pMEC1136

Also referred to as pYPK0-Df. This vector expresses four genes and was assembled from four single gene expression cassettes:

Gene Enzyme Acronym Cassette
SsXYL1 D-xylose reductase XR pYPK0_TEF1_PsXYL1_TDH3
SsXYL2 xylitol dehydrogenase XDH pYPK0_TDH3_PsXYL2_PGI1
ScXKS1 Xylulokinase XK pYPK0_PGI1_ScXKS1_FBA1
ScTAL1 Transaldolase tal1p pYPK0_FBA1_ScTAL1_PDC1

The systematic name of this vector is : pYPK0-ScTEF1-XR-ScTDH3-XDH-ScPGI1-XK-ScFBA1-TAL1-ScPDC1 The vector pMEC1135 is identical to this vector, but has a point mutation in XYL1.


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_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]:
(pYPK0_E_Z, cas1, cas2, cas3, cas4)


Out[10]:
(Dseqrecord(-5681),
 Amplicon(2432),
 Amplicon(2889),
 Amplicon(3532),
 Amplicon(2754))

In [11]:
asm =Assembly( [pYPK0_E_Z, cas1, cas2, cas3, cas4] , limit = 61)

In [12]:
asm


Out[12]:
Assembly
fragments..: 5681bp 2432bp 2889bp 3532bp 2754bp
limit(bp)..: 61
G.nodes....: 10
algorithm..: common_sub_strings

In [13]:
candidate = asm.assemble_circular()[0]

In [14]:
candidate.figure()


Out[14]:
 -|name|98
|       \/
|       /\
|       98|2432bp_PCR_prod|712
|                          \/
|                          /\
|                          712|2889bp_PCR_prod|1013
|                                              \/
|                                              /\
|                                              1013|3532bp_PCR_prod|644
|                                                                   \/
|                                                                   /\
|                                                                   644|2754bp_PCR_prod|61
|                                                                                       \/
|                                                                                       /\
|                                                                                       61-
|                                                                                          |
 ------------------------------------------------------------------------------------------

In [15]:
pw = candidate.synced("tcgcgcgtttcggtgatgacggtgaaaacctctg")

In [16]:
len(pw)


Out[16]:
14760

In [17]:
pw.cseguid()


Out[17]:
vjorydf8wV9TuwIkIydwg5qFSHw

In [18]:
pw.id = "pMEC1136"
pw.description="pYPK0-ScTEF1-XR-ScTDH3-XDH-ScPGI1-XK-ScFBA1-TAL1-ScPDC1 (alt name pYPK0_Df)"
pw.stamp()


Out[18]:
cSEGUID_vjorydf8wV9TuwIkIydwg5qFSHw

In [19]:
pw.write("pMEC1136.gb")




Download

pMEC1136


In [20]:
reloaded =read("pMEC1136.gb")

In [21]:
reloaded.cseguid()


Out[21]:
vjorydf8wV9TuwIkIydwg5qFSHw