TrnspsnPlsmd Tn5F|rbsGenR|SpecR|P_T7|Tn5R||CmR|TnP|OriTr|R6K
Transposon bearing spectinomycin resistance on a suicide plasmid bearing chloramphenicol resistance. The parts are not actually colinear, as in a normal composite assembly. The coding strand changes at the || indicated in the name.
This plasmid is not stable in cells. (must be transformed and used within a couple days)
Construction intermediate for a transposon bearing chloramphenicol resistance in the plasmid.
Transposable element with spectinomycin resistance marker and (if it worked better) a promoterless gentimicin marker that can be used to select for transposons that have fallen inside a transcribed region.
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.
Construction intermediate for a transposon with a spectinomycin selection marker inside of the transposed region.
Plasmid seems to be stable in DH10B, but not TG1.
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}
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.
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
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.
Conversion of the iGEM parts registry to SBOL2.1