Antimicrobial resistance (AMR) has emerged as an escalating health issue in the global public health arena. To evaluate and predict AMR, it is of utmost importance to identify and characterize both the known and unknown genes responsible for AMR. While known genes can be readily detected, identifying unknown genes present a challenge. In this study, we developed the plasmid K fragment (pKF) by modifying the pUC19 vector, specifically by removing the multiple cloning site and introducing a Prom-RBS sequence. pKF was used for amplification of K fragment that contains a ribosomal binding site (RBS), and promoter at both ends and plasmid origin of replication. The functionality of added Prom-RBS sequence and K fragment as a cloning vector was tested by cloning chloramphenicol resistance gene amplicon and erythromycin resistance gene from genomic DNA, respectively. The cloning experiment demonstrated the usability of this newly developed cloning method with K fragment. K fragment is an innovative vector that can be easily obtained through amplification by polymerase chain reaction and lacks antibiotic resistance markers. This novel approach is convenient to use since it allows cloning of resistance genes at all orientations and this flexibility can be maneuvered by changing restriction enzymes for primers and fragments. With these distinctive features, this vector stands out to be a versatile tool for cloning both known and unknown resistance genes, and the improved method with K fragment enables the microbiological and molecular characterization of cloned genes. K fragment can be utilized for cloning of resistance genes in bacteria originated in different environments without having to perform bacterial culture. We believe that the convenience brought by this technique could lend itself efficient in the battle against the growing AMR crisis through pre-emptive identification of resistance genes.