|
|
| 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 | [http://partsregistry.org/Part:BBa_K337036 BBa_K337036] | pSMB_miTuner Plasmid HD3
|
| K4 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV_TetO2/Luc2_sv40/CMV/Kozag_hRluc_BGH | [http://partsregistry.org/Part:BBa_K337038 BBa_K337038] | pSMB_miTuner Plasmid HD4
|
| miM | sv40ter(rc)/eBFP(rc)/biCMV/eGFP(fw)/sv40ter(fw) | [http://partsregistry.org/Part:BBa_K337049 BBa_K337049] | pSMB_miMeasure
|
Synthetic Single Binding Sites
(Physical DNA not submitted)
| Design | Content | Registry link | Name
|
| KD:97% | perfect binding site | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337052 BBa_K337052] | shRNA miRhaat
|
| KD:69% | imperfect binding site: point mut 11 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337053 BBa_K337053] | shRNA miRhaat
|
| KD:28% | imperfect binding site: bulge 16-18 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337054 BBa_K337054] | shRNA miRhaat
|
| KD:96% | perfect binding site | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337055 BBa_K337055] | miR122
|
| KD:64% | imperfect binding site: | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337056 BBa_K337056] | miR122
|
| KD:24% | imperfect binding site: | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337057 BBa_K337057] | miR122
|
Endogenous Binding Site Patterns
| ID | Content | Registry link
|
| 1.3 | hsa-miR-122 Binding site pattern (3BS) | [http://partsregistry.org/Part:BBa_K337000 BBa_K337000]
|
| 1A | hsa-miR-122 Binding site pattern (4BS) | [http://partsregistry.org/Part:BBa_K337003 BBa_K337003]
|
| 1-5 | hsa-miR-122 Binding site pattern (2BS) | [http://partsregistry.org/Part:BBa_K337004 BBa_K337004]
|
| 3.7 | hsa-miR-122 Binding site pattern (2BS - additional 10bp Spacer) | [http://partsregistry.org/Part:BBa_K337005 BBa_K337005]
|
| 1.5 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | [http://partsregistry.org/Part:BBa_K337006 BBa_K337006]
|
| 1.8 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | [http://partsregistry.org/Part:BBa_K337007 BBa_K337007]
|
| 3.1 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | [http://partsregistry.org/Part:BBa_K337008 BBa_K337008]
|
| 4.5 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | [http://partsregistry.org/Part:BBa_K337009 BBa_K337009]
|
| 4.6 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | [http://partsregistry.org/Part:BBa_K337010 BBa_K337010]
|
| mir221-10L2 | hsa-miR-221 Binding site pattern (2BS) | [http://partsregistry.org/Part:BBa_K337011 BBa_K337011]
|
miTunig Kit - Basic Parts
| ID | Content | Registry link | Comment
|
| F1 | RSV fw | [http://partsregistry.org/Part:BBa_K337012 BBa_K337012] | -
|
| F2 | SV40 rc | [http://partsregistry.org/Part:BBa_K337013 BBa_K337013] | -
|
| F3 | RSV rc | [http://partsregistry.org/Part:BBa_K337014 BBa_K337014] | -
|
| F4 | BGH fw | [http://partsregistry.org/Part:BBa_K337001 BBa_K337001] | -
|
| F5 | BGH rc | [http://partsregistry.org/Part:BBa_K337002 BBa_K337002] | leads to insertion of BamHI site
|
| F7 | microRNA 10HD | [http://partsregistry.org/Part:BBa_K337016 BBa_K337016] | template for creation of shRNA-like miRNA
|
| F8 | CMV fw | [http://partsregistry.org/Part:BBa_K337018 BBa_K337018] | -
|
| F9 | FRT site | [http://partsregistry.org/Part:BBa_K337019 BBa_K337019] | -
|
| F10 | hRluc | [http://partsregistry.org/Part:BBa_K337025 BBa_K337025] | -
|
| F16 | TetR | [http://partsregistry.org/Part:BBa_K337028 BBa_K337028] | -
|
| F17 | Luc2 | [http://partsregistry.org/Part:BBa_K337030 BBa_K337030] | compatible for insertion of microRNA binding sites
|
