Supplementary Note
Vectors are available upon request by non-commercial users. GFP 1–10 and GFP 11 can also be prepared by primer-directed mutation of GFPs on commercially available plasmids or by ordering coding sequences from gene synthesis companies.
Amino acid and sequence of the GFP 11 cassette:
SSLKRRKIPMGSSHHHHHHSSGLVPRGSHM*(frame shift +1)
*LIGSDGGSGGGSTSRDHMVLHEYVNAAGIT*GT*LEHHHHHH*DPAANKARKEAELAAATAEQ*LA*PLEA
DNA sequence of the GFP 11 cassette
0421007
TAGAGATACTGAGCACATCAGCAGGACGCACTGACCGAGTTCATTAAAGAGGAGAAAGATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGTAATTAATTAATTGGATCCGATGGAGGGTCTGGTGGCGGATCAACAAGTCGTGACCACATGGTCCTTCATGAGTACGTAAATGCTGCTGGGATTACATAAGGTACCTAACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCTCTAGAGGCCTC
02129811
Amino acid sequence of the GFP 1-10 cassette
DRDLDPAKLIRLTIHMGGTSMSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK*GTLEHHHHHH*DPAANKARKEAELAAATAEQ*LA*PLEA
DNA sequence of the GFP 1-10 cassette
01079936
AGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATACATATGGGTGGCACTAGTATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGAGGAGAGGGTGAAGGTGATGCTACAATCGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAAAGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAAATACAAGACGCGTGCTGTAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTACTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCACAGTTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAAACTGTCCTTTCGAAAGATCCCAACGAAAAGTAAGGTACCCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCTCTAGAGGCCTC
02129811
Vector primers to reamplify the split GFP fragments cassette
Top primer for pTET GFP11 plasmid:
0421007 5’-TAGAGATACTGAGCACATCAGCAGGACGCACTGACC-3’
Bottom primer for pTET GFP11 and pET GFP1-10 plasmids:
02129811 5’-GAGGCCTCTAGAGGTTATGCTAGTTATTGC-3’
Top primer for pET GFP1-10 plasmid:
01079936 5’-AGGATCGAGATCTCGATCCCGCGAAATTAATAC-3’
The GFP 11 cassette can be amplified from pTET plasmid using 0421007 and 02129811 primers. The restriction sites for cloning insert proteins is indicated in bold in the DNA sequence. A frameshift stuffer with three translational stops (one in each frame) prevents false positives from a self-religated plasmid. A 6His tag followed by a thrombin cleavage site is located at the N terminus. The initiator methionine is indicated in bold in the amino-acid sequence of both GFP 11 and GFP 1-10 cassettes. The GFP 1-10 cassette can be amplified using 01079936 and 02129811 primers.