Construction of pYPK0_TEF1_PsXYL1_TDH3 (pMEC1126)



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")

Yeast Pathway Kit Standard Primers



In [3]:

    
from Bio.Restriction import ZraI, AjiI, EcoRV



In [4]:

    
pYPK0 = read("pYPK0.gb")



In [5]:

    
promoter_clone = pYPKa_Z_TEF1 = read("pYPKa_Z_TEF1.gb")

Genbank record



In [6]:

    
# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://www.ncbi.nlm.nih.gov/pubmed/1756986', width='100%', height=250)









    Out[6]:



In [7]:

    
gb =Genbank("bjornjobbb@gmail.com")
gene_template = gb.nucleotide("X59465.1")



In [8]:

    
gene_template.list_features()









    Out[8]:





+-----+------------------+-----+------+------+------+--------------+------+
| Ft# | Label or Note    | Dir | Sta  | End  |  Len | type         | orf? |
+-----+------------------+-----+------+------+------+--------------+------+
|   0 | nd               | --> | 0    | 2052 | 2052 | source       |  no  |
|   1 | nd               | --> | 0    | 355  |  355 | regulatory   |  no  |
|   2 | nd               | --> | 192  | 199  |    7 | regulatory   |  no  |
|   3 | nd               | --> | 272  | 281  |    9 | regulatory   |  no  |
|   4 | nd               | --> | 287  | 301  |   14 | stem_loop    |  no  |
|   5 | N:label:xyl1_cDN | --> | 342  | 1366 | 1024 | misc_feature |  no  |
|   6 | nd               | --> | 355  | 1312 |  957 | gene         | yes  |
|   7 | nd               | --> | 355  | 1312 |  957 | CDS          | yes  |
|   8 | nd               | --> | 1334 | 1339 |    5 | polyA_site   |  no  |
+-----+------------------+-----+------+------+------+--------------+------+



In [9]:

    
orf = gene_template.extract_feature(7)



In [10]:

    
orf.isorf()









    Out[10]:





True



In [11]:

    
print(orf.seq)









    



ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTTCGGCTGTTGGAAAGTCGACGTCGACACCTGTTCTGAACAGATCTACCGTGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCCAACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGATCTATCGGTGTTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATCGAATTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTCCATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCATCATTCCAAAGTCCAACACTGTCCCAAGATTGTTGGAAAACAAGGACGTCAACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGACATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTTCGTCTAA



In [12]:

    
print(len(orf))
orf.seguid()









    



957






    Out[12]:





lGWKlZ3EDAyY2V-7tTQoJRqgW6w



In [13]:

    
print(str(orf.seq.translate()))









    



MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQIYRAIKTGYRLFDGAEDYANEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGKSPAQVLLRWSSQRGIAIIPKSNTVPRLLENKDVNSFDLDEQDFADIAKLDINLRFNDPWDWDKIPIFV*



In [14]:

    
pf, pr =parse('''

>XR_YPK_rec_fwd 
gtcgaggaacgccaggttgcccactttctcactagtgaAAATGCCTTCTATTAAGTTGAACTCTGG
>XR_YPK_rec_rev 
ATTTAAatcctgatgcgtttgtctgcacagatggcgcgTTAGACGAAGATAGGAATCTTGTCCC

''')



In [15]:

    
g =pcr( pf, pr, orf)



In [16]:

    
g.figure()









    Out[16]:





                                        5ATGCCTTCTATTAAGTTGAACTCTGG...GGGACAAGATTCCTATCTTCGTCTAA3
                                                                      |||||||||||||||||||||||||| tm 58.7 (dbd) 66.4
                                                                     3CCCTGTTCTAAGGATAGAAGCAGATTgcgcggtagacacgtctgtttgcgtagtcctaAATTTA5
5gtcgaggaacgccaggttgcccactttctcactagtgaAAATGCCTTCTATTAAGTTGAACTCTGG3
                                         |||||||||||||||||||||||||| tm 57.9 (dbd) 64.7
                                        3TACGGAAGATAATTCAACTTGAGACC...CCCTGTTCTAAGGATAGAAGCAGATT5



In [17]:

    
terminator_clone = pYPKa_E_TDH3 =read("pYPKa_E_TDH3.gb")



In [18]:

    
p  = pcr( p167, p567, promoter_clone)
t  = pcr( p568, p166, terminator_clone)



In [19]:

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









    Out[19]:





(Dseqrecord(-5681), Dseqrecord(-81))



In [20]:

    
(pYPK0_E_Z, p, g, t)









    Out[20]:





(Dseqrecord(-5681), Amplicon(741), Amplicon(1035), Amplicon(804))



In [21]:

    
asm =Assembly((pYPK0_E_Z, p, g, t), limit=29)



In [22]:

    
asm









    Out[22]:





Assembly
fragments..: 5681bp 741bp 1035bp 804bp
limit(bp)..: 29
G.nodes....: 8
algorithm..: common_sub_strings



In [23]:

    
candidate = asm.assemble_circular()[0]
candidate.figure()









    Out[23]:





 -|name|98
|       \/
|       /\
|       98|741bp_PCR_prod|38
|                         \/
|                         /\
|                         38|1035bp_PCR_prod|35
|                                            \/
|                                            /\
|                                            35|804bp_PCR_prod|61
|                                                              \/
|                                                              /\
|                                                              61-
|                                                                 |
 -----------------------------------------------------------------



In [24]:

    
pYPK0_TEF1_PsXYL1_TDH3 = candidate.synced(pYPK0)

The pYPK0_TEF1_PsXYL1_TDH3 has cseguid ak608NPPLpMmn_YIHGwQPcQzee0 and 8015 bp



In [25]:

    
print(len(pYPK0_TEF1_PsXYL1_TDH3))
pYPK0_TEF1_PsXYL1_TDH3.cseguid()









    



8015






    Out[25]:





ak608NPPLpMmn_YIHGwQPcQzee0



In [26]:

    
pYPK0_TEF1_PsXYL1_TDH3.name="pMEC1126"
#pYPK0_TEF1_PsXYL1_TDH3.description = "pYPK0_TEF1_PsXYL1_TDH3tp"
pYPK0_TEF1_PsXYL1_TDH3.stamp()









    Out[26]:





cSEGUID_ak608NPPLpMmn_YIHGwQPcQzee0



In [27]:

    
pYPK0_TEF1_PsXYL1_TDH3.write("pYPK0_TEF1_PsXYL1_TDH3.gb")









    




pYPK0_TEF1_PsXYL1_TDH3.gb

Download sequence

pYPK0_TEF1_PsXYL1_TDH3



In [28]:

    
reloaded =read("pYPK0_TEF1_PsXYL1_TDH3.gb")



In [29]:

    
reloaded.cseguid()









    Out[29]:





ak608NPPLpMmn_YIHGwQPcQzee0



In [30]: