Construction of pYPK0_TDH3_PsXYL2_PGI1 (pMEC1128)

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]:

    

from Bio.Restriction import ZraI, AjiI, EcoRV


    

In [4]:

    

pYPK0 =read("pYPK0.gb")


    

In [5]:

    

promoter_clone = pYPKa_Z_TDH3 =read("pYPKa_Z_TDH3.gb")


    

In [6]:

    

# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://google.com/patents/EP0450430A2?cl=es', width='100%', height=250)


    

    
Out[6]:





        
        

In [7]:

    

gb =Genbank("bjornjobbb@gmail.com")
gene_template = gb.nucleotide("A16166.1")


    

In [8]:

    

gene_template.list_features()


    

    
Out[8]:





+-----+---------------+-----+-----+------+------+--------+------+
| Ft# | Label or Note | Dir | Sta | End  |  Len | type   | orf? |
+-----+---------------+-----+-----+------+------+--------+------+
|   0 | nd            | --> | 0   | 1963 | 1963 | source |  no  |
|   1 | nd            | --> | 318 | 1410 | 1092 | CDS    | yes  |
+-----+---------------+-----+-----+------+------+--------+------+

In [9]:

    

orf = gene_template.extract_feature(1)


    

In [10]:

    

print(orf.seq)


    

    




ATGACTGCTAACCCTTCCTTGGTGTTGAACAAGATCGACGACATTTCGTTCGAAACTTACGATGCCCCAGAAATCTCTGAACCTACCGATGTCCTCGTCCAGGTCAAGAAAACCGGTATCTGTGGTTCCGACATCCACTTCTACGCCCATGGTAGAATCGGTAACTTCGTTTTGACCAAGCCAATGGTCTTGGGTCACGAATCCGCCGGTACTGTTGTCCAGGTTGGTAAGGGTGTCACCTCTCTTAAGGTTGGTGACAACGTCGCTATCGAACCAGGTATTCCATCCAGATTCTCCGACGAATACAAGAGCGGTCACTACAACTTGTGTCCTCACATGGCCTTCGCCGCTACTCCTAACTCCAAGGAAGGCGAACCAAACCCACCAGGTACCTTATGTAAGTACTTCAAGTCGCCAGAAGACTTCTTGGTCAAGTTGCCAGACCACGTCAGCTTGGAACTCGGTGCTCTTGTTGAGCCATTGTCTGTTGGTGTCCACGCCTCCAAGTTGGGTTCCGTTGCTTTCGGCGACTACGTTGCCGTCTTTGGTGCTGGTCCTGTTGGTCTTTTGGCTGCTGCTGTCGCCAAGACCTTCGGTGCTAAGGGTGTCATCGTCGTTGACATTTTCGACAACAAGTTGAAGATGGCCAAGGACATTGGTGCTGCTACTCACACCTTCAACTCCAAGACCGGTGGTTCTGAAGAATTGATCAAGGCTTTCGGTGGTAACGTGCCAAACGTCGTTTTGGAATGTACTGGTGCTGAACCTTGTATCAAGTTGGGTGTTGACGCCATTGCCCCAGGTGGTCGTTTCGTTCAAGTTGGTAACGCTGCTGGTCCAGTCAGCTTCCCAATCACCGTTTTCGCCATGAAGGAATTGACTTTGTTCGGTTCTTTCAGATACGGATTCAACGACTACAAGACTGCTGTTGGAATCTTTGACACTAACTACCAAAACGGTAGAGAAAATGCTCCAATTGACTTTGAACAATTGATCACCCACAGATACAAGTTCAAGGACGCTATTGAAGCCTACGACTTGGTCAGAGCCGGTAAGGGTGCTGTCAAGTGTCTCATTGACGGCCCTGAGTAA

In [11]:

    

orf.isorf()


    

    
Out[11]:





True

In [12]:

    

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


    

    




1092






    
Out[12]:





CyliZ1T5FR7RZCWuSzPm9dOHmM0

In [13]:

    

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


    

    




MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFYAHGRIGNFVLTKPMVLGHESAGTVVQVGKGVTSLKVGDNVAIEPGIPSRFSDEYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLVKLPDHVSLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAAAVAKTFGAKGVIVVDIFDNKLKMAKDIGAATHTFNSKTGGSEELIKAFGGNVPNVVLECTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPITVFAMKELTLFGSFRYGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRYKFKDAIEAYDLVRAGKGAVKCLIDGPE*

In [14]:

    

pf, pr =parse('''>XDH_YPK_rec_fwd 
                        gtcgaggaacgccaggttgcccactttctcactagtgaAAATGACTGCTAACCCTTCCTTGGTG
                        >XDH_YPK_rec_rev 
                        ATTTAAatcctgatgcgtttgtctgcacagatggcgcgTTACTCAGGGCCGTCAATGAGACA''')


    

In [15]:

    

g =pcr( pf, pr, orf)


    

In [16]:

    

g.figure()


    

    
Out[16]:





                                        5ATGACTGCTAACCCTTCCTTGGTG...TGTCTCATTGACGGCCCTGAGTAA3
                                                                    |||||||||||||||||||||||| tm 62.8 (dbd) 71.8
                                                                   3ACAGAGTAACTGCCGGGACTCATTgcgcggtagacacgtctgtttgcgtagtcctaAATTTA5
5gtcgaggaacgccaggttgcccactttctcactagtgaAAATGACTGCTAACCCTTCCTTGGTG3
                                         |||||||||||||||||||||||| tm 61.8 (dbd) 69.3
                                        3TACTGACGATTGGGAAGGAACCAC...ACAGAGTAACTGCCGGGACTCATT5

In [17]:

    

terminator_clone = pYPKa_E_PGI1 =read("pYPKa_E_PGI1.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))


    

In [20]:

    

(pYPK0_E_Z, p, g, t)


    

    
Out[20]:





(Dseqrecord(-5681), Amplicon(860), Amplicon(1170), Amplicon(1105))

In [21]:

    

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


    

In [22]:

    

asm


    

    
Out[22]:





Assembly
fragments..: 5681bp 860bp 1170bp 1105bp
limit(bp)..: 31
G.nodes....: 8
algorithm..: common_sub_strings

In [23]:

    

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


    

    
Out[23]:





 -|name|98
|       \/
|       /\
|       98|860bp_PCR_prod|38
|                         \/
|                         /\
|                         38|1170bp_PCR_prod|35
|                                            \/
|                                            /\
|                                            35|1105bp_PCR_prod|61
|                                                               \/
|                                                               /\
|                                                               61-
|                                                                  |
 ------------------------------------------------------------------

In [24]:

    

pYPK0_TDH3_PsXYL2_PGI1 = candidate.synced(pYPK0)


    

In [25]:

    

print(len(pYPK0_TDH3_PsXYL2_PGI1))
pYPK0_TDH3_PsXYL2_PGI1.cseguid()


    

    




8570






    
Out[25]:





ycHQ8EOwqQihZOUFmIbCiwHhMZ0

In [26]:

    

pYPK0_TDH3_PsXYL2_PGI1.name="pMEC1128"
#pYPK0_TDH3_PsXYL2_PGI1.description = "pYPK0_TDH3_PsXYL2_PGI1tp"
pYPK0_TDH3_PsXYL2_PGI1.stamp()


    

    
Out[26]:





cSEGUID_ycHQ8EOwqQihZOUFmIbCiwHhMZ0

In [27]:

    

pYPK0_TDH3_PsXYL2_PGI1.write("pYPK0_TDH3_PsXYL2_PGI1.gb")


    

    





pYPK0_TDH3_PsXYL2_PGI1.gb

In [28]:

    

reloaded =read("pYPK0_TDH3_PsXYL2_PGI1.gb")


    

In [29]:

    

reloaded.cseguid()


    

    
Out[29]:





ycHQ8EOwqQihZOUFmIbCiwHhMZ0

In [30]:

    

reloaded.description


    

    
Out[30]:





'cSEGUID_ycHQ8EOwqQihZOUFmIbCiwHhMZ0_2018-05-15T07:38:25.887910'

In [31]: