人源P450 CYP11A1笔记.docVIP

1. CYP11A1在微生物细胞中并不需要定位于线粒体，以成熟蛋白的形式定位于细胞溶质或内质网膜上也能正常表达功能；有研究表明牛源CYP11A1可以有效催化22R-羟胆甾-4烯-3酮类底物生成黄体酮.2. CYP11A1Report for CCDS32291.1 (current version)CCDS Sequence DataBluehighlighting indicates alternating exons.Redhighlighting indicates amino acids encoded across a splice junction.NM_000781.2NP_000772.2cholesterol side-chain cleavage enzyme, mitochondrial isoform a precursorDescription: Transcript Variant: This variant (1) encodes the longer isoform (a).Nucleotide Sequence (1566 nt):ATGCTGGCCAAGGGTCTTCCCCCACGCTCAGTCCTGGTCAAAGGCTGCCAGACCTTTCTGAGTGCCCCCAGGGAGGGGCTGGGGCGTCTCAGGGTGCCCACTGGCGAGGGAGCTGGC39甘氨酸ATC40异亮氨酸TCCACCCGCAGTCCTCGCCCCTTCAATGAGATCCCCTCTCCTGGTGACAATGGCTGGCTAAACCTGTACCATTTCTGGAGGGAGACGGGCACACACAAAGTCCACCTTCACCATGTCCAGAATTTCCAGAAGTATGGCCCGATTTACAGGGAGAAGCTCGGCAACGTGGAGTCGGTTTATGTCATCGACCCTGAAGATGTGGCCCTTCTCTTTAAGTCCGAGGGCCCCAACCCAGAACGATTCCTCATCCCGCCCTGGGTCGCCTATCACCAGTATTACCAGAGACCCATAGGAGTCCTGTTGAAGAAGTCGGCAGCCTGGAAGAAAGACCGGGTGGCCCTGAACCAGGAGGTGATGGCTCCAGAGGCCACCAAGAACTTTTTGCCCCTGTTGGATGCAGTGTCTCGGGACTTCGTCAGTGTCCTGCACAGGCGCATCAAGAAGGCGGGCTCCGGAAATTACTCGGGGGACATCAGTGATGACCTGTTCCGCTTTGCCTTTGAGTCCATCACTAACGTCATTTTTGGGGAGCGCCAGGGGATGCTGGAGGAAGTAGTGAACCCCGAGGCCCAGCGATTCATTGATGCCATCTACCAGATGTTCCACACCAGCGTCCCCATGCTCAACCTTCCCCCAGACCTGTTCCGTCTGTTCAGGACCAAGACCTGGAAGGACCATGTGGCTGCATGGGACGTGATTTTCAGTAAAGCTGACATATACACCCAGAACTTCTACTGGGAATTGAGACAGAAAGGAAGTGTTCACCACGATTACCGTGGCATCCTCTACAGACTCCTGGGAGACAGCAAGATGTCCTTCGAGGACATCAAGGCCAACGTCACAGAGATGCTGGCAGGAGGGGTGGACACGACGTCCATGACCCTGCAGTGGCACTTGTATGAGATGGCACGCAACCTGAAGGTGCAGGATATGCTGCGGGCAGAGGTCTTGGCTGCGCGGCACCAGGCCCAGGGAGACATGGCCACGATGCTACAGCTGGTCCCCCTCCTCAAAGCCAGCATCAAGGAGACACTAAGACTTCACCCCATCTCCGTGACCCTGCAGAGATATCTTGTAAATGACTTGGTTCTTCGAGATTACATGATTCCTGCCAAGACACTGGTGCAAGTGGCCATCTATGCTCTGGGCCGAGAGCCCACCTTCTTCTTCGACCCGGAAAATTTTGACCCAACCCGATGGCTGAGCAAAGACAAGAACATCACCTACTTCCGGAACTTGGGCTTTGGCTGGGGTGTGCGGCAGTGTCTGGGACGGCGGATCGCTGAGCTAGAGATGACCATCTTCCTCATCAATATGCTGGAGAACTTCAGAGTTGAAATCCAACACCTCAGCGATGTGGGCACCACATTCAACCTCATTCTGATGCCTGAAAAGCCCATCTCCTTCACCTTCTGGCCCTTTAACCAGGAAGCAACCCAGCAGTGATranslation(521aa):MLAKGLPPRSVLVKGCQTFLSAPREGLGRLRVPTGEGAGISTRSPRPFNEIPSPGDNGWLNLYHFWRETGTHKVHLHHVQNFQKYGPIYREKLGNVESVYVIDPEDVALLFKSEGPNPERFLIPPWVAYHQYYQRPIGVLLKKSAAWKKDRVALNQEVMAPEATKNFLPLLDAVSRDFVSVLHRRIKKAGSGNYSGDISDDLFRFAFESITNVIFGERQGMLEEVVNPEAQRFIDAIYQMFHTSVPMLNLPPDLFRLFRTKTWKDHVAAWDVIFSKADIYTQNFYWELRQKGSVHHDYRGILYRLLGDSKMSFEDIKANVTEMLAGGVDTTSMTLQWHLYEMARNLKVQDMLRAEVLAARHQAQGDMATMLQLVPLLKASIKETLRLHPISVTLQRYLVNDLVLRDYMIPAKTLVQVAIYALGREPTFFFDPENFDPTRWLSKDKNITYFRNLGFGWGVRQCLGRRIAELEMTIFLINMLENFRVEIQHLSDVGTTFNLILMPEKPISFTFWPFNQEATQQReport