BBa_K143007 1 BBa_K143007 5??? Integration Sequence for the PyrD locus of B.subtilis 2008-09-15T11:00:00Z 2015-05-08T01:10:23Z The 5??? integration sequence was taken from the B.subtilis chromosome and is homologous to the chromosome from a few hundred bp upstream of the gene's start codon until 300 bp into the gene. It was produced by PCR cloning with Pfu DNA polymerase Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (<bbpart> BBa_K143004</bbpart>) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The PyrD gene has been a target for numerous integration vectors, including the shuttle vectors pPyr-Cm (GenBank Accession number AY464558) and pPyr-Kan (GenBank Accession number AY464559) <cite>1</cite>. Integration into the PyrD locus makes the ''B.subtilis'' auxotrophs for uracil and transformants require about 40&#956;g/ml to allow for growth. This allows us to assay for integration by growing a replica plate with no supplemented uracil to negativly select for transformants. false false _128_ 0 2090 9 No part sequence false The PyrD integration sequences were designed from the PyrD gene's Genbank entry<cite>#1</cite> and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and PyrD gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined. The integration sequences were then produced by PCR cloning with Pfu DNA polymerase false James Chappell igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z