pYPKa_E_TDH3

Freezer ID

Strain#..: ?
Box......: ?
Position.: ?

Importing the pydna package. Pydna is open source, documentated here and has a support forum as well as a publication:

Pereira F, Azevedo F, Carvalho Â, Ribeiro GF, Budde MW, Johansson B: Pydna: a simulation and documentation tool for DNA assembly strategies using python. BMC Bioinformatics 2015, 16:142.



In [1]:

    
from pydna.all import *

Read the cloning vector from a local file file, description of pYPKa.



In [2]:

    
pYPKa =read("pYPKa.gb")

This vector should be a circular 3128 bp DNA molecule.



In [3]:

    
pYPKa









    Out[3]:




pYPKa.gb

The circular seguid checksum of pYPKa should be

aV1eIrzOiCjvw01yvKkxDXHKLMk



In [4]:

    
pYPKa.cseguid()









    Out[4]:





aV1eIrzOiCjvw01yvKkxDXHKLMk

Importing the restriction enzyme to be used for cloning from Biopython. EcoRV



In [5]:

    
from Bio.Restriction import EcoRV

This enzyme should cut only once in pYPKa.



In [6]:

    
pYPKa_EcoRV = pYPKa.linearize(EcoRV)

The template below comes from a Genbank record. Access to Genbank is needed in order to download the template. If you execute this script, change the email address below to your own. Always tell Genbank who you are, when using their web service.



In [7]:

    
gb =Genbank("bjornjobb@gmail.com")

The template is downloaded from Genbank below.



In [8]:

    
template = gb.nucleotide("BK006941.2 REGION: complement(883811..884508)")

The template is a 698 bp linear DNA fragment.



In [9]:

    
template









    Out[9]:




BK006941.2  883811-884508

The insert has the sequence shown below.



In [10]:

    
print(template.seq)









    



ATAAAAAACACGCTTTTTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAA

The seguid checksum of the template should be

7mYN7gVjDcNrIyVWTW02290Vtk0



In [11]:

    
template.seguid()









    Out[11]:





7mYN7gVjDcNrIyVWTW02290Vtk0

Two primers are used to amplify the insert:



In [12]:

    
f,r =parse("""

>415_ScTDH3tpf
TTAAATAATAAAAAACACGCTTTTTCAGT
>623_ScTDH3tpr_PacI
taattaaTTTGTTTGTTTATGTGTGTTTATTCG

""", ds=False)

insert =pcr(f, r, template)

The primers anneal on the template like this.



In [13]:

    
insert.figure()









    Out[13]:





       5ATAAAAAACACGCTTTTTCAGT...CGAATAAACACACATAAACAAACAAA3
                                 |||||||||||||||||||||||||| tm 55.0 (dbd) 63.7
                                3GCTTATTTGTGTGTATTTGTTTGTTTaattaat5
5TTAAATAATAAAAAACACGCTTTTTCAGT3
        |||||||||||||||||||||| tm 53.4 (dbd) 59.9
       3TATTTTTTGTGCGAAAAAGTCA...GCTTATTTGTGTGTATTTGTTTGTTT5

A recombinant plasmid is formed by ligating the insert PCR product to the linear vector.



In [14]:

    
plasmid = (pYPKa_EcoRV + insert).looped()

The plasmid sequence is rotated so that the origin is in the same position as for the cloning vector sequence.



In [15]:

    
pYPKa_E_TDH3 = plasmid.synced(pYPKa)

Calculate cseguid checksum for the resulting plasmid. Should be

h1clDle64wo9AsT3Cie2P3KpSeE



In [16]:

    
pYPKa_E_TDH3.cseguid()









    Out[16]:





h1clDle64wo9AsT3Cie2P3KpSeE

The file is named pYPKa_E_TDH3tp



In [17]:

    
pYPKa_E_TDH3.name = "pYPKa_E_TDH3tp"
pYPKa_E_TDH3.description = "pYPKa_E_TDH3tp"

Stamp sequence with cSEGUID checksum



In [18]:

    
pYPKa_E_TDH3.stamp()









    Out[18]:





cSEGUID_h1clDle64wo9AsT3Cie2P3KpSeE

Write sequence to a local file.



In [19]:

    
pYPKa_E_TDH3.write("pYPKa_E_TDH3.gb")









    




pYPKa_E_TDH3.gb

Download pYPKa_E_TDH3



In [20]:

    
from pydna.all import *
reloaded =read("pYPKa_E_TDH3.gb")
assert reloaded.cseguid() in reloaded.definition