Team:Heidelberg/Parts

From 2010.igem.org

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|R4||RSV(rc)/CMV_TetO2(fw)||[http://partsregistry.org/Part:BBa_K337050 BBa_K337050]
|R4||RSV(rc)/CMV_TetO2(fw)||[http://partsregistry.org/Part:BBa_K337050 BBa_K337050]
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|-
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|R5||BGH(rc)/shRNA10(rc)||[http://partsregistry.org/Part:BBa_K337051 BBa_K337051]
+
|R5||BGH(rc)/shRNA10(rc)||[http://partsregistry.org/Part:BBa_K337051 BBa_K337051]| synthetic microRNA template
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|R13||BGH(rc)/shRNA10(rc)/sv40(rc)/RSV(fw)||[http://partsregistry.org/Part:BBa_K337021 BBa_K337021]||rowspan="7"|tuning construct cloning into reporter plasmid backbone containing a binding site against synthetic shRNA-like miRNA
|R13||BGH(rc)/shRNA10(rc)/sv40(rc)/RSV(fw)||[http://partsregistry.org/Part:BBa_K337021 BBa_K337021]||rowspan="7"|tuning construct cloning into reporter plasmid backbone containing a binding site against synthetic shRNA-like miRNA

Revision as of 02:23, 27 October 2010

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

table 1: Our miBricks. Listed ID refers to internal nomenclature.
IDContentRegistry linkComment
F1RSV fw[http://partsregistry.org/Part:BBa_K337012 BBa_K337012]-
F2SV40 rc[http://partsregistry.org/Part:BBa_K337013 BBa_K337013]-
F3RSV rc[http://partsregistry.org/Part:BBa_K337014 BBa_K337014]-
F4BGH fw[http://partsregistry.org/Part:BBa_K337001 BBa_K337001]-
F5BGH rc[http://partsregistry.org/Part:BBa_K337002 BBa_K337002] leads to insertion of BamHI site
F7microRNA 10HD[http://partsregistry.org/Part:BBa_K337016 BBa_K337016] template for creation of shRNA-like miRNA
F8CMV fw[http://partsregistry.org/Part:BBa_K337018 BBa_K337018]-
F9FRT site[http://partsregistry.org/Part:BBa_K337019 BBa_K337019]-
F10hRluc[http://partsregistry.org/Part:BBa_K337025 BBa_K337025]-
F16TetR[http://partsregistry.org/Part:BBa_K337028 BBa_K337028]-
F17Luc2[http://partsregistry.org/Part:BBa_K337030 BBa_K337030] compatible for insertion of microRNA binding sites
biCMVbiCMV[http://partsregistry.org/Part:BBa_K337030 BBa_K337017]-

composite parts

table 2: Our composite parts from subsequent assembling steps. Listed ID refers to internal nomenclature.
IDContentRegistry linkPurpose of the cassette
F6luc2_sv40ter[http://partsregistry.org/Part:BBa_K337015 BBa_K337015] Luc2 reporter gene with terminator referring to SV40 promoter
F15CMV_TetO2[http://partsregistry.org/Part:BBa_K337027 BBa_K337027] CMV promoter under control of Tet Operator for regulation of gene expression
R1Sv40(rc)/RSV(fw)[http://partsregistry.org/Part:BBa_K337045 BBa_K337045] bidirectional hybrid promoter
R2Sv40(rc)/CMV(fw)[http://partsregistry.org/Part:BBa_K337047 BBa_K337047]
R3RSV(rc)/CMV(fw)[http://partsregistry.org/Part:BBa_K337048 BBa_K337048]
R4RSV(rc)/CMV_TetO2(fw)[http://partsregistry.org/Part:BBa_K337050 BBa_K337050]
R5BGH(rc)/shRNA10(rc) synthetic microRNA template
R13BGH(rc)/shRNA10(rc)/sv40(rc)/RSV(fw)[http://partsregistry.org/Part:BBa_K337021 BBa_K337021]tuning construct cloning into reporter plasmid backbone containing a binding site against synthetic shRNA-like miRNA
R14BGH(rc)/shRNA10(rc)/sv40(rc)/CMV(fw)[http://partsregistry.org/Part:BBa_K337022 BBa_K337022]
R15BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw)[http://partsregistry.org/Part:BBa_K337023 BBa_K337023]
R16BGH(rc)/shRNA10(rc)/RSV(rc)/CMV(fw)_TetO2(fw)[http://partsregistry.org/Part:BBa_K337024 BBa_K337024]
R17BGH(rc)/shRNA6(rc)/sv40(rc)/RSV(fw)[http://partsregistry.org/Part:BBa_K337026 BBa_K337026]
R18BGH(rc)/shRNA6(rc)/sv40(rc)/CMV(fw)[http://partsregistry.org/Part:BBa_K337029 BBa_K337029]
R19BGH(rc)/shRNA6(rc)/RSV(rc)/CMV(fw)[http://partsregistry.org/Part:BBa_K337031 BBa_K337031]
R20BGH(rc)/shRNA6(rc)/RSV(rc)/CMV_TetO2(fw)[http://partsregistry.org/Part:BBa_K337033 BBa_K337033]tuning construct core containing Tet Operator for On-Targeting together with repressor construct
R21Luc2(rc)_sv40(rc)/CMV(fw)[http://partsregistry.org/Part:BBa_K337034 BBa_K337034] bidirectional hybrid promoter with Luc2 reference gene under control of SV40 promoter
R22Luc2(rc)_sv40(rc)/RSV(fw)[http://partsregistry.org/Part:BBa_K337039 BBa_K337039]
R32Kozag_hRluc/BGH(fw)[http://partsregistry.org/Part:BBa_K337037 BBa_K337037]reference reporter
R33TetR_mut(XhoI/XbaI)[http://partsregistry.org/Part:BBa_K337039 BBa_K337039]mutagenized TetR including cutting sites to paste miRNA binding sites
T1BGH(rc)_CMV/TetR/BGH(fw)/BGH(rc)[http://partsregistry.org/Part:BBa_K337041 BBa_K337041]repressor construct for On-Targeting together with tuning construct
T2BGH(rc)_RSV/TetR/BGH(fw)/BGH(rc)[http://partsregistry.org/Part:BBa_K337043 BBa_K337043]

