Recombineering is a homologous-based DNA cloning and modification technique. Recombineering-mediated chromosomal gene knock-in usually involves a selectable/counterselectable cassette. Though a variety of selectable/counterselectable cassettes were developed; however, a specifically designed gene deletion strain or minimal medium is often required. Herein, we describe a novel selectable/counterselectable cassette Plac-ccdB-aacC1 in which aacC1 (gentamicin resistance gene) is used as the selectable marker for the homologous arm-flanked cassette knock-in, while the IPTG inducible ccdB gene is used as the counterselectable marker for chromosomal gene knock-in. The counterselection is achieved via supplementing 1 mM IPTG in the LB agar medium. An oligonucleotide designed to evade the mismatch repair system was utilized to engineer an Escherichia coli DH10B-derived gyrA462 strain that was used to as the host for the plasmid harboring the P-lac-ccdB-aacC1 cassette. By using the P-lac-ccdB-aacC1 cassette, a linear-linear homologous recombination (LLHR) system was generated by knocking a 6.2 kb araC-P-BAD-red gamma-recET-recA DNA fragment into the E. coli DH10B chromosome. The functional of the LLHR recombineering system was characterized by cloning of the target DNA from PCR product as well as from the genomic DNA mixture. The P-lac-ccdB-aacC1 cassette will be a useful tool in E. coli recombineering.