Prefix homologous recombination for Synechocystis ("Left flank")
Synechocystis sp. PCC 6803 possesses a annotated open reading frame (slr0168) with no known function. This open reading frame is used as a ‘neutral site’, i. e. a part of the genome that can be manipulated without apparent effect on the phenotype. For example this ‘neutral site’ has been used to evaluate mRNA expression of promoter probes using the lux and GFP system (Kunert et al, 2000).
This is a prefix region of DNA for homologous recombination into a neutral site within the genome of Synechocystis sp. PCC 6803.
NB: To be used with BBa_K1424001 , where the insert is positioned between BBa_K142000 (left flank) and BBa_K142001 (right flank).
Usage and Biology
BBa_K1424000 & BBa_K1424001 [] were used (flanking a kanamycin resistance cassette designed for Synechocystis sp. PCC 6803; BBa_K1424003 []) as double homologous recombination sequences for insertion of exogenous DNA into the genome of Synechocystis sp. PCC 6803. Colonies were picked and segregated on kanamycin containing BG11 growth media. The inclusion of exogenous DNA into the host genome was verified by performing a band-shift colony PCR. Using left homologous region forward primer and right homologous region reverse primer, PCR amplification of this genomic region can be performed. The increase in band size (band shift) of the mutant strain respective of the wild type strain confirms integration of exogenous kanamycin resistance cassette into the host genome.
ADVANTAGES IN GENOME ENGINEERING APPLICATIONS: This part can be used to integrate exogenous DNA into the genome of Synechocystis sp. PCC 6803 at the site of gene slr0168 (gene of unknown function).
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25INCOMPATIBLE WITH RFCIllegal NgoMIV site found at 441
- 1000COMPATIBLE WITH RFC