pMEC1137

Also referred to as pYPK0-T. 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]:

    
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)

p411, p413, p419, p537, p539, p621, p625, p647, p648, p651, p652, p660  =parse('''
>411_ScENO2tpf (23-mer)
TTAAATACGCGGCGTTATGTCAC
>413_ScPDC1tpf (24-mer)
TTAAATAAGGGTAGCCTCCCCATA
>419_ScTPI1tpf (32-mer)
TTAAATTGTTTAAAGATTACGGATATTTAACT
>537_ScRPS19btpf (27-mer)
ttaaatCTTTATTATCTTTGGTTCTAT
>539_ScRPS19atpf (26-mer)
ttaaatTTTTACTCTATTTGTCGATC
>621_ScTPI1tpr_PacI (35-mer)
taattaaTTTTAGTTTATGTATGTGTTTTTTGTAG 
>625_ScPDC1tpr_PacI (34-mer)
taattaaTTTGATTGATTTGACTGTGTTATTTTG
>647_ScRPS19btpr_PacI
taattaaCTTTATTATCTTTGGTTCTAT
>648_ScRPS19btpf2
ttaaatTCTAGTATGGTTTGAAACCT
>651_ScRPS19atpr_PacI
taattaaTTTTACTCTATTTGTCGATC
>652_ScRPS19atpf2
ttaaatGTTAACTGAAATGAAAATTT
>660_ScENO2tpr_PacI
taattaaTTATTATTGTATGTTATAGTATTAGTTGCTTG''')



In [7]:

    
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 [8]:

    
cas1  =pcr( p167, p647, p1)
cas2  =pcr( p648, p651, p2)
cas3  =pcr( p652, p621, p3)
cas4  =pcr( p419, p166, p4)



In [9]:

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



In [10]:

    
cas1, cas2, cas3, cas4









    Out[10]:





(Amplicon(3821), Amplicon(2005), Amplicon(2022), Amplicon(2906))



In [11]:

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



In [12]:

    
asm









    Out[12]:





Assembly
fragments..: 5681bp 3821bp 2005bp 2022bp 2906bp
limit(bp)..: 61
G.nodes....: 12
algorithm..: common_sub_strings



In [13]:

    
candidate = asm.assemble_circular()[0]



In [14]:

    
candidate.figure()









    Out[14]:





 -|name|98
|       \/
|       /\
|       98|3821bp_PCR_prod|639
|                          \/
|                          /\
|                          639|2005bp_PCR_prod|557
|                                              \/
|                                              /\
|                                              557|2022bp_PCR_prod|596
|                                                                  \/
|                                                                  /\
|                                                                  596|2906bp_PCR_prod|61
|                                                                                      \/
|                                                                                      /\
|                                                                                      61-
|                                                                                         |
 -----------------------------------------------------------------------------------------



In [15]:

    
pw = candidate.synced("tcgcgcgtttcggtgatgacggtgaaaacctctg")



In [16]:

    
len(pw)









    Out[16]:





14484



In [17]:

    
pw.cseguid()









    Out[17]:





6E0AGs78NqyqjtuAWRWhkIGlLnI



In [18]:

    
pw.name = "pMEC1137"
pw.description=" pYPK0-PDC1-ScTKL1-RPS19b-ScRPE1-RPS19a-ScRKI1-TPI1-CiGXF1-ScENO2"
pw.stamp()









    Out[18]:





cSEGUID_6E0AGs78NqyqjtuAWRWhkIGlLnI



In [19]:

    
pw.write("pMEC1137.gb")









    




pMEC1137.gb