The CEN6/ARS4 S. cerevisiae single copy origin of replication was PCR amplified from p413TEF with primers CEN6ARSH_fwd and CEN6ARSH_rev pair of primers and the dominant marker gene bleMX4 was amplified from pUG66 with bleMX4_fwd and bleMX4_rev pair of primers. The primers were designed to allow for homologous recombination with the pSU0 plasmid replacing the 2µ ORI and URA3 gene with the CEN6ARSH and bleMX4, resulting in a plasmid designated pMEC1042.
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from pydna.all import *
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gb =Genbank("bjornjobb@gmail.com")
The p4XX PPP vectors are described in the publication below
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# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://www.sciencedirect.com/science/article/pii/0378111995000377', width='100%', height=250)
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p416TEF = gb.nucleotide("EF210199.1") #relevant sequence is the same as p413TEF
pUG66 = gb.nucleotide("AF298794.1")
pSU0 = gb.nucleotide("AB215109.1")
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from Bio.Restriction import EcoRV
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CEN6ARSH_fwd, CEN6ARSH_rev =parse('''
>476_CEN6ARSHf (58-mer)
TTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAacggatcgcttgcctgta
>475_CEN6ARSHr (30-mer)
cttttcatcacgtgctataaaaataattat''')
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cen =pcr(CEN6ARSH_fwd, CEN6ARSH_rev, p416TEF)
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bleMX4_fwd, bleMX4_rev =parse('''
>478_bleMX4f
aatttaaattataattatttttatagcacgtgatgaaaaggtccccgccgggtc
>477_bleMX4r
agtggaacgaaaactcacgttaagggattttggtcatgaggagctcgttttcga''')
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ble =pcr(bleMX4_fwd, bleMX4_rev, pUG66)
pSU0_EcoRV = pSU0.linearize(EcoRV)
notedown pMEC1042.py > pMEC1042.ipynb
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a=Assembly((pSU0_EcoRV, cen, ble))
pMEC1042 = a.assemble_circular()[0]
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pMEC1042.stamp()
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pMEC1042.locus = "pMEC1042"
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pMEC1042.write("pMEC1042.gb")
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from pydna.all import *
reloaded=read("pMEC1042.gb")
assert reloaded.cseguid() in reloaded.definition
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