CPP sequences were inserted in frame by oligonucleotide insertion in the unique restriction sites SpeÉ and KpnÉ localized between polyhistidine and Cre recombinase coding sequences (see Additional file 1).
Aligned sequences were inserted into the SSURef SILVA guide tree using the parsimony insertion function of ARB [59].
In marine haplosclerids, these extra sequences were inserted within helices E23_1, E23_14 and 43, and in Hexactinellida, the insertions only occurred within helix E23_1 at a different position than in marine haplosclerids (Figs. 3, 4).
Briefly, the codon optimized sequences were inserted into the subgenomic reading frame of a modified DNA plasmid encoding the self-amplifying RNA41.
These sequences were inserted into a movement-deficient version of CPMV RNA-2 which is rescued from cowpea plants (Vigna unguiculota) by inoculation with RNA-1.
Confirmed AtPIP1 4 promoter and cDNA sequences were inserted into pCAMBIA1301 at the front of P35S and the right border to form the genetic complementation union and exclude P35S in the recombinant vector.
Periodic pilot sequences were inserted to divide the data payload into frames, and each frame was artificially partitioned into blocks for processing, as shown in Figure 7.
DNA oligos encoding the corresponding gRNA sequences were inserted into the cloning sites of pSpCas9-2A-Puro-MCS and the construct was transfected into THP-1 cells using a nucleofector (Amaxa).
Then the synthetic sequences were inserted into pUC19 vectors in a ligation system with a final volume of 20 μL containing 1 U T4 DNA Polymerase, Tris acetate 33 mM, CH3COOK 66 mM, (CH3COO 2Mg 10 mM, DTT 0.5 mM, target gene 200 ng and vector DNA 50 ng.
The amplified sequences were inserted into the BamHI/XhoI restriction sites of p415.
Coding sequences were inserted into pMAL-c2x (New England Biolabs) and expressed in E. coli BL21.
