Construction of pYPK0_PDC1_ScTKL1_RPS19b (pMEC1131)



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_PDC1 =read("pYPKa_Z_PDC1.gb")

Genbank record



In [6]:

    
# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://www.ncbi.nlm.nih.gov/pubmed/9169875', width='100%', height=250)









    Out[6]:



In [7]:

    
gb =Genbank("bjornjobbb@gmail.com")
gene_template = gb.nucleotide("NM_001184171.1")



In [8]:

    
gene_template.list_features()









    Out[8]:





+-----+------------------+-----+-----+-------+------+--------+------+
| Ft# | Label or Note    | Dir | Sta | End   |  Len | type   | orf? |
+-----+------------------+-----+-----+-------+------+--------+------+
|   0 | nd               | --> | 0   | 2043  | 2043 | source | yes  |
|   1 | nd               | --> | <0  | >2043 | 2043 | gene   | yes  |
|   2 | N:Transketolase; | --> | 0   | 2043  | 2043 | CDS    | yes  |
+-----+------------------+-----+-----+-------+------+--------+------+



In [9]:

    
orf = gene_template.extract_feature(2)



In [10]:

    
orf.isorf()









    Out[10]:





True



In [11]:

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









    



2043






    Out[11]:





m6HK97EKOlSwRjnm3ayQsKUgsDs



In [12]:

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









    



MTQFTDIDKLAVSTIRILAVDTVSKANSGHPGAPLGMAPAAHVLWSQMRMNPTNPDWINRDRFVLSNGHAVALLYSMLHLTGYDLSIEDLKQFRQLGSRTPGHPEFELPGVEVTTGPLGQGISNAVGMAMAQANLAATYNKPGFTLSDNYTYVFLGDGCLQEGISSEASSLAGHLKLGNLIAIYDDNKITIDGATSISFDEDVAKRYEAYGWEVLYVENGNEDLAGIAKAIAQAKLSKDKPTLIKMTTTIGYGSLHAGSHSVHGAPLKADDVKQLKSKFGFNPDKSFVVPQEVYDHYQKTILKPGVEANNKWNKLFSEYQKKFPELGAELARRLSGQLPANWESKLPTYTAKDSAVATRKLSETVLEDVYNQLPELIGGSADLTPSNLTRWKEALDFQPPSSGSGNYSGRYIRYGIREHAMGAIMNGISAFGANYKPYGGTFLNFVSYAAGAVRLSALSGHPVIWVATHDSIGVGEDGPTHQPIETLAHFRSLPNIQVWRPADGNEVSAAYKNSLESKHTPSIIALSRQNLPQLEGSSIESASKGGYVLQDVANPDIILVATGSEVSLSVEAAKTLAAKNIKARVVSLPDFFTFDKQPLEYRLSVLPDNVPIMSVEVLATTCWGKYAHQSFGIDRFGASGKAPEVFKFFGFTPEGVAERAQKTIAFYKGDKLISPLKKAF*



In [13]:

    
pf, pr =parse('''

>TKL_YPK_rec_fwd 
gtcgaggaacgccaggttgcccactttctcactagtgaAAATGACTCAATTCACTGACATTGATAAG

>TKL_YPK_rec_rev 
ATTTAAatcctgatgcgtttgtctgcacagatggcgcgTTAGAAAGCTTTTTTCAAAGGAGAAATTA

''')



In [14]:

    
g =pcr( pf, pr, orf)



In [15]:

    
g.figure()









    Out[15]:





                                        5ATGACTCAATTCACTGACATTGATAAG...TAATTTCTCCTTTGAAAAAAGCTTTCTAA3
                                                                       ||||||||||||||||||||||||||||| tm 55.6 (dbd) 65.0
                                                                      3ATTAAAGAGGAAACTTTTTTCGAAAGATTgcgcggtagacacgtctgtttgcgtagtcctaAATTTA5
5gtcgaggaacgccaggttgcccactttctcactagtgaAAATGACTCAATTCACTGACATTGATAAG3
                                         ||||||||||||||||||||||||||| tm 56.7 (dbd) 64.4
                                        3TACTGAGTTAAGTGACTGTAACTATTC...ATTAAAGAGGAAACTTTTTTCGAAAGATT5



In [16]:

    
terminator_clone = pYPKa_E_RPS19b =read("pYPKa_E_RPS19b.gb")



In [17]:

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



In [18]:

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



In [19]:

    
(pYPK0_E_Z, p, g, t)









    Out[19]:





(Dseqrecord(-5681), Amplicon(1117), Amplicon(2121), Amplicon(731))



In [20]:

    
asm =Assembly((pYPK0_E_Z, p, g, t), limit=31)



In [21]:

    
asm









    Out[21]:





Assembly
fragments..: 5681bp 1117bp 2121bp 731bp
limit(bp)..: 31
G.nodes....: 8
algorithm..: common_sub_strings



In [22]:

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









    Out[22]:





 -|name|98
|       \/
|       /\
|       98|1117bp_PCR_prod|38
|                          \/
|                          /\
|                          38|2121bp_PCR_prod|35
|                                             \/
|                                             /\
|                                             35|731bp_PCR_prod|61
|                                                               \/
|                                                               /\
|                                                               61-
|                                                                  |
 ------------------------------------------------------------------



In [23]:

    
result = candidate.synced(pYPK0)

The new construct should have cseguid m-7lOIJ60aZl4b7rN4vanlD7OWk and 9404 bp.



In [24]:

    
print(len(result))
result.cseguid()









    



9404






    Out[24]:





m-7lOIJ60aZl4b7rN4vanlD7OWk



In [25]:

    
result.name = "pMEC1131"
result.description = "pYPK0_PDC1_ScTKL1_RPS19btp"
result.stamp()









    Out[25]:





cSEGUID_m-7lOIJ60aZl4b7rN4vanlD7OWk



In [26]:

    
result.write("pYPK0_PDC1_ScTKL1_RPS19b.gb")









    




pYPK0_PDC1_ScTKL1_RPS19b.gb

Download genbank file.



In [27]:

    
from pydna.all import *



In [28]:

    
reloaded = read("pYPK0_PDC1_ScTKL1_RPS19b.gb")



In [29]:

    
reloaded.cseguid()









    Out[29]:





m-7lOIJ60aZl4b7rN4vanlD7OWk



In [30]:

    
reloaded.description









    Out[30]:





'pYPK0_PDC1_ScTKL1_RPS19btp cSEGUID_m-7lOIJ60aZl4b7rN4vanlD7OWk_2018-05-15T08:08:54.210154'