pYPKa_Z_TDH1_rev

This plasmid is the same as pYPKa_Z_TDH1 but with the insert inverted

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. ZraI



In [5]:

    
from Bio.Restriction import ZraI

This enzyme should cut only once in pYPKa.



In [6]:

    
pYPKa_ZraI = pYPKa.linearize(ZraI)

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("BK006943.2 REGION: 337038..338270")

The template is a 267 bp linear DNA fragment.



In [9]:

    
template









    Out[9]:




BK006943.2  337038-338270

The insert has the sequence shown below.



In [10]:

    
str(template.seq)









    Out[10]:





'AAAAGCTCGACCTTAAATAAGGTGAAGATGTGGTTCTCTGTTCTTTCAACATCTCGATATTTAACTTTTCGTTTGGCATTATAGTGTTATACATAACAATATCTAGATTTCAGTAGAAGACTTTTGCGTAAGTTATAACGAACTAAACAAGCAAGAGGTAAAATACGATATAGTAAGAAGCTCAAGTGCTAAAACTTTTTTTCTCTACGAAAGGAGGAATAGCATATGGTTTTCTTCCCTTTTACAGTGCTTCGGAAAAGCACAGCGTTGTCCAAGGGAACAATTTTTCTTCAAGTTAATGCATAAGAAATATCTTTTTTTATGTTTAGCTAAGTAAAAGCAGCTTGGAGTAAAAAAAAAAATGAGTAAATTTCTCGATGGATTAGTTTCTCACAGGTAACATAACAAAAACCAAGAAAAGCCCGCTTCTGAAAACTACAGTTGACTTGTATGCTAAAGGGCCAGACTAATGGGAGGAGAAAAAGAAACGAATGTATATGCTCATTTACACTCTATATCACCATATGGAGGATAAGTTGGGCTGAGCTTCTGATCCAATTTATTCTATCCATTAGTTGCTGATATGTCCCACCAGCCAACACTTGATAGTATCTACTCGCCATTCACTTCCAGCAGCGCCAGTAGGGTTGTTGAGCTTAGTAAAAATGTGCGCACCACAAGCCTACATGACTCCACGTCACATGAAACCACACCGTGGGGCCTTGTTGCGCTAGGAATAGGATATGCGACGAAGACGCTTCTGCTTAGTAACCACACCACATTTTCAGGGGGTCGATCTGCTTGCTTCCTTTACTGTCACGAGCGGCCCATAATCGCGCTTTTTTTTTAAAAGGCGCGAGACAGCAAACAGGAAGCTCGGGTTTCAACCTTCGGAGTGGTCGCAGATCTGGAGACTGGATCTTTACAATACAGTAAGGCAAGCCACCATCTGCTTCTTAGGTGCATGCGACGGTATCCACGTGCAGAACAACATAGTCTGAAGAAGGGGGGGAGGAGCATGTTCATTCTCTGTAGCAGTAAGAGCTTGGTGATAATGACCAAAACTGGAGTCTCGAAATCATATAAATAGACAATATATTTTCACACAATGAGATTTGTAGTACAGTTCTATTCTCTCTCTTGCATAAATAAGAAATTCATCAAGAACTTGGTTTGATATTTCACCAACACACACAAAAAACAGTACTTCACTAAATTTACACACAAAACAAA'

Two primers are used to amplify the insert:



In [11]:

    
f,r =parse(""">547_ScTDH1tpf (27-mer)
                     ttaaatAAAAGCTCGACCTTAAATAAG
                     >653_ScTDH1tpr_PacI
                     taattaaTTTGTTTTGTGTGTAAATTTAG""", ds=False)

insert =pcr(f, r, template)
insert









    Out[11]:




Amplicon(1246)

The primers anneal on the template like this.



In [12]:

    
insert.figure()









    Out[12]:





      5AAAAGCTCGACCTTAAATAAG...CTAAATTTACACACAAAACAAA3
                               |||||||||||||||||||||| tm 49.5 (dbd) 55.4
                              3GATTTAAATGTGTGTTTTGTTTaattaat5
5ttaaatAAAAGCTCGACCTTAAATAAG3
       ||||||||||||||||||||| tm 51.8 (dbd) 57.6
      3TTTTCGAGCTGGAATTTATTC...GATTTAAATGTGTGTTTTGTTT5

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



In [13]:

    
plasmid = (pYPKa_ZraI + insert.rc()).looped()

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



In [14]:

    
pYPKa_Z_TDH1_rev = plasmid.synced(pYPKa)



In [15]:

    
pYPKa_Z_TDH1_rev.cseguid()









    Out[15]:





D8abxjpwv03qaCdXDn6T8JZYjR4

The file is named pYPKa_Z_TDH1_rev



In [16]:

    
pYPKa_Z_TDH1_rev.name = "pYPKa_Z_TDH1_rev"
pYPKa_Z_TDH1_rev.description = "pYPKa_Z_TDH1_rev"

Stamp sequence with cSEGUID checksum



In [17]:

    
pYPKa_Z_TDH1_rev.stamp()









    Out[17]:





cSEGUID_D8abxjpwv03qaCdXDn6T8JZYjR4

Write sequence to a local file.



In [18]:

    
pYPKa_Z_TDH1_rev.write("pYPKa_Z_TDH1_rev.gb")









    




pYPKa_Z_TDH1_rev.gb

Download pYPKa_Z_TDH1_rev



In [19]:

    
from pydna.all import *
reloaded =read("pYPKa_Z_TDH1_rev.gb")
assert reloaded.cseguid() in reloaded.definition