]:

    
candidate = asm.assemble_circular()[0]



In [12]:

    
candidate.figure()









    Out[12]:





 -|name|98
|       \/
|       /\
|       98|2432bp_PCR_prod|712
|                          \/
|                          /\
|                          712|2889bp_PCR_prod|1013
|                                              \/
|                                              /\
|                                              1013|3532bp_PCR_prod|644
|                                                                   \/
|                                                                   /\
|                                                                   644|2754bp_PCR_prod|61
|                                                                                       \/
|                                                                                       /\
|                                                                                       61-
|                                                                                          |
 ------------------------------------------------------------------------------------------



In [13]:

    
pw = candidate.synced(pYPK0)



In [14]:

    
len(pw)









    Out[14]:





14760



In [15]:

    
pw.cseguid()









    Out[15]:





KsItUghJ26gWmTx7JAcZTSjEXas



In [16]:

    
pw.name = "pMEC1135"
pw.description="pYPK0-TEF1-SsXR_N272D-TDH3-SsXDH-PGI1-XK-FBA1-ScTAL1-PDC1 pYPK0_C4"
pw.stamp()









    Out[16]:





cSEGUID_KsItUghJ26gWmTx7JAcZTSjEXas



In [17]:

    
pw.write("pMEC1135.gb")









    




pMEC1135.gb