The ccd operon having a constitutive promoter and the ccdB coding region. ccdB protein is lethal in normal cloning strains. This part is used to aid the process of moving a BioBrick part to a new plasmid. A target plasmid carrying P1010 is cut and mixed with the cut insert. Plasmids that recombine are selected against because they kill the cell that picks them up. P1010 is only provided as plasmid or in DB3.1 which is ccdB resistant.
Kanamycin Resistance Cassette in Cyanobacteria
Ampicillin resistance cassette for modular BioBricks vector construction.
This provides the entire chloramphenicol resistance operon and will express CmR.
same part as BBa_P1002 except in reverse orientation.
This contains CmR, a terminator and then AmpR and a terminator.
gentamycin/kanamycin resistance cassette
The aacA-aphD gene from the transposon Tn4001 conveys resistance to both Gentamycin and Kanamycin with a single two-domain resistance protein.
;primers to make Biobrick version of GmR gene cassette (P1012) gtttcttcgaattcgcggccgcttctagagTGGACTTGACTGAGTTTATGGAA, P3aph-F gtttcttcctgcagcggccgctactagtaTCTGGACTTGACTCACTTCCTTT, P3aph-R ;P3aph-R (3' - 5') ;aaaggaagtgagtcaagtccagatactagtagcggccgctgcaggaagaaac
Originally from Tn4001 transposon; sequence M18086. Created from plasmid pISM2062 from Chris Minion, Iowa State University.
This cassette provides the entire tetracycline resistance operon and will express TetR.
Kanamycin resistance cassette and a double terminator
This part contains a kanamycin resistance cassette and a double terminator (BBa_P1003 + BBa_B0014). This allows the kanamycin cassette to be functional. The cassette can be used to design a recombinant vector by gene insertion in a non lytic bacteriophage as the Lambda phage. In fact it allows us to know if a bacteriophage is modified or not and if the inserted gene is expressed or not. When the virus is modified the kanamycin cassette induces a kanamycin resistance, so the bacteria become resistance, and when the virus is not modified bacteria are killed by kanamycin. This part is completed by B0014 + P1003 + B0014 to finish the transcription before the resistance cassette.
When we design the bio-brick it was impossible to obtain a functional bio-brick by using the plasmid SB1AK3. But it's worked with a pSB1A1.
Part:BBa_P1003, Designed by Reshma Shetty Group: Knight Lab, MIT (2006-01-23) Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16)
ccdB and minimal pUC19 derived high copy origin
{| |- |width='35%'| |width='65%'| The CcdB protein, constitutively expressed by P1016, is lethal to most of the [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell BioBrick cell strains], only DB3.1 is resistant. |} This part is designed to be located in the multiple cloning site of low copy BioBrick vectors. It is composed of two parts BBa_P1016 and BBa_I50022. BBa_P1016 provides selection against non-insert containing clones (see below). BBa_I50022 is based on the high copy pUC19 origin. It enables the base vector to be propagated at high copy for ease of purification. P1016 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or religated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. I50020 is useful because it is a high copy origin located within the BioBricks cloning site. Any plasmids containing this part will be propagated (and thus purified) at high copy. Once the insert of interest is cloned into the plasmid, the I50022 part is eliminated and the plasmid returns to the copy number of the primary origin in the plasmid (usually low copy). A new set of low copy BioBrick plasmids are available with the I52001 insert. See [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=I52001 I52001 Physical DNA] for the current list of plasmids that are available in this form.
This part is a composite part of BBa_P1016 and BBa_I50022. It is designed to be placed in the multiple cloning site of BioBricks vectors. Vectors with this part in the multiple cloning site will be selected against during BioBricks assembly because BBa_P1016 is lethal to most cell strains. Also, BBa_I50022 enables the vector to be propagated at high copy regardless of the other origins on the vector.
ccdB and nonfunctional pUC19 derived high copy origin
This part is designed to be located in the multiple cloning site of low copy BioBrick vectors. BBa_P1016 provides selection against non-insert containing clones and BBa_I50020 enables the base vector to be propagated at high copy for ease of purification
This part is a composite part of BBa_P1016 and BBa_I50020. It is designed to be placed in the multiple cloning site of BioBricks vectors. Vectors with this part in the multiple cloning site will be selected against during BioBricks assembly because BBa_P1016 is lethal to most cell strains. Also, BBa_I50020 enables the vector to be propagated at high copy regardless of the other origins on the vector.
Derived from luxICDABEG operon terminator of Vibrio fischeri <genbank>AF170104</genbank>.
Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.
In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A->G mutation (at position 40 -- sequence coordinate/not MFOLD coordinate). The above sequence does not reflect this mutation (but the MFOLD image does). This terminator's location cannot be found using some inverted repeat detectors like PALINDROME because it is too short and contains a mismatch. This one was found with the help of Tom Knight. It lies between two coding regions that point towards eachother.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Minimal pUC19-derived high copy replication origin
High copy origin of replication derived from pSB1A3. Designed to omit antibiotic resistance markers and primer binding sites. Lacks mutations that render BBa_I50020 nonfunctional as a replication origin.
