Streptoalloteichus hindustanus, PDB: 1BYL
Zeocoin and bleomycin resistance cassette including promoter and coding sequence. It lacks a terminator.
Codon optimized for E coli by GenScript.
This cassette provides the entire tetracycline resistance operon and will express TetR.
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
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.
gentamycin/kanamycin resistance cassette
Originally from Tn4001 transposon; sequence M18086. Created from plasmid pISM2062 from Chris Minion, Iowa State University.
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
Ampicillin resistance cassette for modular BioBricks vector construction.
Kanamycin resistance cassette for modular BioBricks vector construction.
OriKan: a composite part to turn any pSB1C3 into a Bacillus plasmid
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.
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.
Double terminator + Kanamycin resistance cassette + Double ter
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)
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.
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.
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.
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
Chloramphenicol resistance gene including its native promoter and ribosome binding site. Confers resistance to 25 ug/mL chloramphenicol. {pSB1A2-Bca1016}
This provides the entire chloramphenicol resistance operon and will express CmR.
same part as BBa_P1002 except in reverse orientation.
Kanamycin Resistance Cassette in Cyanobacteria
Kanamycin resistance cassette and a double terminator
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)
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.
This contains CmR, a terminator and then AmpR and a terminator.
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
Conversion of the iGEM parts registry to SBOL2.1