for CCDS45303.1 (current version)cholesterol side-chain cleavage enzyme, mitochondrial isoform bDescription: Transcript Variant: This variant (2) uses an alternate first exon compared to variant 1, resulting in the translation start site being located at a downstream AUG. The resulting isoform (b) is shorter at the N-terminus compared to isoform a.Summary: This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the mitochondrial inner membrane and catalyzes the conversion of cholesterol to pregnenolone, the first and rate-limiting step in the synthesis of the steroid hormones.Two transcript variants encoding different isoforms have been found for this gene. The cellular location of the smaller isoform is unclear since it lacks the mitochondrial-targeting transit peptide.CCDS Sequence DataBluehighlighting indicates alternating exons.Redhighlighting indicates amino acids encoded across a splice junction.Nucleotide Sequence (1092 nt):ATGGCTCCAGAGGCCACCAAGAACTTTTTGCCCCTGTTGGATGCAGTGTCTCGGGACTTCGTCAGTGTCCTGCACAGGCGCATCAAGAAGGCGGGCTCCGGAAATTACTCGGGGGACATCAGTGATGACCTGTTCCGCTTTGCCTTTGAGTCCATCACTAACGTCATTTTTGGGGAGCGCCAGGGGATGCTGGAGGAAGTAGTGAACCCCGAGGCCCAGCGATTCATTGATGCCATCTACCAGATGTTCCACACCAGCGTCCCCATGCTCAACCTTCCCCCAGACCTGTTCCGTCTGTTCAGGACCAAGACCTGGAAGGACCATGTGGCTGCATGGGACGTGATTTTCAGTAAAGCTGACATATACACCCAGAACTTCTACTGGGAATTGAGACAGAAAGGAAGTGTTCACCACGATTACCGTGGCATCCTCTACAGACTCCTGGGAGACAGCAAGATGTCCTTCGAGGACATCAAGGCCAACGTCACAGAGATGCTGGCAGGAGGGGTGGACACGACGTCCATGACCCTGCAGTGGCACTTGTATGAGATGGCACGCAACCTGAAGGTGCAGGATATGCTGCGGGCAGAGGTCTTGGCTGCGCGGCACCAGGCCCAGGGAGACATGGCCACGATGCTACAGCTGGTCCCCCTCCTCAAAGCCAGCATCAAGGAGACACTAAGACTTCACCCCATCTCCGTGACCCTGCAGAGATATCTTGTAAATGACTTGGTTCTTCGAGATTACATGATTCCTGCCAAGACACTGGTGCAAGTGGCCATCTATGCTCTGGGCCGAGAGCCCACCTTCTTCTTCGACCCGGAAAATTTTGACCCAACCCGATGGCTGAGCAAAGACAAGAACATCACCTACTTCCGGAACTTGGGCTTTGGCTGGGGTGTGCGGCAGTGTCTGGGACGGCGGATCGCTGAGCTAGAGATGACCATCTTCCTCATCAATATGCTGGAGAACTTCAGAGTTGAAATCCAACACCTCAGCGATGTGGGCACCACATTCAACCTCATTCTGATGCCTGAAAAGCCCATCTCCTTCACCTTCTGGCCCTTTAACCAGGAAGCAACCCAGCAGTGATranslation (363 aa):MAPEATKNFLPLLDAVSRDFVSVLHRRIKKAGSGNYSGDISDDLFRFAFESITNVIFGERQGMLEEVVNPEAQRFIDAIYQMFHTSVPMLNLPPDLFRLFRTKTWKDHVAAWDVIFSKADIYTQNFYWELRQKGSVHHDYRGILYRLLGDSKMSFEDIKANVTEMLAGGVDTTSMTLQWHLYEMARNLKVQDMLRAEVLAARHQAQGDMATMLQLVPLLKASIKETLRLHPISVTLQRYLVNDLVLRDYMIPAKTLVQVAIYALGREPTFFFDPENFDPTRWLSKDKNITYFRNLGFGWGVRQCLGRRIAELEMTIFLINMLENFRVEIQHLSDVGTTFNLILMPEKPISFTFWPFNQEATQQ3. FDX1ferredoxin 1 Homo sapiens(human) ADX ID:2230 adrenodoxinReport for CCDS8344.1 (current version)CCDSStatusSpeciesChrom.GeneCCDS ReleaseNCBI Annotation ReleaseEnsembl Annotation ReleaseLinks8344.1PublicHomo sapiens11FDX11710676CCDS Sequence DataBluehighlighting indicates alternating exons.Redhighlighting indicates amino acids encoded across a splice junction.Nucleotide Sequence (555 nt):ATGGCTGCCGCTGGGGGCGCCCGGCTGCTGCGCGCCGCTTCTGCTGTCCTCGGCGGCCCGGCCGGCCGGTGGCTGCACCACGCTGGGTCCCGCGCTGGATCCAGCGGCCTGCTGAGGAACCGGGGGCCGGGCGGGAGCGCGGAGGCGAGCCGGTCGCTGAGCGTGTCGGCGCGGGCCCGGAGC丝氨酸AGCTCAGAAGATAAAATAACAGTCCACTTTATAAACCGTGATGGTGAAACATTAACAACCAAAGGAAAAGTTGGTGATTCTCTGCTAGATGTTGTGGTTGAAAATAATCTAGATATTGATGGCTTTGGTGCATGTGAGGGAACCCTGGCTTGTTCAACCTGTCACCTCATCTTTGAAGATCACATATATGAGAAGTTAGATGCAATCACTGATGAGGAGAATGACATGCTCGATCTGGCATATGGACTAACAGACAGATCACGGTTGGGCTGCCAAATCTGTTTGACAAAATCTATGGACAATATGACTGTTCGAGTGCCTGAAACAGTGGCTGATGCCAGACAATCCATTGATGTGGGCAAGACCTCCTGATranslation (184 aa):MAAAGGARLLRAASAVLGGPAGRWLHHAGSRAGSSGLLRNRGPGGSAEASRSLSVSARARSSSEDKITVHFINRDGETLTTKGKVGDSLLDVVVENNLDIDGFGACEGTLACSTCHLIFEDHIYEKLDAITDEENDMLDLAYGLTDRSRLGCQICLTKSMDNMTVRVPETVADARQSIDVGKTSthe cytochrome P450scc (CYP11A1), flavoprotein (NADPH-adrenodoxin reductase, AdR) iron-sulfur protein (adrenodoxin, Adx).CCDS Sequence DataBluehighlighting indicates alternating exons.Redhighlighting indicates amino acids encoded across a splice junction.Mouse over the nucleotide or protein sequence below and click on the highlighted codon or residue to select the pair.FDXRferredoxin reductase Homo sapiens(human) Nucleotide Sequence (1605 nt):Translation (534 