Construction of pYPK0_TPI1_CiGXF1_ENO2 (pMEC1134)



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_TPI1 =read("pYPKa_Z_TPI1.gb")

Genbank record



In [6]:

    
# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://www.ncbi.nlm.nih.gov/pubmed/16402921', width='100%', height=250)









    Out[6]:



In [7]:

    
gb =Genbank("bjornjobbb@gmail.com")
gene_template = gb.nucleotide("AJ937350.1") # plasmid pGXF1 Leandro et al. 2006



In [8]:

    
gene_template.list_features()









    Out[8]:





+-----+---------------+-----+-----+------+------+--------+------+
| Ft# | Label or Note | Dir | Sta | End  |  Len | type   | orf? |
+-----+---------------+-----+-----+------+------+--------+------+
|   0 | nd            | --> | 0   | 1644 | 1644 | source | yes  |
|   1 | nd            | --> | 0   | 1644 | 1644 | gene   | yes  |
|   2 | nd            | --> | 0   | 1644 | 1644 | CDS    | yes  |
+-----+---------------+-----+-----+------+------+--------+------+



In [9]:

    
orf = gene_template.extract_feature(2)



In [10]:

    
orf.isorf()









    Out[10]:





True



In [11]:

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









    



1644






    Out[11]:





nRdsznd4WZFAGP2u0IIooDFQS90



In [12]:

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









    



MSQDSHSSGAATPVNGSILEKEKEDSPVLQVDAPQKGFKDYIVISIFCFMVAFGGFVFGFDTGTISGFVNMSDFKDRFGQHHADGTPYLSDVRVGLMISIFNVGCAVGGIFLCKVADVWGRRIGLMFSMAVYVVGIIIQISSSTKWYQFFIGRLIAGLAVGTVSVVSPLFISEVSPKQIRGTLVCCFQLCITLGIFLGYCTTYGTKTYTDSRQWRIPLGLCFAWAILLVVGMLNMPESPRYLVEKHRIDEAKRSIARSNKIPEEDPFVYTEVQLIQAGIEREALAGQASWKELITGKPKIFRRVIMGIMLQSLQQLTGDNYFFYYGTTIFQAVGLKDSFQTSIILGIVNFASTFVGIYVIERLGRRLCLLTGSAAMFICFIIYSLIGTQHLYKQGYSNETSNTYKASGNAMIFITCLYIFFFASTWAGGVYCIISESYPLRIRSKAMSIATAANWLWGFLISFFTPFITSAIHFYYGFVFTGCLAFSFFYVYFFVYETKGLSLEEVDEMYASGVLPLKSASWVPPNLEHMAHSAGYAGADKATDEQV*



In [13]:

    
pf, pr =parse('''>580_GXF1_YPK_fwd (83-mer)
                        gtcgaggaacgccaggttgcccactttctcactagtgaAAatgtcacaagattcgcattctt

                        >579_GXF1_YPK_rev (83-mer)
                        ATTTAAatcctgatgcgtttgtctgcacagatggcgcgttaaacctgttcgtcggtggcctt''')



In [14]:

    
g =pcr( pf, pr, orf)



In [15]:

    
g.figure()









    Out[15]:





                                        5atgtcacaagattcgcattctt...aaggccaccgacgaacaggtttaa3
                                                                  |||||||||||||||||||||||| tm 64.0 (dbd) 72.9
                                                                 3ttccggtggctgcttgtccaaattgcgcggtagacacgtctgtttgcgtagtcctaAATTTA5
5gtcgaggaacgccaggttgcccactttctcactagtgaAAatgtcacaagattcgcattctt3
                                         |||||||||||||||||||||| tm 56.4 (dbd) 63.9
                                        3tacagtgttctaagcgtaagaa...ttccggtggctgcttgtccaaatt5



In [16]:

    
terminator_clone = pYPKa_E_ENO2 =read("pYPKa_E_ENO2.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(744), Amplicon(1722), Amplicon(625))



In [20]:

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



In [21]:

    
asm









    Out[21]:





Assembly
fragments..: 5681bp 744bp 1722bp 625bp
limit(bp)..: 31
G.nodes....: 8
algorithm..: common_sub_strings



In [22]:

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









    Out[22]:





 -|name|98
|       \/
|       /\
|       98|744bp_PCR_prod|38
|                         \/
|                         /\
|                         38|1722bp_PCR_prod|35
|                                            \/
|                                            /\
|                                            35|625bp_PCR_prod|61
|                                                              \/
|                                                              /\
|                                                              61-
|                                                                 |
 -----------------------------------------------------------------



In [23]:

    
result = candidate.synced(pYPK0)

The new construct should have cseguid 0t3SB9dac_rF8FmwUiA95T8KlRA and 8526 bp.



In [24]:

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









    



8526






    Out[24]:





0t3SB9dac_rF8FmwUiA95T8KlRA



In [25]:

    
result.name="pMEC1134"
result.description = "pYPK0_TPI1_CiGXF1_ENO2tp"
result.stamp()









    Out[25]:





cSEGUID_0t3SB9dac_rF8FmwUiA95T8KlRA



In [26]:

    
result.write("pYPK0_TPI1_CiGXF1_ENO2.gb")









    




pYPK0_TPI1_CiGXF1_ENO2.gb

Download genbank file.



In [27]:

    
reloaded =read("pYPK0_TPI1_CiGXF1_ENO2.gb")



In [28]:

    
reloaded.cseguid()









    Out[28]:





0t3SB9dac_rF8FmwUiA95T8KlRA



In [29]:

    
reloaded.description









    Out[29]:





'pYPK0_TPI1_CiGXF1_ENO2tp cSEGUID_0t3SB9dac_rF8FmwUiA95T8KlRA_2018-05-15T07:38:21.736145'

old



In [30]: