pMJ509: pLM005-Cre10.g430350[W47A/R48A/W51A/R52A/W110A/R111A/W148A/R149A]-Venus-3xFLAG

Description: Previously synthesized pMJ507 (CC-5652) was modified by site-directed mutagenesis. All four triplet pairs coding for a Tryptophan-Arginine dipeptide were mutagenized to code for a double Alanine (W47A/R48A/W51A/R52A/W110A/R111A/W148A/R149A).

Selection in E. coli: ampicillin
Selection in C. reinhardtii: paromomycin
Host strain: DH5α
Transformed into C. reinhardtii strain cMJ030 to produce CC-5654

Plasmid: 8646 bp DNA circular
CDS	0784..1587	“APHVIII”
CDS	2699..4644	“Cre10.g430350W47A/R48A/W51A/R52A/W110A/R111A/W148A/R149A”
CDS	4645..5469	“VENUS-3xFLAG fusion protein”
TRANSLATION: NGDLGGSGGRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELIKGGGDYKDHDGDYKDHDIDYKDDDDK-

Plasmid sequence: 1..8646
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGATATCCTTGACATGATTGGTGCGTATGTTTGTATGAAGCTACAGGACTGATTTGGCGGGCTATGAGGGCGGGGGAAGCTCTGGAAGGGCCGCGATGGGGCGCGCGGCGTCCAGAAGGCGCCATACGGCCCGCTGGCGGCACCCATCCGGTATAAAAGCCCGCGACCCCGAACGGTGACCTCCACTTTCAGCGACAAACGAGCACTTATACATACGCGACTATTCTGCCGCTATACATAACCACTCAGCTAGCTTAAGATCCCATCAAGCTTGCATGCCGGGCGCGCCAGAAGGAGCGCAGCCAAACCAGGATGATGTTTGATGGGGTATTTGAGCACTTGCAACCCTTATCCGGAAGCCCCCTGGCCCACAAAGGCTAGGCGCCAATGCAAGCAGTTCGCATGCAGCCCCTGGAGCGGTGCCCTCCTGATAAACCGGCCAGGGGGCCTATGTTCTTTACTTTTTTACAAGAGAAGTCACTCAACATCTTAAAATGGCCAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGGATGGCCACTCCGCCCTCCCCGGTGCTGAAGAATTTCGAAGCATGGACGATGCGTTGCGTGCACTGCGGGGTCGGTATCCCGGTTGTGAGTGGGTTGTTGTGGAGGATGGGGCCTCGGGGGCTGGTGTTTATCGGCTTCGGGGTGGTGGGCGGGAGTTGTTTGTCAAGGTGGCAGCTCTGGGGGCCGGGGTGGGCTTGTTGGGTGAGGCTGAGCGGCTGGTGTGGTTGGCGGAGGTGGGGATTCCCGTACCTCGTGTTGTGGAGGGTGGTGGGGACGAGAGGGTCGCCTGGTTGGTCACCGAAGCGGTTCCGGGGCGTCCGGCCAGTGCGCGGTGGCCGCGGGAGCAGCGGCTGGACGTGGCGGTGGCGCTCGCGGGGCTCGCTCGTTCGCTGCACGCGCTGGACTGGGAGCGGTGTCCGTTCGATCGCAGTCTCGCGGTGACGGTGCCGCAGGCGGCCCGTGCTGTCGCTGAAGGGAGCGTCGACTTGGAGGATCTGGACGAGGAGCGGAAGGGGTGGTCGGGGGAGCGGCTTCTCGCCGAGCTGGAGCGGACTCGGCCTGCGGACGAGGATCTGGCGGTTTGCCACGGTGACCTGTGCCCGGACAACGTGCTGCTCGACCCTCGTACCTGCGAGGTGACCGGGCTGATCGACGTGGGGCGGGTCGGCCGTGCGGACCGGCACTCCGATCTCGCGCTGGTGCTGCGCGAGCTGGCCCACGAGGAGGACCCGTGGTTCGGGCCGGAGTGTTCCGCGGCGTTCCTGCGGGAGTACGGGCGCGGGTGGGATGGGGCGGTATCGGAGGAAAAGCTGGCGTTTTACCGGCTGTTGGACGAGTTCTTCTGAGGGACCTGATGGTGTTGGTGGCTGGGTAGGGTTGCGTCGCGTGGGTGACAGCACAGTGTGGACGTTGGGATCCCCGCTCCGTGTAAATGGAGGCGCTCGTTGATCTGAGCCTTGCCCCCTGACGAACGGCGGTGGATGGAAGATACTGCTCTCAAGTGCTGAAGCGGTAGCTTAGCTCCCCGTTTCGTGCTGATCAGTCTTTTTCAACACGTAAAAAGCGGAGGAGTTTTGCAATTTTGTTGGTTGTAACGATCCTCCGTTGATTTTGGCCTCTTTCTCCATGGGCGGGCTGGGCGTATTTGAAGCGGGTACCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGACCAATCGTCACACGAGCCCTCGTCAGAAACACGTCTCCGCCACGCTCTCCCTCTCACGGCCGACCCCGCAGCCCTTTTGCCCTTTCCTAGGCCACCGACAGGACCCAGGCGCTCTCAGCATGCCTCAACAACCCGTACTCGTGCCAGCGGTGCCCTTGTGCTGGTGATCGCTTGGAAGCGCATGCGAAGACGAAGGGGCGGAGCAGGCGGCCTGGCTGTTCGAAGGGCTCGCCGCCAGTTCGGGTGCCTTTCTCCACGCGCGCCTCCACACCTACCGATGCGTGAAGGCAGGCAAATGCTCATGTTTGCCCGAACTCGGTGTCCTTAAAAAGCCGCTTCTTGTCGTCGTTCCGAGACATGTTAGCAGATCGCAGTGCCACCTTTCCTGACGCGCTCGGCCCCATATTCGGACGCAATTGTCATTTGTAGCACAATTGGAGCAAATCTGGCGAGGCAGTAGGCTTTTAAGTTGCAAGGCGAGAGAGCAAAGTGGGACGCGGCGTGATTATTGGTATTTACGCGACGGCCCGGCGCGTTAGCGGCCCTTCCCCCAGGCCAGGGACGATTATGTATCAATATTGTTGCGTTCGGGCACTCGTGCGAGGGCTCCTGCGGGCTGGGGAGGGGGATCTGGGAATTGGAGGTACGACCGAGATGGCTTGCTCGGGGGGAGGTTTCCTCGCCGAGCAAGCCAGGGTTAGGTGTTGCGCTCTTGACACGTTGTGCATTCTAGGACCCCACTGCTACTCACAACAAGCCCAGTTATGCCTATGCTTGGCTCTCGTCTTTTGGGTGCGCCCAGCACAAGCGCGCCAGCAAGGGTAAGTGGATCGACAGGTTGAATGCGATAGGCGGCATTCATACTGGCTCCGAAAGTTCGGTAGTGCTTCGGCGAGGTTCGGGACCCGAATGTTATCTGGAACCTGTGGTAACTGCTCACCGCGCGGCGTGATGAATCATCACAGGCTCCTCTGGCGCAGCATGGGCGACTTCTGGTGGCCTGCCGCGCGACTGTGAGGTCCAGGCAAGTGAAGCCGATTATTTCTTGTTGACTTCGCATATTCACGCGCGTTTCGGCATCAAAACGCGCTGGTGTGGGACCTTGGCTTCTCAGCTATTGCATAGCGCGCGGTGCGGGCGCCATCCAGGGCCTCTTCCTGTGTCCCGACCGCGCGGCTTCCAGCCACCGTGTGCACCGCAAACCTTGCTCCACGGTCTCCCTTAACAATTCCCGACTCGGCCCCGGCCCCAAACACAGCTCGCCACGCTCTACTGGCGACGCGGCCAGCTCCGCGGCAACGGAGCTTGAGGGCGGCGCCAACGGCGCGGCGCACCACGACCACGACCACGAGGAAGTCGACGTCCACGCCAACGGCCACAGCTACAGCAACGGCGTCTCCCATTCTGACAGCTACAGCAACGGCGCCAGCTACAACGGCACCAGTTACGCCACGGGCATCAGCGCCAGCGCGGCCAACGGCTCCGCCGCCGCCAACGGTAGCAGCTATGCGGCTCGCGCCGCGCAGCTGGTTCGCGAGCAGGACGAGGACGAGGACCACGACTTCCACCGGCGTGCAGCGGACGCGGCCACGGAGCACTCGCCGCAGGGCTCCCGCTGGGGCTCCGAGGACGACGATGCCGCCTCCTCCTCAGCCAACGGGCACCACCACGACCAGCAAGAGGAGGAGCAACAAGGTGAGCAGGAGGAAGGCGGCGGCGGTTTCTTCACGAGTGCCTTCAAGGACGCGCTGAAAGACGCCCTAGCGCGCAGTCCACTGTTCGGGTCGCAGTACGGCGGCGGGTCGGGCGACGAGGAGAGCATTCCGCCGCAGCCCACGGAGAACGAGGCGGTGGAGGCAACCACGGGTATGTGGTATTGTAGGGTGGTCTCTGCAGGGTCAAGGCAGGGGGTTGCGGCTTGGTGGGGCCAAAGGGCACGCAGGGCCTTCTCAACGACCATGAGCACGCCGTTGTAGGCTTTCCGCAATCCTGGTAAACTCCTGACAAGGTCATGACTGCCATACATCGCAACCTAAACACTAAACTCATCTTTCCTTCTGCGTGCAAACGTACTGTGGCAGGCTCGTCGCTGTTCAGCAACAGCTTCCGGTCTGCGCTAAAGGAGTCTCTGGAGCGGCAGCAGACGGAGATGGACGTCAAGTACGGGTACGGCAGCGATGACGAGTACATCGCCACGCCGTACAAGTCAGCGGGCCCGGCGCGCCGGTGAGTTAGAAGAAGCGGGCAGGGAGGGGGAGGAGGAGCAGGGGCAGGGAGGAGGACTGGTGGCGGGGGAGCGGGGCCTGGTCGTTCCGCACCCGAGGGCTCCAGGTGAGAGGTTGGCAGTACGACAGGGGACTGCCGCTGGCCTTGGGGTGTTTCCGAACACCGACCGGTCCTCTCAGCCGTCGCAAACGCTGGATACTGCACCACCGCGCTCACCCACCCGGCCCTCTGTCTGTTGCCCCTGCCTCTCCCTCTCCCACGTAGGGAGTTGTCCCCCATGGAGGAGCTGCTCTCTTCCGCGCTGAGCGCCAGCCAGCACACACACCGCCGCCTGACGCTCATAAAGAACGAGATCGATGCGGCGCTGGAGCGCGAGGCCAAGCAGATTGAGCGGCTGGATTTCGCTCTCAAGAAGTGCGAGAACGACGCGGCCTACTATGCGACGCTCGAGCGCATGATGCGCGAGCGCGAGCGGGAGGGCAAGAACGGAGATCTGGGTGGCTCCGGCGGCCGGATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTACGGCAAGCTGACCCTGAAGCTGATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGATCAAGGGCGGTGGCGACTACAAGGACCATGACGGTGACTATAAGGATCACGACATCGACTACAAGGACGATGACGACAAGTAAGATCTCTAACTGCACGAATTCTCAGGTCGGGACATCCCCGACCAGGGGCGGCGGGGATGTTGCTGGGCCGATGGAAAGTAGCAACCCAGCCAGCGGCTTCCAGCGCACTCCAGCTGCTCACGGTTGCGACATTGCGCGTGCACGCTTGCGCGTCCCTCACTCGGCCAGCTTGTCGCCGCAGACATCCCTAGCATTGTGCGGACTGCGGTCGTCAGTTAGCGTAGTGGCGGGGCTCAAAGCGTGATGCAGCTGGTGGCTGATTGCATGTGCTACATATGCTGTTATGTTTTGCATGAACTTCGATGCATTGGATGCTGGGTGCACGCGTTTGCATGTGTTTGTGCCGGCATGCTGCCGTCGTCGGCCGTACGTTTACGTTTCTGTGTGCCGGGGTCTTTATTTCCGCCTGCAGACCGACGTCGGTTCCACTAGTTCTAGATGGCAGCAGCTGGACCGCCTGTACCATGGAGAAGAGCTTTACTTGCCGGGATGGCCGATTTCGCTGATTGATACGGGATCGGAGCTCGGAGGCTTTCGCGCTAGGGGCTAGGCGAAGGGCAGTGGTGACCAGGGTCGGTGTGGGGTCGGCCCACGGTCAATTAGCCACAGGAGGATCAGGGGGAGGTAGGCACGTCGACTTGGTTTGCGACCCCGCAGTTTTGGCGGACGTGCTGTTGTAGATGTTAGCGTGTGCGTGAGCCAGTGGCCAACGTGCCACACCCATTGAGAAGACCAACCAACTTACTGGCAATATCTGCCAATGCCATACTGCATGTAATGGCCAGGCCATGTGAGAGTTTGCCGTGCCTGCGCGCGCCCCGGGGGCGAAGCTTGGTACCGCTAGCGTCAGCTGAACTCCTGCTGCCACGCTGACAGAGACGCGCCCATGACGTAGCGCCGACTGCTGCTCCTCTGCTCGTGGAGCTCTGAATCTGCTGCCGGCTGCCCCCGCCCCCGCTGCTGAGGCTGAATCTACTCCGCTCTCCACCCCAGTCCCAGGAAGAGAGGCGCATTTACTTCGCACAGACGTTACAGCACACCCTTGATCATCATCAGCTGCTCTTCCCTGCCGCTGCAACACGCCCCGCGCGATATCCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC