Below are descriptions and data files for several vectors that we have used for RMCE. We will continue to add unpublished vectors that may also be of use to the community. The data files below were assembled using sequence that we generated across the junctions of cloned segments, which has been combined with the published sequences of vector backbones.
pUASTP2.1 contains a P-element target for RMCE, the mini-white gene flanked by inverted attP sites. It is a derivative of PUASTP2 in which the UAS and TATA sequences flanking the cassette have been removed. (Specifically, PUASTP2 was digested with BamHI and StuI to remove UAS-TATA-attP, and an attP-containing BamHI-EcoRV fragment from pTA-attP was inserted). We have generated several insertions of this target into the genome, but this vector can be used to generate more target lines by standard P element transgenesis. Sequence is available here.
pUASTP2 contains a P-element target for RMCE, the mini-white gene flanked by inverted attP sites. This target cassette was contructed in the parent vector pUAST. We have generated several insertions of this target into the genome, but this vector can be used to generate more target lines by standard P element transgenesis. Sequence is available here.
*Please note that the UAS binding sites and hsp70 promoter of pUAST are outside of the exchange cassette in this vector - i.e. following RMCE, these elements will remain flanking the cassette. We are currently generating insertions of a new target site that lacks these elements.
piB-GFP is a donor vector that contains an hsp70 TATA box fused to an enhanced GFP open reading frame and an SV40 polyA tail. This gene construct is flanked by inverted attB sites in the vector pBluescript. piB-GFP is a useful vector for cloning a gene of interest into a donor cassette; the GFP gene can be excised and new sequences can be inserted between the inverted attB sites using BamHI, SalI, or HindIII. Sequence is available here, and a map can be found here.
*One note on preparing this vector: we had difficulty growing piB-GFP in a larger 25 ml culture for midiprepping, but there were no problems in smaller 3 ml cultures suitable for minipreps. For all of our GFP injections, we used mini-prepped piB-GFP that was further purified on a pcr-purification column (we used qiagen columns, but I'm sure any brand would do. The miniprepped DNA itself contained enough RNAse contaminant to completely digest the phiC31 RNA, but passage over the second column fixed this problem). It is unclear whether this issue will persist with other inserts.
**Andrey and Art from the Kuroda lab alerted us to the fact that piB-GFP contains a small piece of sequence derived from a 3' P element end, which immediately flanks the 3' UTR (SV40 tail) of the GFP gene (this came along for the ride in our cloning strategy). The 100 bp sequence extends from 2906-3004 in the piB-GFP vector sequence. Popping out GFP with BamHI removes this region, while cutting with SalI or HindIII does not, meaning that following an RMCE event, this 100bp sequence will be present both in the cassette and in the P element end of the target site. We don't expect this to have an effect on gene expression, but this sequence should be avoided when designing primers to confirm insertions.
pCiB-yin is a donor vector that contains an intronless yellow gene flanked by inverted attB sites. The cassette was constructed in the parent vector pCar4. In terms of use, there are no convenient restriction sites available to use pCiB-yin as a starting point for cloning (piB-GFP is more useful for that), but pCiB-yin provides a useful positive control for injections since insertion of the cassette produces a dominant pigmentation phenotype in a yellow background. Sequence is available here.