|
|
Line 248: |
Line 248: |
| |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends | | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |2||CMV_TetO2||[http://partsregistry.org/Part:BBa_K337027 BBa_K337027]|| CMV promoter under control of Tet Operator for regulation of gene expression | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |3||Sv40(rc)/RSV(fw)||[http://partsregistry.org/Part:BBa_K337045 BBa_K337045]||rowspan="4"| bidirectional hybrid promoter | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |4||Sv40(rc)/CMV(fw)||[http://partsregistry.org/Part:BBa_K337047 BBa_K337047] | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |5||RSV(rc)/CMV(fw)||[http://partsregistry.org/Part:BBa_K337048 BBa_K337048] | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |6||RSV(rc)/CMV_TetO2(fw)||[http://partsregistry.org/Part:BBa_K337050 BBa_K337050] | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |7||BGH(rc)/shRNA10(rc)||[http://partsregistry.org/Part:BBa_K337051 BBa_K337051]||rowspan="1"| synthetic microRNA template part | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |8||BGH(rc)/shRNA10(rc)/sv40(rc)/RSV(fw)||[http://partsregistry.org/Part:BBa_K337020 BBa_K337020]||rowspan="7"|tuning construct cloning into reporter plasmid backbone containing a binding site against synthetic shRNA-like miRNA | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |9||BGH(rc)/shRNA10(rc)/sv40(rc)/CMV(fw)||[http://partsregistry.org/Part:BBa_K337021 BBa_K337021] | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |R15||BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw)||[http://partsregistry.org/Part:BBa_K337022 BBa_K337022] | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |R16||BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw)_TetO2(fw)||[http://partsregistry.org/Part:BBa_K337023 BBa_K337023] | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |R17||BGH(rc)/shRNA6(rc)/sv40(rc)/RSV(fw)||[http://partsregistry.org/Part:BBa_K337024 BBa_K337024] | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |R18||BGH(rc)/shRNA6(rc)/sv40(rc)/CMV(fw)||[http://partsregistry.org/Part:BBa_K337026 BBa_K337026] | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |R19||BGH(rc)/shRNA6(rc)/RSV(rc)/CMV(fw)||[http://partsregistry.org/Part:BBa_K337029 BBa_K337029] | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
| |- | | |- |
- | |R20||BGH(rc)/shRNA6(rc)/RSV(rc)/CMV_TetO2(fw)||[http://partsregistry.org/Part:BBa_K337031 BBa_K337031]||tuning construct core containing Tet Operator for On-Targeting together with repressor construct | + | |1||Fragment 1 of wt AAV1||[http://partsregistry.org/Part:BBa_K337058 BBa_K337058]||Bsa1 sites on both ends |
- | |-
| + | |
- | |R21||Luc2(rc)_sv40(rc)/CMV(fw)||[http://partsregistry.org/Part:BBa_K337033 BBa_K337033]||rowspan="2"| bidirectional hybrid promoter with Luc2 reference gene under control of SV40 promoter
| + | |
- | |-
| + | |
- | |R22||Luc2(rc)_sv40(rc)/RSV(fw)||[http://partsregistry.org/Part:BBa_K337034 BBa_K337034]
| + | |
- | |- | + | |
- | |R32||Kozag_hRluc/BGH(fw)||[http://partsregistry.org/Part:BBa_K337037 BBa_K337037]||reference reporter | + | |
- | |-
| + | |
- | |R33||TetR_mut(XhoI/XbaI)||[http://partsregistry.org/Part:BBa_K337039 BBa_K337039]||mutagenized TetR including cutting sites to paste miRNA binding sites
| + | |
- | |-
| + | |
- | |T1||BGH(rc)_CMV/TetR/BGH(fw)/BGH(rc)||[http://partsregistry.org/Part:BBa_K337041 BBa_K337041]||rowspan="2"|repressor construct for On-Targeting together with tuning construct
| + | |
- | |-
| + | |
- | |T2||BGH(rc)_RSV/TetR/BGH(fw)/BGH(rc)||[http://partsregistry.org/Part:BBa_K337043 BBa_K337043]
| + | |
| |- | | |- |
| |} | | |} |
miBricks - Parts submitted to the registry
The parts we submit belong to the two core aims our project: Reaching regulatory control (1) and specificity (2) of gene expression of any gene of interest in any target cell or tissue of choice. Therefore we engineered parts, that address these two aims on two different regulatory levels.
First, we engineered gene therapy vectors based on synthetic adeno associated viruses (AAVs). On parts level, we provide about 50 plasmids that can be used for creating shuffled AAV libraries or even rationally designed, recombinant AAV vectors. Those parts we refer to as virobytes, are designed in a format directly applicable for the Virobytes Assembly protocol we develop.
On RNA level we provide the miTuner toolkit consisting of roughly 60 parts enabling gene expression control based on synthetic or cell-specific endogenous microRNAs. This toolkit consists of three main constructs: The pSMB_miMeasure binding site characterization standard and two pSMB_miTuner expression controlling plasmids. Furthermore, it contains 12 basic and 28 intermediate construction parts, synthetic, single microRNA binding sites as well as binding site patterns in BB-2 (RFC 12, Tom Knight) standard. This enables maximum flexibility for applications in many different contexts.
