MluI restriction enzyme site with flanking nucleotides
NsiI restriction enzyme site (PstI compatible overhang)
NsiI recognition site. Digestion with NsiI generates a compatible cohesive end to PstI.
Contains an MfeI restriction site. Cut this part with MfeI+Spe and the other part with Eco+Xba. Ligation will form two mixed sites, neither of which can be recut. This effectively fixes the left side of the Biobrick part
HindIII restriction enzyme site with extra flanking nucleotides
HindIII restriction site with three flanking base pairs
SacII restriction enzyme site with extra flanking nucleotides
A E. coli strain that carries the BsiWI gene from Bacillus species (D. Clark).
SalI restriction enzyme site with extra flanking nucleotides
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. 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.
XmaI restriction enzyme site with extra flanking nucleotides
XmaI restriction site with 3 flanking base pairs
I-SceI homing endonuclease site Restriction site for the homing endonuclease I-SceI. Sequence will be cut as follows: 5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3' 3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5'
Design was very simple. The recognition site was copied from NEB and synthesised
I-SceI homing endonuclease restriction site
XbaI restriction site with extra flanking nucleotides
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
MfeI restriction enzyme site (EcoRI compatible overhang)
MfeI recognition site. Digestion with MfeI yields a compatible cohesive end to EcoRI.
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.
Restriction site for the homing endonuclease I-PpoI.
Muscarella,D.E., Ellison,E.L., Ruoff,B.M. and Vogt,V.M. (1990) Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum. Mol. Cell. Biol., 10, 3386???3396.
This is a '''BamHI'''restriction site. This part might not be useful as a physical biobrick. It has been created to follow modularization in parts X
ApaI restriction site with extra flanking nucleotides
NheI restriction enzyme site (XbaI, SpeI compatible overhang)
NheI recognition site. Digestion with NheI generates a compatible cohesive end to XbaI and SpeI.
BmtI restriction enzyme site with extra flanking nucleotides
BmtI restriction site with three base pairs before site
AclI restriction enzyme site with extra nucleotides
AclI restriction site with 3 flanking bp
Bacillus stearothermophilus GR75 (Z. Chen)
SapI restriction enzyme site (offset cutter)
SapI is an offsite cutter restriction site. The recognition sequence for this enzyme is the 8 bp sequence GCTCTTCN (on the 5' strand). Cleavage at this site results in a 3 bp, 3' overhang (right side, bottom strand) directly downstream of this sequence. This specific part has a T in the 8th position.
This part was designed for use with Heather Keller's alternative assembly method for blunt cloning RBS sequences upstream of a coding sequence.
PvuI restriction enzyme site with extra flanking nucleotides
PvuI restriction site with 3 flanking base pairs
AvrII restriction enzyme site (XbaI, SpeI compatible overhang)
AvrII recognition site. Digestion with AvrII generates a compatible cohesive end to XbaI and SpeI.
SapI restriction enzyme site, reversed (offset cutter)
SapI restriction site in the reverse orientation (recognition sequence on the 3' strand). SapI is an offsite cutter. From left to right this part includes the designed 3 bp 5' overhang (ATG), a single bp spacer, the 7 bp recognition sequence (5'-GCTCTTCT-3'), and two bp spacer. Cleavage at this site results in a 3 bp, 5' overhang (left side, top strand), which makes it compatible with Heather Keller's RBS assembly scheme for creating RBS/CDS junctions without a mixed site. This part is specific for CDSs with an ATG start site and is not compatible with GTG start sites (via HKs assembly scheme). The single bp spacer on the 3' end allows RBSes construct with this part to be used in standard biobricks assembly and is necessary to maintain the reading frame of a downstream CDS. Note that Standard Biobricks Assembly with this site results in the creation of a new ATG start site, and a 7 amino acid fusion at the N terminus of the downstream protein.
This part only exists in composite parts within the registry. This restriction site is used by Heather Keller for creating blunt junctions between RBS sequences and coding regions (i.e. no biobricks scar in between).
KpnI restriction enzyme site with extra flanking nucleotides
KpnI restriction site with 3bp flanking each side
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).
An E. coli strain that carries the cloned Acc65I gene from Acinetobacter calcoaceticus 65
BamHI restriction site with extra flanking nucleotides