Transposon bearing chloramphenicol resistance on a suicide plasmid bearing spectinomycin resistance. The parts are not actually colinear, as in a normal composite assembly. The coding strand changes at the || indicated in the name.
Intermediate for construction of a transposon with a plasmid bearing a spectinomycin resistance marker.
Transposable piece of a transposon bearing suicide plasmid, containing chloramphenicol resistance, an origin of replication for decoding, and a selectable gentimicin marker that only activates when the transposon lands in a transcribed region.
Spectinomycin R open-reading-frame basic part
PCR JL1/JL2 on wt. pSPlam (917bp, EcoRI/SpeI, Neb2) Paste into pSB1A2-I13521 (EcoRI/SpeI, Neb2, Large 2062)
JL1 Forward EcoRI oligo to bioBrick SpecR: gacttgaattcgcggccgcttctagagtccaagcgagctcgatatc JL2 Reverse SpeI oligo to bioBrick SpecR: ccgctactagtCAGCAACGATGTTACGCAGC
Tn5 Transposase [Tnp1-PN] (with PstI and NotI)
PCR ca1014F/R on pRL27 (1763 bp, EcoRI/SpeI) Sub into pSB1A2-I13521 (EcoRI/SpeI) Product is Bca1014 *internal PstI and NotI sites
Tn5 transposase-expressing cassette from pRL. Cassette includes a complete promoter/rbs/orf cassette. The promoter is Ptet, but not derived from r0040. There are PstI and NotI sites in this part, so care must be taken when doing assembly not to use those sites. pSB1A2-Bca1014
There are PstI and NotI sites in this part, so care must be taken when doing assembly not to use those sites.
Construction intermediate for a transposon selectable for insertion into a transcribed region.
Intermediate for a transposon with a chloramphenicol resistance marker and a replication origin inside of the transposed region.
OriT-R (Origin of transfer for the R-plasmid nic region)
OriTR, the R plasmid nic region, is where the relaxosome nicks the plasmid and conjugative transfer by R plasmid machinery begins.
Overlap extension of synthetic oligonucleotides
DNA substrate for Tn5 transposase. Flanking a sequence with J61021 on the 5' end and J61022 on the 3' end makes it a functional transposon.
DNA substrate for Tn5 transposase. Flanking a sequence with J61021 on the 5' end and J61022 on the 3' end makes it a functional transposon. pSB1A2-Bca1013
Gentamicin resistance gene including a medium/weak ribosome binding site, but NO promoter. Intended for use in transposons, to select for elements that fall inside of a transcribed region. The RBS does not appear to be strong enough for this purpose.
The T7 promoter should only produce PoPS when the T7 polymerase is also being expressed.
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}
PCR ca1011F/R on pG80ko (445 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Product is Bca1011 ---- ca1011F Forward Biobricking of R6K GGACTgaattcgcggccgcttctagagtgattcgcacgggcccatg ca1011R Reverse biobricking of R6K ccgctactagtaGCAGTTCAACCTGTTGATAG
R6K origin of replication requires a pir+ or pir116 strain for replication in the absence of a second origin. In most E. coli strains, this is a silent feature. {pSB1A2-Bca1011}
Extend ca1010F/R (73 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Product is pSB1A2-Bca1010 ---- ca1010F Forward (universal biobrick) EcoRI for FRT GGACTgaattcgcggccgcttctagag ca1010R Reverse SpeI oligo for FRT cctatactagtagaagttcctattctctaAaaagtataggaacttcctctagaagcggccgcg
Site for recombination by flp recombinase. Genes flanked by FRT sites (oriented in the same direction) in the genome can be excised with the introduction of flp recombinase-expressing helper plasmid pCP20. Note that the original FRT sequence contained an XbaI site that has been removed with a point mutation for compatibility with standard assembly. See Datsenko and Wanner for details of its use in markerless knockouts and knockins.
Conversion of the iGEM parts registry to SBOL2.1