Main Measurement Constructs - Engineered
ID | Content | Registry link | Name
|
K3 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGH | BBa_K337036 | pSMB_miTuner Plasmid HD3
|
K4 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV_TetO2/Luc2_sv40/CMV/Kozag_hRluc_BGH | BBa_K337038 | pSMB_miTuner Plasmid HD4
|
miM | sv40ter(rc)/eBFP(rc)/biCMV/eGFP(fw)/sv40ter(fw) | BBa_K337049 | pSMB_miMeasure
|
Synthetic Single Binding Sites
(Physical DNA not submitted)
Design | Content | Registry link | Name
|
KD:97% | perfect binding site | BBa_K337052 | shRNA miRhaat
|
KD:69% | imperfect binding site: point mut 11 | BBa_K337053 | shRNA miRhaat
|
KD:28% | imperfect binding site: bulge 16-18 | BBa_K337054 | shRNA miRhaat
|
KD:96% | perfect binding site | BBa_K337055 | miR122
|
KD:64% | imperfect binding site: | BBa_K337056 | miR122
|
KD:24% | imperfect binding site: | BBa_K337057 | miR122
|
Endogenous Binding Site Patterns
ID | Content | Registry link
|
1.3 | hsa-miR-122 Binding site pattern (3BS) | BBa_K337000
|
1A | hsa-miR-122 Binding site pattern (4BS) | BBa_K337003
|
1-5 | hsa-miR-122 Binding site pattern (2BS) | BBa_K337004
|
3.7 | hsa-miR-122 Binding site pattern (2BS - additional 10bp Spacer) | BBa_K337005
|
1.5 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337006
|
1.8 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337007
|
3.1 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337008
|
4.5 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337009
|
4.6 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337010
|
mir221-10L2 | hsa-miR-221 Binding site pattern (2BS) | BBa_K337011
|
miTunig Kit - Basic Parts
miTuning Kit - Intermediate Parts 1
ID | Content | Registry link | Purpose of the cassette
|
F6 | luc2_sv40ter | BBa_K337015 | Luc2 reporter gene with terminator referring to SV40 promoter
|
F15 | CMV_TetO2 | BBa_K337027 | CMV promoter under control of Tet Operator for regulation of gene expression
|
R1 | Sv40(rc)/RSV(fw) | BBa_K337045 | bidirectional hybrid promoter
|
R2 | Sv40(rc)/CMV(fw) | BBa_K337047
|
R3 | RSV(rc)/CMV(fw) | BBa_K337048
|
R4 | RSV(rc)/CMV_TetO2(fw) | BBa_K337050
|
R5 | BGH(rc)/shRNA10(rc) | BBa_K337051 | synthetic microRNA template part
|
R13 | BGH(rc)/shRNA10(rc)/sv40(rc)/RSV(fw) | BBa_K337020 | tuning construct cloning into reporter plasmid backbone containing a binding site against synthetic shRNA-like miRNA
|
R14 | BGH(rc)/shRNA10(rc)/sv40(rc)/CMV(fw) | BBa_K337021
|
R15 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw) | BBa_K337022
|
R16 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw)_TetO2(fw) | BBa_K337023
|
R17 | BGH(rc)/shRNA6(rc)/sv40(rc)/RSV(fw) | BBa_K337024
|
R18 | BGH(rc)/shRNA6(rc)/sv40(rc)/CMV(fw) | BBa_K337026
|
R19 | BGH(rc)/shRNA6(rc)/RSV(rc)/CMV(fw) | BBa_K337029
|
R20 | BGH(rc)/shRNA6(rc)/RSV(rc)/CMV_TetO2(fw) | BBa_K337031 | tuning construct core containing Tet Operator for On-Targeting together with repressor construct
|
R21 | Luc2(rc)_sv40(rc)/CMV(fw) | BBa_K337033 | bidirectional hybrid promoter with Luc2 reference gene under control of SV40 promoter
|
R22 | Luc2(rc)_sv40(rc)/RSV(fw) | BBa_K337034
|
R32 | Kozag_hRluc/BGH(fw) | BBa_K337037 | reference reporter
|
R33 | TetR_mut(XhoI/XbaI) | BBa_K337039 | mutagenized TetR including cutting sites to paste miRNA binding sites
|
T1 | BGH(rc)_CMV/TetR/BGH(fw)/BGH(rc) | BBa_K337041 | repressor construct for On-Targeting together with tuning construct
|
T2 | BGH(rc)_RSV/TetR/BGH(fw)/BGH(rc) | BBa_K337043
|
Tuning Kit - Intermediate Parts 2
ID | Content | Registry link | Name
|
K1 | BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/CMV/Kozag_hRluc_BGH | BBa_K337032 | pSMB_miTuner Plasmid HD1
|
K2 | BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGH | BBa_K337035 | pSMB_miTuner Plasmid HD2
|
K5 | BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/RSV/Kozag_hRluc_BGH | BBa_K337040 | pSMB_miTuner Plasmid HD5
|
K6 | BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/RSV/Luc2_sv40/RSV/Kozag_hRluc_BGH | BBa_K337042 | pSMB_miTuner Plasmid HD6
|
K7 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/RSV/Kozag_hRluc_BGH | BBa_K337044 | pSMB_miTuner Plasmid HD7
|
K8 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV_TetO2/Luc2_sv40/RSV/Kozag_hRluc_BGH | BBa_K337046 | pSMB_miTuner Plasmid HD8
|
ViroBytes
ID | Part name | Registry link | Description
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
1 | Fragment 1 of wt AAV1 | BBa_K337058 | Bsa1 sites on both ends
|
|