Homemade maps are often unreliable as they omit “unimportant” features that may be critical for your experiment. The pTLNX vector also has a gene for plasmid selection (ccdB), along with virus SV40 nuclear localization signal and Xenopus globine 3’ UTR, that allows for high expression levels of cloned genes. Downstream from SP6 promoter, the rrnBT2 terminator allows efficient termination of genes cloned into the multiple cloning site 2 (Figure 2). In addition to the familiar pBR322 origin and antibiotic resistance genes AmpR and CmR (chloramphenicol resistance), there are also SP6 and lacUV promoters present. They require phage polymerases and are therefore inactive in vivo.īelow is a map of pTLNX, a Xenopus oocyte expression vector (Figure 2). Promoters from phages SP6 and T7 are often used for in vitro RNA amplification. In addition to genes, plasmids often include transcription promoters and terminators derived from E.coli phages. And, as we all know, the disruption in a gene will lead to inactivation of gene function – in this case, antibiotic resistance. For instance, PvuI cuts in the middle of Amp R, and BamHI cuts in the middle of Tet R. When using restriction enzyme sites to clone your gene of interest into your plasmid, be careful to look at which sites fall within your antibiotic resistance gene. So even if you have two plasmids with different origins of replication, you will not be able to select two plasmids at the same time if one expresses a methicillin resistance gene and the other expresses an ampicillin resistance gene. Tet and amp are read in different directions.īear in mind that enzyme beta-lactamase is not specific in detoxifying penicillin-derived antibiotics. These genes encode an efflux pump ( tet R) and beta-lactamase ( amp R) to excrete tetracycline and ampicillin from the cell, respectively. PBR322 has two antibiotic resistance genes: tet (tetracycline resistance) and amp (ampicillin resistance). The sites should be unique, but it pays to check, as derivative vectors often contain additional forgotten sequences. Restriction sites for corresponding enzymes are shown as vertical lines with the position of the starting nucleotides. The pBR322 ori is also used in pUC18, which is the second most common backbone used in eukaryotic vectors. This means that you will not be able to maintain two pBR323- derived vectors in one cell even if they have genes for different antibiotic resistances on them. The other thing to remember about ori is that plasmids with the same origin are often incompatible. Whatever you do, don’t change it! Once a plasmid is unable to replicate, it is useless.

“ Ori” means the origin of plasmid replication. In gene sequence databases, such Entrez-PubMed, plasmid sequences are diagrammed as linear sequences starting from the ori. Otherwise, the native promoter can interfere with your gene expression. If you clone your gene of interest in a middle of another gene, make sure that both of them are transcribed in the same direction. The black arrows show the direction of transcription, which is essential for cloning. Image by Avacop & Yikrazuul via Wikimedia Commons

Serial cloner linear to circular