Single-gRNA expression adenoviral vector
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.
U6 Promoter: Drives expression of the downstream gRNA sequence. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.
gRNA: Guide RNA compatible with the Cas9 variant being used.
Terminator: Terminates transcription of the gRNA.
hPGK promoter: Human phosphoglycerate kinase 1 promoter. It drives the ubiquitous expression of the downstream marker gene.
Marker: A visually detectable fluorescent gene (such as EGFP). This allows cells transduced with the vector to be selected and/or visualized.
TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.
3' ITR: 3' inverted terminal repeat.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.
Dual-gRNA expression adenoviral vector
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.
U6 Promoter: Drives expression of the downstream gRNA sequence. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.
gRNA #1: The first guide RNA compatible with the Cas9 variant being used.
gRNA #2: The second guide RNA compatible with the Cas9 variant being used.
Terminator: Terminates transcription of the gRNA.
hPGK promoter: Human phosphoglycerate kinase 1 promoter. It drives the ubiquitous expression of the downstream marker gene.
Marker: A visually detectable fluorescent gene (such as EGFP). This allows cells transduced with the vector to be selected and/or visualized.
TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.
3' ITR: 3' inverted terminal repeat.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.