| biCMV | biCMV | [http://partsregistry.org/Part:BBa_K337017 BBa_K337017] | -
|
miTuning Kit - Intermediate Parts 1
| ID | Content | Registry link | Purpose of the cassette
|
| F6 | luc2_sv40ter | [http://partsregistry.org/Part:BBa_K337015 BBa_K337015] | Luc2 reporter gene with terminator referring to SV40 promoter
|
| F15 | CMV_TetO2 | [http://partsregistry.org/Part:BBa_K337027 BBa_K337027] | CMV promoter under control of Tet Operator for regulation of gene expression
|
| R1 | Sv40(rc)/RSV(fw) | [http://partsregistry.org/Part:BBa_K337045 BBa_K337045] | bidirectional hybrid promoter
|
| R2 | Sv40(rc)/CMV(fw) | [http://partsregistry.org/Part:BBa_K337047 BBa_K337047]
|
| R3 | RSV(rc)/CMV(fw) | [http://partsregistry.org/Part:BBa_K337048 BBa_K337048]
|
| R4 | RSV(rc)/CMV_TetO2(fw) | [http://partsregistry.org/Part:BBa_K337050 BBa_K337050]
|
| R5 | BGH(rc)/shRNA10(rc) | [http://partsregistry.org/Part:BBa_K337051 BBa_K337051] | synthetic microRNA template part
|
| R13 | BGH(rc)/shRNA10(rc)/sv40(rc)/RSV(fw) | [http://partsregistry.org/Part:BBa_K337020 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) | [http://partsregistry.org/Part:BBa_K337021 BBa_K337021]
|
| R15 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw) | [http://partsregistry.org/Part:BBa_K337022 BBa_K337022]
|
| R16 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw)_TetO2(fw) | [http://partsregistry.org/Part:BBa_K337023 BBa_K337023]
|
| R17 | BGH(rc)/shRNA6(rc)/sv40(rc)/RSV(fw) | [http://partsregistry.org/Part:BBa_K337024 BBa_K337024]
|
| R18 | BGH(rc)/shRNA6(rc)/sv40(rc)/CMV(fw) | [http://partsregistry.org/Part:BBa_K337026 BBa_K337026]
|
| R19 | BGH(rc)/shRNA6(rc)/RSV(rc)/CMV(fw) | [http://partsregistry.org/Part:BBa_K337029 BBa_K337029]
|
| 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
|
| R21 | Luc2(rc)_sv40(rc)/CMV(fw) | [http://partsregistry.org/Part:BBa_K337033 BBa_K337033] | 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] | 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]
|
Tuning Kit - Intermediate Parts 2
| ID | Content | Registry link | Name
|
| K1 | BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/CMV/Kozag_hRluc_BGH | [http://partsregistry.org/Part:BBa_K337032 BBa_K337032] | pSMB_miTuner Plasmid HD1
|
| K2 | BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGH | [http://partsregistry.org/Part:BBa_K337035 BBa_K337035] | pSMB_miTuner Plasmid HD2
|
| K5 | BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/RSV/Kozag_hRluc_BGH | [http://partsregistry.org/Part:BBa_K337040 BBa_K337040] | pSMB_miTuner Plasmid HD5
|
| K6 | BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/RSV/Luc2_sv40/RSV/Kozag_hRluc_BGH | [http://partsregistry.org/Part:BBa_K337042 BBa_K337042] | pSMB_miTuner Plasmid HD6
|
| K7 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/RSV/Kozag_hRluc_BGH | [http://partsregistry.org/Part:BBa_K337044 BBa_K337044] | pSMB_miTuner Plasmid HD7
|
| K8 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV_TetO2/Luc2_sv40/RSV/Kozag_hRluc_BGH | [http://partsregistry.org/Part:BBa_K337046 BBa_K337046] | pSMB_miTuner Plasmid HD8
|
ViroBytes
| ID | Part name | Registry link | Description
|
| 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
|
| 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
|
| 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
|
| 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
|
| 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
|
| 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
|
| 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
|
| 1 | Fragment 1 of wt AAV1 | [http://partsregistry.org/Part:BBa_K337058 BBa_K337058] | Bsa1 sites on both ends
|
|
"
