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Box......: ?
Position.: ?
Importing the pydna package. Pydna is open source, documentated here and has a support forum as well as a publication:
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from pydna.all import *
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pYPKa =read("pYPKa.gb")
This vector should be a circular 3128 bp DNA molecule.
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pYPKa
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The circular seguid checksum of pYPKa should be
aV1eIrzOiCjvw01yvKkxDXHKLMk
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pYPKa.cseguid()
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Importing the restriction enzyme to be used for cloning from Biopython. EcoRV
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from Bio.Restriction import EcoRV
This enzyme should cut only once in pYPKa.
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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.
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gb =Genbank("bjornjobb@gmail.com")
The template is downloaded from Genbank below.
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template = gb.nucleotide("BK006948.2 REGION: complement(92851..93394)")
The template is a 544 bp linear DNA fragment.
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template
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The insert has the sequence shown below.
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str(template.seq)
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The seguid checksum of the template should be
CqNHbKdfVC5vap_jjDXfiQIyBvo
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template.seguid()
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Two primers are used to amplify the insert:
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f,r =parse(""">652_ScRPS19atpf2
ttaaatGTTAACTGAAATGAAAATTT
>651_ScRPS19atpr_PacI
taattaaTTTTACTCTATTTGTCGATC""", ds=False)
insert =pcr(f, r, template)
The primers anneal on the template like this.
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insert.figure()
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A recombinant plasmid is formed by ligating the insert PCR product to the linear vector.
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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.
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pYPKa_E_RPS19a = plasmid.synced(pYPKa)
Calculate cseguid checksum for the resulting plasmid. Should be
DxlDnby_v4TXT2ecK1SKMKQDbIQ
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pYPKa_E_RPS19a.cseguid()
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The file is named pYPKa_E_RPS19atp
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pYPKa_E_RPS19a.name = "pYPKa_E_RPS19atp"
pYPKa_E_RPS19a.description = "pYPKa_E_RPS19atp"
Stamp sequence with cSEGUID checksum
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pYPKa_E_RPS19a.stamp()
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Write sequence to a local file.
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pYPKa_E_RPS19a.write("pYPKa_E_RPS19a.gb")
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from pydna.all import *
reloaded =read("pYPKa_E_RPS19a.gb")
assert reloaded.cseguid() in reloaded.definition
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