Mike's Moss Resources
New: Moss/Arabidopsis Golden Gate vector set
I have created some Golden Gate destination vectors to provide more flexibility in plasmid design and construction and to get away from the Gateway system. They were designed to be a simplified system catering to the types of transformations we tend to do most often rather than a super-flexible but multi-step system such as GoldenBraid, MoClo, or Loop Assembly.They allow use of either the BsaI or BsmBI type IIS restriction enzymes, and they have a lacZ'-GFP fusion that is removed in the final construct. Some vectors already contain selection cassettes plus neutral-target homology arms, some only selection cassettes, and others neither.
Moss Golden Gate (& GG CRISPR) Quick Guide
Moss Golden Gate Assembling (& CRISPR sgRNA loading) instructions
Moss Selection cassettes
Plasmids with moss selectable marker flanked by loxP sites (derived from pBNRF)G418 (20 µg/ml) and Hyg (15 µg/ml) selections work better than others. Zeocin (25 µg/ml) also works fairly well, but seems to induce some unrelated growth phenotypes occasionally (via DNA damage?). Gentamicin (50-100 µg/ml) selection can tricky, but we have had good luck lately. We use Gentamicin Sulfate from Chem Impex (cat 00149). Kanamycin (100 µg/ml) takes a while to kill sensitive moss cells, so we have only used it in dual selections with another antibiotic. pMP1109 (pBiLox+35S:nptII:camv) G418/Kanamycin-res*. Seq
pMP1159 (pBiLox+2x35S:aph(4)-Ia:camv) Hygromycin-res. Seq
pMP1169 (pBiLox+35S:Zeo:camv) Zeocin-res. Seq
pMP1119 (pBiLox+35S:aacC1:camv) Gentamycin/G418-res*. Seq
*Expression of aacC1 in moss confers resistance to gentamicin and G418 but not kanamycin, while nptII expression confers resistance to G418 and kanamycin but not gentamicin.
Old Gateway vectors
Note. The "Pp108B" targeting locus used in most of these plasmids contains repetitive DNA that may explain variability in expression levels. It also seems to preclude using CRISPR to facilitate integration: transformants always seem to acquire the resistance conferred by the cassette on the CRISPR plasmid suggesting that it is integrating into the genome somewhere.Further, the GFP, YFP, and CFP tags (except mVenus) are all the outdated versions that dimerize and are dimmer than modern versions. I do not recommend using them.
Gateway Destination vectors for over-expression/inducible expression
pMP1155 (pUK-Pp108B-nptII-Ubi:Gateway:ocs) Seq
pMP1228 (pUK-Pp108B-nptII-Ubix:Gateway:ocs)(no intron) Seq
pMP1180 (pUK-Pp108B-nptII-GmHsp17.3B:Gateway:e9) Seq
pMP1432 (pUK-Pp108B-Gent-GmHsp17.3B:Gateway:e9) Seq
Gateway Destination vectors for over-expression with tags
pMP1300 (pUK-Pp108B-nptII-Ubi:Gateway:GUS:ocs) Seq
pMP1367 (pUK-Pp108B-nptII-Ubi:Gateway:TAP:ocs) Seq
pMP1302 (pUK-Pp108B-nptII-Ubi:Gateway:TAP2:ocs) Seq
pMP1298 (pUK-Pp108B-nptII-Ubi:Gateway:GFPhdel:ocs) Seq
pMP1382 (pUK-Pp108B-nptII-Ubi:Gateway:GFP-His:ocs) Seq
pMP1379 (pUK-Pp108B-nptII-Ubi:Gateway:YFP-HA:ocs) Seq
pMP1574 (pUK-Pp108B-nptII-Ubi:Gateway:CFP-HA:ocs) Seq
pMP1472 (pUK-Pp108B-nptII-Ubi:Gateway:YFPnls-HA:ocs) Seq
pMP1442 (pUK-Pp108B-nptII-Ubi:Gateway:mVenus-HA:ocs) Seq
pMP1474 (pUK-Pp108B-nptII-Ubi:Gateway:mVenusnls-HA:ocs) Seq
pMP1445 (pUK-Pp108B-nptII-Ubi:Gateway:DsRED2-HA:ocs) Seq
pMP1475 (pUK-Pp108B-nptII-Ubi:Gateway:DsRED2nls-HA:ocs) Seq
pMP1377 (pUK-Pp108B-nptII-Ubi:Gateway:4xMyc:nos) Seq
pMP1373 (pUK-Pp108B-nptII-Ubi:Gateway:1xMyc:ocs) Seq
pMP1369 (pUK-Pp108B-nptII-Ubi:Gateway:HA:ocs) Seq
pMP1371 (pUK-Pp108B-nptII-Ubi:Gateway:FLAG:ocs) Seq
pMP1375 (pUK-Pp108B-nptII-Ubi:Gateway:AcV5:ocs) Seq
pMP1575 (pUK-Pp108B-nptII-Ubi:Gateway:GRlbd:ocs) Seq
pMP1578 (pUK-Pp108B-nptII-Ubi:Gateway:ESR1lbd:ocs) Seq
pMP1335 (pUK-Pp108B-nptII-Ubi:GFP6:Gateway:ocs) Seq
pMP1361 (pUK-Pp108B-nptII-Ubi: YFP:Gateway:ocs) Seq
pMP1363 (pUK-Pp108B-nptII-Ubi:TAP2:Gateway:ocs) Seq
pMP1359 (pUK-Pp108B-nptII-Ubi:1xMyc:Gateway:ocs) Seq
pMP1355 (pUK-Pp108B-nptII-Ubi:HA:Gateway:ocs) Seq
pMP1357 (pUK-Pp108B-nptII-Ubi:FLAG:Gateway:ocs) Seq
pMP1365 (pUK-Pp108B-nptII-Ubi:AcV5:Gateway:ocs) Seq
Physcomitrella neutral target loci plasmids
pMP1141 (pGEM+Pp108A) hydrolase target Seq
pMP1033 (pUK+Pp108B) adjacent intergenic region target Seq
Endogenous Pp108 target locus Seq