A BioScaffold Part (Uses PsrI), Protein Head Remover see Part Design page
BioScaffold Linker - Removes Stop Codons & scars & replaces with a Gly-Ser linker
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein** ====N.B.==== CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion. The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. Updates will soon follow on this. This version accounts for cleaving DNA 12/10bp (top/bottom) on the 5' of the recognition sequence and 10/12bp (top/bottom) on the 3' site.
CspCI enzyme has different variations in the distance of restricting DNA from the recognition site.This biobrick is almost identical to part BBa_K259004 but modified to account for this discrepancy.
This part is completely designed de novo. This sequence does not occur naturally.
A BioScaffold Part (Uses PpiI), Protein Tail Remover see Part Design Page
When placed in a BioBrick assembly and cut with the restriction enzyme PpiI, this part will direct the restriction enzymes to cut outside the BioBrick scar still in testing, uses restriction enzyme sites PpiI (2) PacI (2) MabI (2), will place in pSB1AK3 vector
Can be used to removed protein tails for protein fusions, see BBF RFC 15 XXXXX^ XXX TACTAGAG BioScaffold Part BBa_J70030 TACTAGAT XXXXX^ ^XXXXX XXX ATGATCAC ATGATCTA^XXXXX XXXXX^ TAA TACTAGAG BioScaffold Part BBa_J70030 TACTAGAT XXXXX^ ^XXXXX ATT ATGATCAC ATGATCTA^XXXXX
A BioScaffold Part (Uses PpiI), for offset see Part Design page
A BioScaffold Part (Uses AarI) for Library Vector Preparation (see Part Design page)
When placed in a BioBrick assembly and cut with the restriction enzyme AarI, this part will direct the restriction enzyme to cut within the BioBrick scar still in testing, uses AarI (2) XmaI (1) See part design page or BBFRFC15, for details about use
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions==== *Create an Assembly Standard 10 fusion giving ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part *Use the BioScaffold Specific Enzymes **Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. **Ligate and you will get the intermediate product; Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein **Restrict with BseRI to remove downstream scar. **Ligate to get the final product; Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein
Part was designed taking into account RFC10 assembly standard and as an attempt to be upwards compatible with RFC10.
This part was designed ''de novo''. The sequence does not occur naturally.
Tyr Residue involved in Restriction/Ligation
A BioScaffold Part (Uses AarI) for Library Insert Preparation (see Part Design page)
tetO Array Contains lots of tetO where bind YFP:tetR (<partinfo>BBa_K606025</partinfo>).
Reduce sequence to avoid restriction sites
Andrew Wright, Dave Lane, Fran??ois-Xavier Barre, pDAG479 plasmid.
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein** ====N.B.==== CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion. The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. This version is for when CspCI cuts 13/10b.p. (top/bottom) on the 5' of the recognition sequence and 10/13bp (top/bottom) on the 3' of the recognition sequence.
The CspCI discrepancy had to be taken into account
This part is completely designed ''de novo''. This sequence does not occur naturally.
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein**
This part was designed taking into consideration the requirements by Assembly Standard 10 (RFC10).
Right Library Insert Preparation Part (To use with J70050, and J70040)
Still in Testing Surround a part with J70050 and J70052, digest with AarI Insert into a vector that contained J70040 and had J70040 removed by AarI and MabI
Not sure if one AarI site on each site will be enough.
A BioScaffold Part (Uses AarI, MabI) for Library Vector Preparation (see Part Design page)
When placed in a BioBrick assembly and cut with the restriction enzyme AarI, this part will direct the restriction enzyme to cut within the BioBrick scar still in testing, uses AarI (2), MabI(2), XmaI (1) * XmaI does not work in vector for BioScaffold part manipulations See part design page or BBFRFC15, for details about use
See BBa_J70034, this is an analogous part except it uses MabI
A Composite BioScaffold Part (PpiI and PsrI) for Protein Fusions (see Design Page)
Brings together a BioScaffold part that removes protein tails with one that removes protein heads in order to create a BioScaffold part that can help create protein fusions using standard assembly
The part that links the two BioScaffold parts can be altered as desired--here I just wanted to leave at least 7 bp beyond the internal scars.
Left Library Insert Preparation Part (To use with J70052, and J70040)
Still in Testing Surround a part with J70050 and J70052, digest with AarI Insert into a vector that contained J70040, and had J70040 removed by AarI and MabI
Not sure if single AarI site will be good enough