aa):ATGGCTTCGCGCTGCTGGCGCTGGTGGGGCTGGTCGGCGTGGCCTCGGACCCGGCTGCCTCCCGCCGGGAGCACCCCGAGCTTCTGCCACCATTTC前32个氨基酸TCCACACAGGAGAAGACCCCCCAGATCTGTGTGGTGGGCAGTGGCCCAGCTGGCTTCTACACGGCCCAACACCTGCTAAAGAGGGTGGAAGCCTTGTGTTCTCAGCCCAGGGTCCTGAACTCTCCTGCTCTGTCTGGGGAAGGGGAGGACCTGGGGGCGTCCCAGCCTCTCTCTCTCGGCCCCACCAGCTGCCACCCTGTTCCCCAGCAGCACCCCCAGGCCCACGTGGACATCTACGAGAAACAGCCTGTGCCCTTTGGCCTGGTGCGCTTTGGTGTGGCGCCTGATCACCCCGAGGTGAAGAATGTCATCAACACATTTACCCAGACGGCCCATTCTGGCCGCTGTGCCTTCTGGGGCAACGTGGAGGTGGGCAGGGACGTGACGGTGCCGGAGCTGCAGGAGGCCTACCACGCTGTGGTGCTGAGCTACGGGGCAGAGGACCATCGGGCCCTGGAAATTCCTGGTGAGGAGCTGCCAGGTGTGTGCTCCGCCCGGGCCTTCGTGGGCTGGTACAACGGGCTTCCTGAGAACCAGGAGCTGGAGCCAGACCTGAGCTGTGACACAGCCGTGATTCTGGGGCAGGGGAACGTGGCTCTGGACGTGGCCCGCATCCTACTGACCCCACCTGAGCACCTGGAGAGAACGGACATCACGAAGGCAGCCCTGGGTGTACTGAGGCAGAGTCGAGTGAAGACAGTGTGGCTAGTGGGCCGGCGTGGACCCCTGCAAGTGGCCTTCACCATTAAGGAGCTTCGGGAGATGATTCAGTTACCGGGAGCCCGGCCCATTTTGGATCCTGTGGATTTCTTGGGTCTCCAGGACAAGATCAAGGAGGTCCCCCGCCCGAGGAAGCGGCTGACGGAACTGCTGCTTCGAACGGCCACAGAGAAGCCAGGGCCGGCGGAAGCTGCCCGCCAGGCATCGGCCTCCCGTGCCTGGGGCCTCCGCTTTTTCCGAAGCCCCCAGCAGGTGCTGCCCTCACCAGATGGGCGGCGGGCAGCAGGTGTCCGCCTAGCAGTCACTAGACTGGAGGGTGTCGATGAGGCCACCCGTGCAGTGCCCACGGGAGACATGGAAGACCTCCCTTGTGGGCTGGTGCTCAGCAGCATTGGGTATAAGAGCCGCCCTGTCGACCCAAGCGTGCCCTTTGACTCCAAGCTTGGGGTCATCCCCAATGTGGAGGGCCGGGTTATGGATGTGCCAGGCCTCTACTGCAGCGGCTGGGTGAAGAGAGGACCTACAGGTGTCATAGCCACAACCATGACTGACAGCTTCCTCACCGGCCAGATGCTGCTGCAGGACCTGAAGGCTGGGTTGCTCCCCTCTGGCCCCAGGCCTGGCTACGCAGCCATCCAGGCCCTGCTCAGCAGCCGAGGGGTCCGGCCAGTCTCTTTCTCAGACTGGGAGAAGCTGGATGCCGAGGAGGTGGCCCGGGGCCAGGGCACGGGGAAGCCCAGGGAGAAGCTGGTGGATCCTCAGGAGATGCTGCGCCTCCTGGGCCACTGA文献整合Cytochrome P450scc (P450scc) catalyzes the first step in steroid hormone synthesis, the conversion ofcholesterol to pregnenolone.The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin.The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach.2.2.1. Starting plasmidsThe vector for expression of bovine AdR, plasmid pBAR1607 (Sagara et al., 1993), was kindly provided by Yasuhiro Sagara. This vector is a derivative of pCWori+, containing a AdR cDNA fragment inserted between the NdeI and KpnI sites of the polylinker.The plasmids pKKHC-Adx (Uhlmann et al., 1992) and pKKHC-CYP106A2 (Simgen et al., 2000) provided the templates for PCR amplification of Adx andCYP106A2, respectively. Expression vector pTrc99a (Amann et al., 1988) and cloning vector pACYC184(Chang and Cohen, 1978) were used for constructing the new expression vector for CYP106A2. The vector for expression of bovine AdR, plasmid pBAR1607 (Sagara et al., 1993), was kindly provided by Yasuhiro Sagara. This vector is a