Construction of pYPK0_PGI1_ScXKS1_FBA1 (pMEC1129)



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_PGI1 =read("pYPKa_Z_PGI1.gb")

Genbank record



In [6]:

    
# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://www.ncbi.nlm.nih.gov/pubmed/9169869', width='100%', height=250)









    Out[6]:



In [7]:

    
gb =Genbank("bjornjobbb@gmail.com")
gene_template = gb.nucleotide("NM_001181323.3")



In [8]:

    
gene_template.list_features()









    Out[8]:





+-----+------------------+-----+-----+-------+------+--------+------+
| Ft# | Label or Note    | Dir | Sta | End   |  Len | type   | orf? |
+-----+------------------+-----+-----+-------+------+--------+------+
|   0 | nd               | --> | 0   | 1803  | 1803 | source | yes  |
|   1 | nd               | --> | <0  | >1803 | 1803 | gene   | yes  |
|   2 | N:Xylulokinase;  | --> | 0   | 1803  | 1803 | CDS    | yes  |
+-----+------------------+-----+-----+-------+------+--------+------+



In [9]:

    
orf = gene_template.extract_feature(0)



In [10]:

    
orf.isorf()









    Out[10]:





True



In [11]:

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









    



1803






    Out[11]:





27N2cQNKNSUXfhxo37o-d6AshX0



In [12]:

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









    



MLCSVIQRQTREVSNTMSLDSYYLGFDLSTQQLKCLAINQDLKIVHSETVEFEKDLPHYHTKKGVYIHGDTIECPVAMWLEALDLVLSKYREAKFPLNKVMAVSGSCQQHGSVYWSSQAESLLEQLNKKPEKDLLHYVSSVAFARQTAPNWQDHSTAKQCQEFEECIGGPEKMAQLTGSRAHFRFTGPQILKIAQLEPEAYEKTKTISLVSNFLTSILVGHLVELEEADACGMNLYDIRERKFSDELLHLIDSSSKDKTIRQKLMRAPMKNLIAGTICKYFIEKYGFNTNCKVSPMTGDNLATICSLPLRKNDVLVSLGTSTTVLLVTDKYHPSPNYHLFIHPTLPNHYMGMICYCNGSLARERIRDELNKERENNYEKTNDWTLFNQAVLDDSESSENELGVYFPLGEIVPSVKAINKRVIFNPKTGMIEREVAKFKDKRHDAKNIVESQALSCRVRISPLLSDSNASSQQRLNEDTIVKFDYDESPLRDYLNKRPERTFFVGGASKNDAIVKKFAQVIGATKGNFRLETPNSCALGGCYKAMWSLLYDSNKIAVPFDKFLNDNFPWHVMESISDVDNENWDRYNSKIVPLSELEKTLI*



In [13]:

    
pf, pr =parse('''

>XX_YPK_rec_fwd 
gtcgaggaacgccaggttgcccactttctcactagtgaAAATGTTGTGTTCAGTAATTCAGAG

>XK_YPK_rec_rev 
ATTTAAatcctgatgcgtttgtctgcacagatggcgcgTTAGATGAGAGTCTTTTCCAGTTC

''')



In [14]:

    
g = pcr( pf, pr, orf)



In [15]:

    
g.figure()









    Out[15]:





                                        5ATGTTGTGTTCAGTAATTCAGAG...GAACTGGAAAAGACTCTCATCTAA3
                                                                   |||||||||||||||||||||||| tm 55.3 (dbd) 60.9
                                                                  3CTTGACCTTTTCTGAGAGTAGATTgcgcggtagacacgtctgtttgcgtagtcctaAATTTA5
5gtcgaggaacgccaggttgcccactttctcactagtgaAAATGTTGTGTTCAGTAATTCAGAG3
                                         ||||||||||||||||||||||| tm 54.4 (dbd) 59.0
                                        3TACAACACAAGTCATTAAGTCTC...CTTGACCTTTTCTGAGAGTAGATT5



In [16]:

    
terminator_clone = pYPKa_E_FBA1 =read("pYPKa_E_FBA1.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))
pYPK0_E_Z, stuffer









    Out[18]:





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



In [19]:

    
(pYPK0_E_Z, p, g, t)









    Out[19]:





(Dseqrecord(-5681), Amplicon(1161), Amplicon(1881), Amplicon(736))



In [20]:

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



In [21]:

    
asm









    Out[21]:





Assembly
fragments..: 5681bp 1161bp 1881bp 736bp
limit(bp)..: 31
G.nodes....: 8
algorithm..: common_sub_strings



In [22]:

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









    Out[22]:





 -|name|98
|       \/
|       /\
|       98|1161bp_PCR_prod|38
|                          \/
|                          /\
|                          38|1881bp_PCR_prod|35
|                                             \/
|                                             /\
|                                             35|736bp_PCR_prod|61
|                                                               \/
|                                                               /\
|                                                               61-
|                                                                  |
 ------------------------------------------------------------------



In [23]:

    
pYPK0_PGI1_ScXKS1_FBA1 = candidate.synced(pYPK0)

The pYPK0_PGI1_ScXKS1_FBA1 has cseguid tDAwCS0RGOllmBII5oQTORuFXpw and 9213 bp.



In [24]:

    
print(len(pYPK0_PGI1_ScXKS1_FBA1))
pYPK0_PGI1_ScXKS1_FBA1.cseguid()









    



9213






    Out[24]:





tDAwCS0RGOllmBII5oQTORuFXpw



In [25]:

    
pYPK0_PGI1_ScXKS1_FBA1.name="pMEC1129"
#pYPK0_PGI1_ScXKS1_FBA1.description = "pYPK0_PGI1_ScXKS1_FBA1tp"
pYPK0_PGI1_ScXKS1_FBA1.stamp()









    Out[25]:





cSEGUID_tDAwCS0RGOllmBII5oQTORuFXpw



In [26]:

    
pYPK0_PGI1_ScXKS1_FBA1.write("pYPK0_PGI1_ScXKS1_FBA1.gb")









    




pYPK0_PGI1_ScXKS1_FBA1.gb

Download sequence

pYPK0_PGI1_ScXKS1_FBA1



In [27]:

    
reloaded =read("pYPK0_PGI1_ScXKS1_FBA1.gb")



In [28]:

    
reloaded.cseguid()









    Out[28]:





tDAwCS0RGOllmBII5oQTORuFXpw



In [29]:

    
reloaded.description









    Out[29]:





'cSEGUID_tDAwCS0RGOllmBII5oQTORuFXpw_2018-05-15T07:38:04.498575'