We had to leave several restriction sites in because point mutations that eliminated these sites were found to render the origin nonfunctional.
gentamycin resistance cassette (aacC1 gene)
The aacC1 gene conveys resistance to gentamycin only. It is derived from a Tn21 series transposon (Wohlleben89) found on the R1033 plasmid (Pseudomonas aeruginosa). This part is created by PCR from the pJAT8 plasmid (Keastling lab) which in turn is derived from pJN105 (Newman and Fuqua).
pJAT8 plasmid edited with primers: GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ccttgtcgccttgcgtataa,aacC1-F GTT TCT TCC TGC AGC GGC CGC TAC TAG TA atctcggcttgaacgaattg,aacC1-R
Zeocoin and bleomycin resistance cassette including promoter and coding sequence. It lacks a terminator.
Codon optimized for E coli by GenScript.
Streptoalloteichus hindustanus, PDB: 1BYL
Chloramphenicol resistance gene including its native promoter and ribosome binding site. Confers resistance to 25 ug/mL chloramphenicol. {pSB1A2-Bca1016}
PCR ca1016F/R on pKD3 (934 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Products are Bca1015 and Bca1016 ---- ca1016F Forward EcoRI to biobrick CAT of pKD3 gacttgaattcgcggccgcttctagaggaataggaacttcatttaaatg ca1016R Reverse SpeI to biobrick CAT of pKD3 ccgctactagtggcgcgcctacctgtgacgg
This part is derived from BBa_P1010 but it lacks ccdA-.
This part is a differs from BBa_P1011 in that the mutations introduced in BBa_P1011 to remove restriction sites are not present in this part. It does have the "normal" ccd promoter and ccdB coding region. It also has a double TAA stop codon as per BioBrick conventions.
ccdA- was eliminated since it is an inactive form of ccdA and should not be required for the toxic phenotype of ccdB.
Novel gentamicin resistance gene. Derived from Genbank DQ208936, pTEX5500ts, kind gift of Barbara E. Murray. This, in turn was derived from Genbank AF016483. See pmid=16391062, pmid=9593155
XbaI site at 660 removed by mutating c->t which also improves codon usage. gatgatttttacagggatattttagagaacgaaatctac,P1014-XF gtagatttcgttctctaaaatatccctgtaaaaatcatc,P1014-XR
Kanamycin resistance cassette for modular BioBricks vector construction.
High copy origin of replication derived from pSB1A3. Designed to omit antibiotic resistance markers and primer binding sites.
Default insert of plasmid BBa_I739201, BBa_I739202, BBa_I739204
OriKan: a composite part to turn any pSB1C3 into a Bacillus plasmid
Part: BBa_K1937002 (pSB1C3-OriKan) (Chassis E. coli/B. subtilis, carrier plasmid pSB1C3) Length: 2510 bp While Bacillus subtilis is of huge interest for a growing number of iGEM projects, it is not easy to develop new parts as it is required that they registered after sub-cloning in the E. coli plasmid pSB1C3. During the iGEM Toulouse 2016 project, we had the idea to create a part that could turn any pSB1C3-based plasmid in a shuttle vector (E. coli/B. subtilis). This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France)
We successfully transformed E. coli and selected clones on chloramphenicol. Likewise, we successfully transformed B. subtilis and selected clones on kanamycine. Presence of the pSB1C3-OriKan plasmid was demonstrated in B. subtilis by PCR checking. The part sequence has been verified by sequencing.
The BBa_K1937002 part contains the repU origin of replication of B. subtilis and the kanamycin resistance gene for B. subtilis. It was obtained by amplifying the repU-Kan region of pSBBsOK_P (BBa_K1351040) using primers OriKan forward (5??? cacagaatcaggggataacgcaggaaagaaACATGTAGTTATAAGTGACTAAACAAATAACTAAATAGATGGG) and OriKan reverse (5??? gttcctggccttttgctggccttttgctcaACATGTCGCAAAATGGCCCGATTTAAG). The resulting fragment was sub-cloned in the pSB1C3 between the EcoRI and PstI restriction sites.
Default insert for AarI AD acceptor vectors
Default insert for AarI AD acceptor vectors. It includes the AarI restriction sites but not the 4 bp D or A overhangs.
It includes the AarI restriction sites but not the 4 bp D or A overhangs.
Double terminator + Kanamycin resistance cassette + Double ter
This part contains a double terminator and a functional kanamycin resistance cassette (promoter, resistance against kanamycin and a double terminator, BBa_B0014 + BBa_P1003 + BBa_B0014). It is reliable to design a recombinant vector by gene insertion in a non lytic bacteriophage as the Lambda phage. In fact it allows us to know if a bacteriophage is modified or not and if the inserted gene is expressed or not. When the virus is modified the kanamycin cassette induces a kanamycin resistance, so the bacteria become resistance, and when the virus is not modified bacteria are killed by kanamycin. We can use this part as a reporter, or as a simple resistance cassette. The double terminator allows the RNA translation arrest before the resistance cassette, so this is already created to insert a coding gene with its RBS before the resistance cassette.
Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16) Part:BBa_P1003, Designed by Reshma Shetty Group: Knight Lab, MIT (2006-01-23)
Conversion of the iGEM parts registry to SBOL2.1