Parts
We assembled a lot of miBricks (i. e. basic parts) to composite parts that were then used to built up our final constructs. The submitted parts are listed below. We did not categorize the parts according to different subprojects since they are universally applicable or clearly denoted through their names. Some binding site patterns, which were created via [... miraPCR], are listed separately.
Basic parts
Composite Parts
Table 2: Our composite parts from subsequent assembling steps. Listed ID refers to internal nomenclature.
ID | Content | Registry link | Purpose of the cassette
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F6 | luc2_sv40ter | BBa_K337015 | Luc2 reporter gene with terminator referring to SV40 promoter
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F15 | CMV_TetO2 | BBa_K337027 | CMV promoter under control of Tet Operator for regulation of gene expression
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R1 | Sv40(rc)/RSV(fw) | BBa_K337045 | bidirectional hybrid promoter
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R2 | Sv40(rc)/CMV(fw) | BBa_K337047
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R3 | RSV(rc)/CMV(fw) | BBa_K337048
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R4 | RSV(rc)/CMV_TetO2(fw) | BBa_K337050
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R5 | BGH(rc)/shRNA10(rc) | BBa_K337051 | synthetic microRNA template part
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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
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R14 | BGH(rc)/shRNA10(rc)/sv40(rc)/CMV(fw) | BBa_K337021
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R15 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw) | BBa_K337022
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R16 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw)_TetO2(fw) | BBa_K337023
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R17 | BGH(rc)/shRNA6(rc)/sv40(rc)/RSV(fw) | BBa_K337024
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R18 | BGH(rc)/shRNA6(rc)/sv40(rc)/CMV(fw) | BBa_K337026
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R19 | BGH(rc)/shRNA6(rc)/RSV(rc)/CMV(fw) | BBa_K337029
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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
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R21 | Luc2(rc)_sv40(rc)/CMV(fw) | BBa_K337033 | bidirectional hybrid promoter with Luc2 reference gene under control of SV40 promoter
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R22 | Luc2(rc)_sv40(rc)/RSV(fw) | BBa_K337034
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R32 | Kozag_hRluc/BGH(fw) | BBa_K337037 | reference reporter
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R33 | TetR_mut(XhoI/XbaI) | BBa_K337039 | mutagenized TetR including cutting sites to paste miRNA binding sites
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T1 | BGH(rc)_CMV/TetR/BGH(fw)/BGH(rc) | BBa_K337041 | repressor construct for On-Targeting together with tuning construct
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T2 | BGH(rc)_RSV/TetR/BGH(fw)/BGH(rc) | BBa_K337043
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Final Constructs
Table 3: Our final constructs. Listed ID refers to internal nomenclature.
ID | Content | Registry link | Name
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K1 | BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/CMV/Kozag_hRluc_BGH | BBa_K337032 | pSMB_miTuner Plasmid HD1
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K2 | BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGH | BBa_K337035 | pSMB_miTuner Plasmid HD2
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K3 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGH | BBa_K337036 | pSMB_miTuner Plasmid HD3
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K4 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV_TetO2/Luc2_sv40/CMV/Kozag_hRluc_BGH | BBa_K337038 | pSMB_miTuner Plasmid HD4
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K5 | BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/RSV/Kozag_hRluc_BGH | BBa_K337040 | pSMB_miTuner Plasmid HD5
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K6 | BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/RSV/Luc2_sv40/RSV/Kozag_hRluc_BGH | BBa_K337042 | pSMB_miTuner Plasmid HD6
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K7 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/RSV/Kozag_hRluc_BGH | BBa_K337044 | pSMB_miTuner Plasmid HD7
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K8 | BGH(rc)/shRNA10(rc)/RSV(rc)/CMV_TetO2/Luc2_sv40/RSV/Kozag_hRluc_BGH | BBa_K337046 | pSMB_miTuner Plasmid HD8
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miM | sv40ter(rc)/eBFP(rc)/biCMV/eGFP(fw)/sv40ter(fw) | BBa_K337049 | pSMB_miMeasure
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Binding Site Patterns
Table 4: Binding site patterns generated by miraPCR.
ID | Content | Registry link
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1.3 | hsa-miR-122 Binding site pattern (3BS) | BBa_K337000
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1A | hsa-miR-122 Binding site pattern (4BS) | BBa_K337003
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1-5 | hsa-miR-122 Binding site pattern (2BS) | BBa_K337004
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3.7 | hsa-miR-122 Binding site pattern (2BS - additional 10bp Spacer) | BBa_K337005
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1.5 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337006
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1.8 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337007
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3.1 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337008
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4.5 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337009
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4.6 | hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12 | BBa_K337010
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mir221-10L2 | hsa-miR-221 Binding site pattern (2BS) | BBa_K337011
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