Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Monica Prudencio
Date created: 2016-10-07 11:00:00
Date modified: 2016-10-10 11:42:44
Variant of repClone Red (J100205)
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_J100307_sequence (Version 1) |
Description
This part is a variant of repClone Red (J100205), with some differences in the TetR and GFP.We designed repClone Red to test the function of repressors. Internal BsaI sites allow the insertion of the Ptet promoter and removal of the backwards P2 promoter pointed towards GFP. Successful cloning of the Ptet promoter will change colonies from green to not green. Variations on the Ptet promoter, or introduction of anhydrotetrocycline (aTc), will affect the activity of the repressor, as indicated by the fluorescence of colonies containing the plasmid. This can be used in synthetic biology laboratories or for undergraduate introductory biology lab modules.
Variations from the registry sequence for TetR and GFP were sequence verified, and functionality was experimentally verified.
Notes
The stop codons of the RBS and start codons of the proteins overlap to increase effective translation. This is cloned into pUC-IDT-Amp (BsaI site removed) with modified Bba prefix so that there is only an EcoRI site upstream and a normal Bba suffix.Source
The two main indicator proteins are GFP (BBa_I746916) and RFP (BBa_E1010). There are some SNPs in the GFP sequence due to codon optimization; these have been verified by sequencing and their functionality has been verified experimentally.| Sequence Annotation | Location | Component / Role(s) |
| tetR Repressor C0041 T->G BD24 P5 Promoter GFP Optimized Sequence T->C T->C B0034 BsaI Recognition Site P2 Promoter Terminator L3S2P21 BsaI Recognition Site BD18 RFP | 1,639 243,243 640,727 728,763 764,1483 945,945 953,953 1490,1501 1517,1522 1523,1568 1577,1637 1638,1643 1649,1736 1734,2414 | feature/protein CDS sequence_alteration feature/mutation feature/rbs ribosome_entry_site feature/promoter promoter feature/protein CDS sequence_alteration feature/mutation feature/mutation sequence_alteration feature/rbs ribosome_entry_site sequence_feature feature/misc promoter feature/promoter feature/misc sequence_feature feature/misc sequence_feature ribosome_entry_site feature/rbs feature/protein CDS |