Editing MRNA Products (section)

= <span style="font-size: 13px;">==</span> =

Digestion with Xba1, &nbsp;Mlu1, PacI: make SAM EGFP mRNA

|-
| EGFP mRNA ARCA
| <br/>
| style="width: 66px;" | <br/>
| style="width: 13px;" | <br/>
| <br/>
| style="width: 40px;" | <br/>
| style="width: 375px;" | 
T7AG-HBG-F&nbsp;TAATACGACTCACTATAAGGtcttctggtccccacagac (not the best primer)

VEE-pA-R&nbsp;GAATTAATTCCCCTCGAcgcgttctag

1149 bp

| style="width: 259px;" | <br/>
|-
| <br/>
| T7AG-EGFP<br/>
| style="width: 66px;" | <br/>
| style="width: 13px;" | 2953<br/>
| &nbsp;?
| style="width: 40px;" | <br/>
| style="width: 375px;" | 
T7AG-HBG-F&nbsp;TAATACGACTCACTATAAGGtcttctggtccccacagac

VEE-pA-R&nbsp;GAATTAATTCCCCTCGAcgcgttctag

1149bp

| style="width: 259px;" | <br/>
|-
| <br/>
| T7-VEE-TagGFP2<br/>
| style="width: 66px;" | <br/>
| style="width: 13px;" | 2926
| &nbsp;?
| style="width: 40px;" | <br/>
| style="width: 375px;" | <br/>
| style="width: 259px;" | <br/>
|-
| FLP self amplifying&nbsp; mRNA
| T7-VEE-NLS-Flp<br/>
| style="width: 66px;" | 
EG-1044

EG-1045

| style="width: 13px;" | 2895<br/>
| 33/I3<br/>
| style="width: 40px;" | 1724, 9283<br/>
| style="width: 375px;" | 
to make template by PCR&nbsp; for&nbsp;EG-1044

<s>T7-HBG-F</s>

<s>TAATACGACTCACTATAAGGACATTTGCTTCTGACACAAC&nbsp;</s>

T7-NLS-FLPe

TAATACGACTCACTATAGGGATGGCCCCCAAGAAAAAGCGG

VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

1724 bp

---

for EG-1045, digest plasmid with Xba1, Mlu1, PacI



| style="width: 259px;" | not SAM, if by PCR
Digestion with Xba1,&nbsp;&nbsp;Mlu1, PacI: make SAM EGFP mRNA

|-
| FLuc&nbsp; mRNA
| T7-VEE-FLuc<br/>
| style="width: 66px;" | EG-316<br/>
| style="width: 13px;" | 
2889

2952

| 35/F1<br/>
| style="width: 40px;" | 2023
| style="width: 375px;" | 
to make template for Cleancap by PCR, superHiFi<br/>1st round

2889-PCR-F:&nbsp;<br/>tcttctggtccccacagactcagagagaacccGCCACCATGGAAGACGCCA

+<br/>VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

2023 bp

2nd round, Taq

T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC

+<br/>VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

digestion:

digest 2953 with Mlu1, purify DNA with glycogen + propanol

==

digest 2889 with Mlu1 for self amplify mRNA

| style="width: 259px;" | <br/>
|-
| NanoFluc
| pRham-T7ag-NLuc-2A-EGFP<br/>
| style="width: 66px;" | <br/>
| style="width: 13px;" | 2935<br/>
| 35/H3<br/>
| style="width: 40px;" | <br/>
| style="width: 375px;" | <br/>
| style="width: 259px;" | <br/>
|-
| Rluc
| pT7ag-Rluc<br/>
| style="width: 66px;" | <br/>
| style="width: 13px;" | 2934<br/>
| 35/H1<br/>
| style="width: 40px;" | <br/>
| style="width: 375px;" | <br/>
| style="width: 259px;" | <br/>
|-
| Rluc
| T7ag-VEE-RLuc<br/>
| style="width: 66px;" | <br/>
| style="width: 13px;" | 2946<br/>
| 35/H4<br/>
| style="width: 40px;" | <br/>
| style="width: 375px;" | <br/>
| style="width: 259px;" | <br/>
|-
| <br/>
| eSpCas9-T2A-EGFP<br/>
| style="width: 66px;" | <br/>
| style="width: 13px;" | 2931
| <br/>
| style="width: 40px;" | <br/>
| style="width: 375px;" | <br/>
| style="width: 259px;" | can not use for mRNA production
|-
| Cas9-EGFP, self amplify mRNA
| T7-VEE-eSPCas9-EGFP<br/>
| style="width: 66px;" | 
EG-317,

EG-314

| style="width: 13px;" | 2938<br/>
| 35/G9<br/>
| style="width: 40px;" | <br/>
| style="width: 375px;" | 
for&nbsp;Cas9-EGFP SAM: digest 2938 with Mlu1

For&nbsp; Cas9-EGFP, PCR using&nbsp;T7AG-HBG-F&nbsp;GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC +&nbsp;VEE-pA-R GAATTAATTCCCCTCGACGCGTTCTAG



| style="width: 259px;" | 
EG-317: NLS-eCas9-T2A-EGFP Self-Amplifying mRNA, GSK or331295-1

EG-314:&nbsp;NLS-eCas9-T2A-EGFP mRNA (CleanCap),&nbsp;or331295-2

|-
| eCas9 self amplify mRNA
| T7ag-VEE-eCas9<br/>
| style="width: 66px;" | EG-322<br/>
| style="width: 13px;" | 2947<br/>
| 35/H5<br/>
| style="width: 40px;" | <br/>
| style="width: 375px;" | digest 2947 with Mlu1, very low mRNA yield
| style="width: 259px;" | GSK&nbsp;or331295-4
|-
| eCas9 cleancap<br/>
| T7ag-pA-eCas9<br/>
| style="width: 66px;" | EG-321<br/>
| style="width: 13px;" | 2939<br/>
| 35/H2&nbsp;<br/>
| style="width: 40px;" | <br/>
| style="width: 375px;" | digest 2939 with Mlu1
| style="width: 259px;" | GSK&nbsp;r331295-3
|-
| <br/>
| <br/>
| style="width: 66px;" | <br/>
| style="width: 13px;" | <br/>
| <br/>
| style="width: 40px;" | <br/>
| style="width: 375px;" | <br/>
| style="width: 259px;" | <br/>
|}