<jupyter_start><jupyter_text>Construction of the pLBL3 vectorThe pLBL3 vector is a derivative of the pLBL2 but with a LEU2 marker instead of the URA3 marker.<jupyter_code>from pydna.all import *
gb = Genbank("<EMAIL>")<jupyter_output><empty_output><jupyter_text>The LEU2 marker was taken from YEplac181<jupyter_code>YEplac181 = gb.nucleotide("X75460.1")
YEplac181<jupyter_output><empty_output><jupyter_text>The two primers below were used to amplify the LEU2 marker also providing tails for assembly with pLBL2.<jupyter_code>fp, rp = parse_primers("""

>Fw_LEU2
tccgtggaattaattctcatctttgacagcttatcatcgataagcGAGGCCAACTGTGGGAATAC

>Rv_LEU2
taatacagttttttagttttgctggccgcatcttctcaaatatgcCTACCCTATGAACATATTCC

""")
LEU2_product = pcr(fp,rp,YEplac181)
LEU2_product
LEU2_product.figure()
LEU2_product.name="LEU2_product"
pLBL2 = read("pLBL2.gb")<jupyter_output><empty_output><jupyter_text>The pLBL2 vector was digested with [NcoI](http://rebase.neb.com/rebase/enz/NcoI.html).<jupyter_code>from Bio.Restriction import NcoI
pLBL2_linear = pLBL2.linearize(NcoI)
pLBL2_linear.id = pLBL2_linear.name = "pLBL2_NcoI"
asm = Assembly((LEU2_product, pLBL2_linear))
asm
candidates = asm.assemble_circular()
candidates
candidate = candidates[0]
candidate
candidate.cseguid()
assert candidate.cseguid() == "Joos92IiFIJrDvwqO0z-ZBMGR4g"
pLBL3 = candidate.synced(pLBL2)
pLBL3.id = pLBL3.name = pLBL3.version = "pLBL3"
pLBL3.stamp()
pLBL3.write("pLBL3.gb")<jupyter_output><empty_output>
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