final constructs

table 3: Our final constructs. Listed ID refers to internal nomenclature.
IDContentRegistry linkName
K1BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/CMV/Kozag_hRluc_BGH[http://partsregistry.org/Part:BBa_K337032 BBa_K337032] pSMB_miTuner Plasmid HD1
K2BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGH[http://partsregistry.org/Part:BBa_K337035 BBa_K337035] pSMB_miTuner Plasmid HD2
K3BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGH[http://partsregistry.org/Part:BBa_K337036 BBa_K337036] pSMB_miTuner Plasmid HD3
K4BGH(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
K5BGH(rc)/shRNA10(rc)/sv40(rc)/RSV/Luc2_sv40/RSV/Kozag_hRluc_BGH[http://partsregistry.org/Part:BBa_K337040 BBa_K337040] pSMB_miTuner Plasmid HD5
K6BGH(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
K7BGH(rc)/shRNA10(rc)/RSV(rc)/CMV/Luc2_sv40/RSV/Kozag_hRluc_BGH[http://partsregistry.org/Part:BBa_K337044 BBa_K337044] pSMB_miTuner Plasmid HD7
K8BGH(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
miMsv40ter(rc)/eBFP(rc)/biCMV/eGFP(fw)/sv40ter(fw)[http://partsregistry.org/Part:BBa_K337049 BBa_K337049] pSMB_miMeasure

binding site patterns

table 4: Binding site patterns generated by miraPCR.
IDContentRegistry link
1.3hsa-miR-122 Binding site pattern (3BS)[http://partsregistry.org/Part:BBa_K337000 BBa_K337000]
1Ahsa-miR-122 Binding site pattern (4BS)[http://partsregistry.org/Part:BBa_K337003 BBa_K337003]
1-5hsa-miR-122 Binding site pattern (2BS)[http://partsregistry.org/Part:BBa_K337004 BBa_K337004]
3.7hsa-miR-122 Binding site pattern (2BS - additional 10bp Spacer)[http://partsregistry.org/Part:BBa_K337005 BBa_K337005]
1.5hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12[http://partsregistry.org/Part:BBa_K337006 BBa_K337006]
1.8hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12[http://partsregistry.org/Part:BBa_K337007 BBa_K337007]
3.1hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12[http://partsregistry.org/Part:BBa_K337008 BBa_K337008]
4.5hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12[http://partsregistry.org/Part:BBa_K337009 BBa_K337009]
4.6hsa-miR-122 Binding site pattern (2BS) with randomized nt9-12[http://partsregistry.org/Part:BBa_K337010 BBa_K337010]
mir221-10L2hsa-miR-221 Binding site pattern (2BS)[http://partsregistry.org/Part:BBa_K337010 BBa_K337010]


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