BBa_K1088052 1 BBa_K1088052 GFP reporter with flexible linker at N-terminus for creation of GFP fusions 2015-03-16T12:00:00Z 2015-05-08T01:09:06Z BBa_I13401 and BBa_K1088051 This part will be suffixed to proteins to create protein-GFP fusions with a flexible linker in between to increase chances of both proteins to be functional. GFP fusions have variuos functions e.g. protein localization assays, immunoblotting and protein purification using anti-GFP antibodies. For protein purification, or other applications of seperating the fused proteins, a TEV protease recognition site is included in the flexible linker. Note that standard RFC[10] assembly with a part suffixed to this part will result in a scar-site that includes an unwanted stop-codon. To prevent this, PCR amplification of inserts using appropriate primers is to be employed (see design page for details) false false _1398_ 0 17057 9 Not in stock false The linker was added according to step 1-4 as specified on the "Design" page of the flexible linker Using standard RFC[10] assembly of two parts creates scarsites (tactag). Transcription of the scarsite encodes: "uac uag" of which uag is a stop codon that would render the linker useless. Thus, standard assembly can not be employed for fusing proteins/domains. 1) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site a) Forward primer: 5'-cgctTCTAGAgNNN...NNN-3' - includes XbaI-site and sequence complementary to DNA sequence of N-terminal protein/domain b) Reverse primer: 5'-atatGGATCCNNN...NNN-3' - includes BamHI-site and sequence complementary to DNA sequence of N-terminal protein/domain (OBS! do NOT include stop-codons of coding sequences) 2) PCR amplify BioBrick with designed primer 3) digest pSB1C3-Linker:GFP and PCR product with XbaI and BamHI 4) ligate the digested pSB1C3-Linker:GFP and PCR product to create a brick with your protein/domain fused with GFP false Patrick Rosendahl Andreassen component2430554 1 BBa_K1088051 component2430562 1 BBa_I13401 annotation2430562 1 BBa_I13401 range2430562 1 31 887 annotation2430554 1 BBa_K1088051 range2430554 1 1 30 BBa_B0012 1 BBa_B0012 TE from coliphageT7 2003-01-31T12:00:00Z 2015-08-31T04:07:20Z Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>. Released HQ 2013 Transcription terminator for the <i>E.coli</i> RNA polymerase. false false _1_ 0 24 7 In stock false <P> <P>Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG).<P> Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator. false Reshma Shetty annotation1690 1 polya range1690 1 28 41 annotation1687 1 stop range1687 1 34 34 annotation1686 1 T7 TE range1686 1 8 27 annotation7020 1 BBa_B0012 range7020 1 1 41 BBa_I13401 1 BBa_I13401 GFP reporter for RHS of library test constructs 2005-04-05T11:00:00Z 2015-08-31T04:07:33Z Released HQ 2013 This part will be suffixed to a promoter.RBS library. The purpose of the experiment is to make a first pass at a library-based construction step to identify efficiency issues and also to examine maxPOPS/RIPS level. false true _6_ 0 2 6 In stock false This part was built by Jen as an intermediate to their nuts and bolts parts. true jasonk component1476713 1 BBa_E0040 component1476728 1 BBa_B0012 component1476718 1 BBa_B0010 annotation1476713 1 BBa_E0040 range1476713 1 1 720 annotation1476728 1 BBa_B0012 range1476728 1 817 857 annotation1476718 1 BBa_B0010 range1476718 1 729 808 BBa_E0040 1 GFP green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212 2004-09-29T11:00:00Z 2016-01-26T02:09:38Z Released HQ 2013 GFP (mut3b) [note that this part does not have a barcode] false true _11_1_ 4206 61 7 In stock false true jcbraff annotation1934520 1 GFP protein range1934520 1 1 720 BBa_K1088051 1 Linker 10 aa linker with BamHI restriction site and TEV recognition site 2015-03-16T12:00:00Z 2015-05-08T01:09:06Z BBa_K105012 A flexible 10 amino acid linker that enables for functional protein/domain fusions. A BamHI site is included in order to prevent creation of a scarsite (in-frame scarsites encode stop-codons). Assembly of parts therefore has to be done with PCR amplification of inserts using appropriate primers instead of standard RFC[10] assembly of parts (see design page for further details). The encoded peptide (GSENLYFQSG) includes a recognition site for the TEV protease (ENLYFQS) that enables for seperation of the fused proteins/domains. false false _1398_ 0 17057 9 Not in stock false Using standard RFC[10] assembly of two parts creates scarsites (tactag). Transcription of the scarsite encodes: "uac uag" of which uag is a stop codon that would render the linker useless. Thus, standard assembly can not be employed for fusing proteins/domains. Instead we propose the following for implementing the linker: 1) Design primers for amplification of the C-terminal protein/domain that includes the linker DNA sequence in the primer a) Forward primer: 5'-cgctTCTAGaGGGATCCgaaaatttgtattttcaatctggtNNN...NNN-3' - includes XbaI-site, linker, and sequence complementary to DNA sequence of C-terminal protein/domain b) Reverse primer: 5'-atatCTGCAGCggccgctACTAGTaNNN...NNN-3' - includes PstI-site, SpeI-site and sequence complementary to DNA sequence of C-terminal protein/domain 2) PCR amplify BioBrick with the designed primers 3) digest pSB1C3 (or any other standard backbone) and amplified PCR product with XbaI and PstI 4) ligate digested pSB1C3 and PCR product to create brick with linker at the N-terminus (pSB1C3-Linker:C-terminal_protein/domain). 5) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site a) Forward primer: 5'-cgctTCTAGAgNNN...NNN-3' - includes XbaI-site and sequence complementary to DNA sequence of N-terminal protein/domain b) Reverse primer: 5'-atatGGATCCNNN...NNN-3' - includes BamHI-site and sequence complementary to DNA sequence of N-terminal protein/domain (OBS! do NOT include stop-codons of coding sequences) 6) PCR amplify BioBrick with designed primer 7) digest pSB1C3-Linker:C-terminal_protein/domain and PCR product with XbaI and BamHI 8) ligate the digested pSB1C3-Linker:C-terminal_protein/domain and PCR product to create brick with protein/domain fused with the linker false Patrick Rosendahl Andreassen annotation2430563 1 BamHI-site range2430563 1 1 6 annotation2430564 1 TEV recognition-site range2430564 1 6 24 BBa_B0010 1 BBa_B0010 T1 from E. coli rrnB 2003-11-19T12:00:00Z 2015-08-31T04:07:20Z Transcriptional terminator consisting of a 64 bp stem-loop. false false _1_ 0 24 7 In stock false true Randy Rettberg annotation4184 1 stem_loop range4184 1 12 55 annotation7018 1 BBa_B0010 range7018 1 1 80 BBa_B0010_sequence 1 ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc BBa_K1088051_sequence 1 ggatccgaaaatttgtattttcaatctggt BBa_K1088052_sequence 1 ggatccgaaaatttgtattttcaatctggtatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata BBa_E0040_sequence 1 atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataa BBa_I13401_sequence 1 atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata BBa_B0012_sequence 1 tcacactggctcaccttcgggtgggcctttctgcgtttata igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z