derivative of pCWori+, containing a AdR cDNA fragment inserted between the NdeI and KpnI sites of the polylinker. The plasmids pKKHC-Adx (Uhlmann et al., 1992) and pKKHC-CYP106A2 (Simgen et al., 2000) provided the templates for PCR amplification of and CYP106A2, respectively.2.2.2. Construction of the plasmid pBAR TwinOligonucleotides encoding the amino- and carboxytermini of the truncated bovine Adx4-108 were used as primers for amplification of the cDNA fragment from plasmid pKKHC-Adx. The forward primer was designed with a restriction site (KpnI) immediately upstream of the ribosome binding site and the coding region and the reverse primer contains a stop codon followed by an EcoRI restriction site to facilitate cloning into the expression vector pBAR1607.The 0.4-kb fragment was ligated between the KpnI and EcoRI sites of pBAR1607. In the resulting construct pBAR Twin the cDNAs of AdR and Adx orientated in tandem are under control of a lac and two tac promoters (Fig. 2).Fig. 2. CYP106A2 and AdR/Adx coexpression plasmids. The 6.7-kb vector pBAR Twin contains two copies of the tac and one lacUV5 promoter, the cDNAs from AdR and Adx, the _-lactamase gene, and the high-copy origin of replication of the pUC series of plasmids. CYP106A2 is encoded on the 5.4 kb construct pACYC FHH2 8 under control of a tac promoter and a transcription terminator derived from the rrnB gene. The plasmid features the low-copy pMB1 origin of replication and the sequence of the cat resistance gene.Cytochrome P450scc (P450scc)3 catalyzes the three hydroxylation reactions required to convert cholesterol to pregnenolone, the first step in steroid hormone biosynthesis.Electrons for the hydroxylation reactions are supplied by NADPH via the electron transfer proteins adrenodoxin reductase and adrenodoxin. Bovine and human cytochrome P450scc genes (CYP11A1) encode 520 and 521 amino acid proteins, respectively.Bacterial expression of P450scc.The human placental cDNA library was from Clontech.Initially the library was screened with antiserum to bovine P450scc (1517). The resulting 50 plaques producing a positive signal were screened by hybridization using the synthetic oligonucleotide59-ATGTTCCACACCAGCCTCCCCATG-39, end labeled with 32Pphosphate (17). From this a full-length cDNA was isolated. PCR was used to remove the