Guide to sequences and annotation
|Screen identification code. Assigned when T1 seedlings were islolated.|
|All tag sequences were BLASTed against Genbank. If the gene has been characterized in Arabidopsis, the accepted name is given. If the tag bears signifigant homology to a previously characterized gene, but is not identical to that gene, it is designed as "X-like". If a tag bears no homology to previously characterized genes, it is classified as "novel" or "unknown".|
|The frame is relative to the reading frame of EGFP. "1" means the native reading frame of the tag is in frame with EGFP. In some cases, the reading frame is designated as "unknown". The "unknown" category includes both cases where the reading frame is unknown becasue the tag sequence has not been characterized, and also cases where the frame is irrelevent. This latter group includes fusions to the 3' untranslated region of a cDNA sequence, artifacts generated during creation gof the cDNA library, and in some cases, fusions to 5' untranslated sequences that result in a stop codon prior to the start of an accepted open reading frame.
The cDNA library was created with an EcoRI linker at the 5' end of the cDNA. Each reported tag sequence starts with the EcoRI site and 4 nucleotides of adjacent linker sequence:
|The EcoRI site is in frame with the EGFP coding frame. The linker adds 4 amino acids between the Ala10 protein linker encoded in pEGAD and the peptide encoded by the cloned sequence. The first three amino acids are invariant and are encoded by the first 9 bases in the linker. The fourth amino acid is variable as it is encoded by the last base of the linker and the first two bases of the inserted sequence.|
|If the gene has been characterized, a brief designation of the currently accepted function of that gene is given. Of course, these designations may change.|
|The status line is divided into three parts, separated by commas:|
|T2 indicates that the line showed heritability of the phenotype in the T2 generation. Lines that show T2 only have not been confirmed by recloning and retransformation of the sequence tag.|
|GYC indicates that the tag has been recloned into Green, Cyan, or Yellow fluorescent protein expression vectors. Lines that show T2, GYC only have not been confirmed by retransformation and microscopic analysis.|
|R indicates that the tag has been recloned, Retransformed, and the localization oattern conferred by the tag confirmed by microscopic analysis. Only tags that have been confirmed by retransformation will be submitted to the stock center as cloned sequences. New tags will be added to the stock center as additional confirmations are made. If you are interested in other cloned tags, please contact us directly.|
|Cycle sequencing was performed into the cDNA insert from primers within EGFP or the Ala10 linker. The primary goal in sequencing was to establish the sequence up to the first in frame stop codon. For longer inserts which showed no termination codons within the sequenced portion of the tag, reactions were also run from the downstream side of the cloning site. The cycle sequences were compared with previously deposited sequences in Genbank to derive the sequence of the cDNA insert up the first in frame stop codon. Where it was required, sequencing form internal primers was also performed.
As described above under "frame", all sequences start with the EcoRi site of the linker used to clone the cDNA library. The reported sequences are typically cycle sequence runs that are corrected and edited up through the first in-frame stop codon. The reported sequence after this stop codon is sometimes incomplete and does not extend through the entire cDNA insert.
|See pEGAD information for notes on cloning with pEGAD.|