TPcon6 and MoClo

Plasmid maps available at:
https://drive.google.com/drive/folders/16-0ek2biyB-hI1RY2xgW6p4fe32FbAJh?usp=share_link

MoClo Standard

Figure after addgene.org/cloning/moclo — Weber et al., PMID 21364738

MoClo Standard

UC.TP Variation

UC architecture

   BsaI
5’-GGTCTCN       NNNN…-3’
3’-CCAGAGNNNNN       …-5’

Example:

GGTCTCATACTGTGCAGGAGTACAGGAGCACACATGCCACCGCCGCGACCGCCGGCGACAACGTGAGCTTTGAGACC
 BsaI                 SD/RBS                  CDS/ORF                   BsaI

TPcon6 architecture

Making pP6

PCR        P6libF P6libR pTP1 P6
GoldenGate P6     BsaI        gg
Transform  gg     Mach1  Amp  pP6

P6libF

ccaaaggtctcATTATANNNNNNNNNNNNNNNNNTGTCAANNNNGAacccaggactcctcgaagtcgttcttaagacaac
      BsaI -10 rev                -35 rev              BseRI

P6libR

CagttGGTCTCAATAATNNNNNNANNNNGTtagtatttctcctcgtctacggttaactgatactc
      BsaI   -10      +1               BseRI

pP6

GAGGAGTCCTGGGTTCNNNNTTGACANNNNNNNNNNNNNNNNNTATAATNNNNNNANNNNGTTAGTATTTCTCCTC
 BseRI                -35                    -10      +1               BseRI

pP6-2A

GAGGAGTCCTGGGTTCGTGATTGACATCTGGCCGATCCCAGCCTATAATCTGTCCATGAGGTTAGTATTTCTCCTC

pP6-14C

GAGGAGTCCTGGGTTCGGACTTGACATGAGGTATGAACTCTACTATAATATCTACAGTGGGTTAGTATTTCTCCTC

Tlib2

Synthesized 61 of these cassettes as an oligo pool, everything but the promoter was included, and in its place were BseRI sites

pP6-2A × pT6B3  =  pT6B3P2A

GoldenGate pT6B3 pP6-2A BseRI gg
Transform  gg    Mach1  Amp   pT6B3P2A