precursor sequence from the P450scc cDNA to enable bacterial expression of the mature protein. A synthetic oligonucleotide containing EcoRI and NcoI sites and the initiator codon ATG within the NcoI site (underlined) was used as a 59-primer and an oligonucleotide containing a KpnI site (underlined) was used as the 39-primer. The sequences of the primers were as follows:5 primer, 5-GGGAATTCCATGGCAAGTACAAGAAGTCCTCGCCCCTTCAATGAG-33 primer, 5-GCAGGTACCCCTTGGGCCCCACCCCTGGGCCT-3ID:231 错误Report for CCDS5831.1 (current version) CCDSStatusSpeciesChrom.GeneCCDS ReleaseNCBI Annotation ReleaseEnsembl Annotation ReleaseLinks5831.1PublicHomo sapiens7AKR1B11710676CCDS Sequence DataBluehighlighting indicates alternating exons.Redhighlighting indicates amino acids encoded across a splice junction.Nucleotide Sequence (951 nt):ATGGCAAGCCGTCTCCTGCTCAACAACGGCGCCAAGATGCCCATCCTGGGGTTGGGTACCTGGAAGTCCCCTCCAGGGCAGGTGACTGAGGCCGTGAAGGTGGCCATTGACGTCGGGTACCGCCACATCGACTGTGCCCATGTGTACCAGAATGAGAATGAGGTGGGGGTGGCCATTCAGGAGAAGCTCAGGGAGCAGGTGGTGAAGCGTGAGGAGCTCTTCATCGTCAGCAAGCTGTGGTGCACGTACCATGAGAAGGGCCTGGTGAAAGGAGCCTGCCAGAAGACACTCAGCGACCTGAAGCTGGACTACCTGGACCTCTACCTTATTCACTGGCCGACTGGCTTTAAGCCTGGGAAGGAATTTTTCCCATTGGATGAGTCGGGCAATGTGGTTCCCAGTGACACCAACATTCTGGACACGTGGGCGGCCATGGAAGAGCTGGTGGATGAAGGGCTGGTGAAAGCTATTGGCATCTCCAACTTCAACCATCTCCAGGTGGAGATGATCTTAAACAAACCTGGCTTGAAGTATAAGCCTGCAGTTAACCAGATTGAGTGCCACCCATATCTCACTCAGGAGAAGTTAATCCAGTACTGCCAGTCCAAAGGCATCGTGGTGACCGCCTACAGCCCCCTCGGCTCTCCTGACAGGCCCTGGGCCAAGCCCGAGGACCCTTCTCTCCTGGAGGATCCCAGGATCAAGGCGATCGCAGCCAAGCACAATAAAACTACAGCCCAGGTCCTGATCCGGTTCCCCATGCAGAGGAACTTGGTGGTGATCCCCAAGTCTGTGACACCAGAACGCATTGCTGAGAACTTTAAGGTCTTTGACTTTGAACTGAGCAGCCAGGATATGACCACCTTACTCAGCTACAACAGGAACTGGAGGGTCTGTGCCTTGTTGAGCTGTACCTCCCACAAGGATTACCCCTTCCATGAAGAGTTTTGATranslation (316 aa):MASRLLLNNGAKMPILGLGTWKSPPGQVTEAVKVAIDVGYRHIDCAHVYQNENEVGVAIQEKLREQVVKREELFIVSKLWCTYHEKGLVKGACQKTLSDLKLDYLDLYLIHWPTGFKPGKEFFPLDESGNVVPSDTNILDTWAAMEELVDEGLVKAIGISNFNHLQVEMILNKPGLKYKPAVNQIECHPYLTQEKLIQYCQSKGIVVTAYSPLGSPDRPWAKPEDPSLLEDPRIKAIAAKHNKTTAQVLIRFPMQRNLVVIPKSVTPERIAENFKVFDFELSSQDMTTLLSYNRNWRVCALLSCTSHKDYPFHEEFSCC+GFPCGGGATCCGCCACCATGATC40异亮氨酸TCCACCCGCAGTCCTCGCCCCTTCAATGAGATCCCCTCTCCTGGTGACAATGGCTGGCTAAACCTGTACCATTTCTGGAGGGAGACGGGCACACACAAAGTCCACCTTCACCATGTCCAGAATTTCCAGAAGTATGGCCCGATTTACAGGGAGAAGCTCGGCAACGTGGAGTCGGTTTATGTCATCGACCCTGAAGATGTGGCCCTTCTCTTTAAGTCCGAGGGCCCCAACCCAGAACGATTCCTCATCCCGCCCTGGGTCGCCTATCACCAGTATTACCAGAGACCCATAGGAGTCCTGTTGAAGAAGTCGGCAGCCTGGAAGAAAGACCGGGTGGCCCTGAACCAGGAGGTGATGGCTCCAGAGGCCACCAAGAACTTTTTGCCCCTGTTGGATGCAGTGTCTCGGGACTTCGTCAGTGTCCTGCACAGGCGCATCAAGAAGGCGGGCTCCGGAAATTACTCGGGGGACATCAGTGATGACCTGTTCCGCTTTGCCTTTGAGTCCATCACTAACGTCATTTTTGGGGAGCGCCAGGGGATGCTGGAGGAAGTAGTGAACCCCGAGGCCCAGCGATTCATTGATGCCATCTACCAGATGTTCCACACCAGCGTCCCCATGCTCAACCTTCCCCCAGACCTGTTCCGTCTGTTCAGGACCAAGACCTGGAAGGACCATGTGGCTGCATGGGACGTGATTTTCAGTAAAGCTGACATATACACCCAGAACTTCTACTGGGAATTGAGACAGAAAGGAAGTGTTCACCACGATTACCGTGGCATCCTCTACAGACTCCTGGGAGACAGCAAGATGTCCTTCGAGGACATCAAGGCCAACGTCACAGAGATGCTGGCAGGAGGGGTGGACACGACGTCCATGACCCTGCAGTGGCACTTGTATGAGATGGCACGCAACCTGAAGGTGCAGGATATGCTGCGGGCAGAGGTCTTGGCTGCGCGGCACCAGGCCCAGGGAGACATGGCCACGATGCTACAGCTGGTCCCCCTCCTCAAAGCCAGCATCAAGGAGACACTAAGACTTCACCCCATCTCCGTGACCCTGCAGAGATATCTTGTAAATGACTTGGTTCTTCGAGATTACATGATTCCTGCCAAGACACTGGTGCAAGTGGCCATCTATGCTCTGGGCCGAGAGCCCACCTTCTTCTTCGACCCGGAAAATTTTGACCCAACCCGATGGCTGAGCAAAGACAAGAACATCACCTACTTCCGGAACTTGGGCTTTGGCTGGGGTGTGCGGCAGTGTCTGGGACGGCGGATCGCTGAGCTAGAGATGACCATCTTCCTCATCAATATGCTGGAGAACTTCAGAGTTGAAATCCAACACCTCAGCGATGTGGGCACCACATTCAACCTCATTCTGATGCCTGAAAAGCCCATCTCCTTCACCTTCTGGCCCTTTAACCAGGAAGCAACCCAGCAGATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCTACATACGGAAAGCTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCAGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGCTCAGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAADX+GFPCGGAATTCGCCACCATGAGC丝氨酸AGCTCAGAAGATAAAATAACAGTCCACTTTATAAACCGTGATGGTGAAACATTAACAACCAAAGGAAAAGTTGGTGATTCTCTGCTAGATGTTGTGGTTGAAAATAATCTAGATATTGATGGCTTTGGTGCATGTGAGGGAACCCTGGCTTGTTCAACCTGTCACCTCATCTTTGAAGATCACATATATGAGAAGTTAGATGCAATCACTGATGAGGAGAATGACATGCTCGATCTGGCATATGGACTAACAGACAGATCACGGTTGGGCTGCCAAATCTGTTTGACAAAATCTATGGACAATATGACTGTTCGAGTGCCTGAAACAGTGGCTGATGCCAGACAATCCATTGATGTGGGCAAGACCTCCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCTACATACGGAAAGCTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCAGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGCTCAGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAA ADR+GFPGCCACCATGTCCACACAGGAGAAGACCCCCCAGATCTGTGTGGTGGGCAGTGGCCCAGCTGGCTTCTACACGGCCCAACACCTGCTAAAGAGGGTGGAAGCCTTGTGTTCTCAGCCCAGGGTCCTGAACTCTCCTGCTCTGTCTGGGGAAGGGGAGGACCTGGGGGCGTCCCAGCCTCT