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<figcaption>Fig1. (A) Physical map of pSRKBB-Empty showing pSR1 replicon as a black band. The replicon contains per (a positive effector of replication) and rep (replicase) gene. pK19 backbone (white band) contains pBR322 ORI and Kanamycin resistance gene (KmR) regulated by the P45 promoter. Mutagenic primer binding sites are shown as red arrows (mut1, mut2, mut3, and mut4). The region in the red box was replaced by (B) a BioBrick cloning site flanked by standard verification primer binding sites, VF2 and VR, derived from Iverson et al. (2016), creating pSRKM_Empty. Destination vectors can be made by digestion and ligation with SpeI to insert standardized LacZα flanked by the designated Type IIs restriction enzyme (recognition sites shown as bold) and four base overhangs for Level-1 (C) or Level-2 (D) assembly. (E) Schematic map of the level-1 destination vector pSRKM_NN and (F) the level-2 destination vector pSRCM_NN with chloramphenicol resistance (CmR). X refers to the fusion sites and NNNN to its four base overhangs: A (GGAG), B (TACT), C (AATG), D (AGGT), E (GCTT), F (CGCT), G (TGCC), and H (ACTA).</figcaption> | <figcaption>Fig1. (A) Physical map of pSRKBB-Empty showing pSR1 replicon as a black band. The replicon contains per (a positive effector of replication) and rep (replicase) gene. pK19 backbone (white band) contains pBR322 ORI and Kanamycin resistance gene (KmR) regulated by the P45 promoter. Mutagenic primer binding sites are shown as red arrows (mut1, mut2, mut3, and mut4). The region in the red box was replaced by (B) a BioBrick cloning site flanked by standard verification primer binding sites, VF2 and VR, derived from Iverson et al. (2016), creating pSRKM_Empty. Destination vectors can be made by digestion and ligation with SpeI to insert standardized LacZα flanked by the designated Type IIs restriction enzyme (recognition sites shown as bold) and four base overhangs for Level-1 (C) or Level-2 (D) assembly. (E) Schematic map of the level-1 destination vector pSRKM_NN and (F) the level-2 destination vector pSRCM_NN with chloramphenicol resistance (CmR). X refers to the fusion sites and NNNN to its four base overhangs: A (GGAG), B (TACT), C (AATG), D (AGGT), E (GCTT), F (CGCT), G (TGCC), and H (ACTA).</figcaption> | ||
</figure> | </figure> | ||
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Revision as of 16:24, 19 October 2016
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