Document outline:
1. Plasmid info and references relevant to the creation of the construct
2. Primers used for sequencing
3. Sequencing results combined from multiple primers
4. Blast results from the combined read(to protein and mRNA, comparing the sequence to that of Akt)
1.Plasmid info:
plasmid name: “pSR-alpha Akt kinase-dead rat”
Vector Backbone: pSR-alpha
Gene/Insert name (entrez ID of insert): Kinase-dead Akt (Akt-K179D)
Species of the gene(s): rat
Promoter: SV40?
Antiboitic Resistance: Ampicilline
Description of construct: “We tested several mutants of Akt for dominant negative effects, including a protein in which Lys179 in the kinase domain was replaced by asparate (Akt-K179D). Although Akt-K179D did not exhibit kinase activity, it [could be phosphorylated after insulin treatment and by an upstream kinase and] was less effective than Akt-AA in inhibiting the activity of endogenous Akt when overexpressed by the adenovirus system.”
Pasted from <http://mcb.asm.org/cgi/content/full/18/7/3708> “
Cloning strategy (5’ and 3’ cloning sites): ?
Creator/source: ?
Bacterial host strain: ?
2.Primers used for sequencing:
SV40promoter from McLab: GCCCCTAACTCCGCCCATCC
462F: GCCCAAGCACCGTGTGACCA
481R: TGGTCACACGGTGCTTGGGC
1191R: CAGCCCCGAGAGCAGGGACT
1172F: AGTCCCTGCTCTCGGGGCTG
3.Sequencing Result-combined from all primers reads:
Insert nucleotide sequence (combined from reads from all primers): (in orange: sequence 5’ of the ATG start; in green: the ATG start codon; in pink: the HA tag sequence; in blue: the sequence 3’ of the akt sequence)
TCGCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCTCGATCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTCCTCGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCTGCAGGTCGACTCTAGGGAATTCCCTAGAGGATCCCCAGCTTGCCATGTACCCATACGATGTTCCGGATTACGCTAACGACGTAGCCATTGTGAAGGAGGGCTGGCTGCACAAACGAGGGGAATATATTAAAACCTGGCGGCCACGCTACTTCCTCCTCAAGAATGATGGCACCTTTATTGGCTACAAGGAACGGCCTCAGGATGTGGAGCAGCGCGAGTCCCCACTCAACAACTTCTCAGTGGCACAATGTCAGCTGATGAAGACAGAGCGGCCGAGGCCCAACACCTTCATCATCCGCTGCCTGCAGTGGACCACGGTCATTGAGCGCACCTTCCATGTGGAAACGCCTGAGGAGCGGGAAGAGTGGACCACCGCCATTCAGACTGTGGCTGATGGACTCAAACGGCAGGAGGAGGAGACGATGGACTTCCGGTCAGGTTCACCCAGTGACAACTCAGGTGCTGAGGAGATGGAGGTGGCCCTGGCCAAGCCCAAGCACCGTGTGACCATGAACGAGTTTGAGTACCTGAAGCTACTGGGCAAGGGCACCTTTGGGAAGGTGATCCTGGTGAAGGAGAAGGCCACAGGTCGCTACTATGCCATGGATATCCTCAAGAAGGAGGTCATCGTTGCCAAGGATGAGGTTGCCCACACGCTTACTGAGAACCGTGTCCTGCAGAACTCTAGGCATCCCTTCCTTACAGCCCTCAAGTACTCATTCCAGACCCACGACCGCCTCTGCTTTGTCATGGAGTACGCCAATGGAGGCGAGCTCTTCTTCCACCTGTCTCGTGAGCGCGTGTTTTCAGAGGACCGGGCCCGCTTCTACGGTGCGGAGATTGTGTCCGCCCTGGACTACTTGCACTCCGAGAAGAACGTGGTGTACCGGGACCTCAAGCTGGAGAACCTCATGCTGGACAAGGACGGGCACATCAAGATAACGGACTTCGGGCTGTGCAAGGAGGGTATCAAGGACGGTGCCACCATGAAGACGTTCTGCGGGACACCCGAGTACCTGGCCCCTGAGGTGCTGGAGGACAACGACTATGGCCGTGCAGTGGACTGGTGGGGGCTGGGCGTGGTCATGTACGAGATGATGTGCGGCCGCCTGCCCTTCTACAACCAGGACCATGAGAAGCTGTTCGAGCTCATCCTAATGGAGGAGATCCGCTTCCCACGCACACTCGGGCCGGAGGCCAAGTCCCTGCTCTCGGGGCTGCTCAAGAAGGACCCTACACAGAGGCTCGGTGGGGGCTCCGAGGACGCCAAGGAGATCATGCAGCACCGCTTCTTTGCCAACATCGTGTGGCAAGATGTGTATGAGAAGAAGCTGAGCCCACCTTTCAAGCCCCAGGTCACCTCTGAGACCGACACCAGGTATTTTGATGAGGAGTTCACAGCTCAGATGATCACCATCACACCGCCTGATCAAGATGACAGCATGGAGTGTGTGGACAGTGAACGGAGGCCGCACTTTCCCCAGTTCTCCTACTCAGCCAGTGGCACAGCCTGAGGCCTGGGGTGGTGGCTGGCAGCCCTGCACCCCTCTGCATTGCCGAGTCCAGAAGCCCCGCATGGATCATCTGTACCTGATGGTTTTGTTTCTCGGATGTGCTGGGGAGGAACCTCGCCAGCCTCGGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGGGGTACCTTCTGAGGCGGAAAGAACCAGCCGGATCCCTCGAGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGGGTGGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCGGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAG
Construct peptide sequence (combined from all 4 reads):
MYPYDVPDYANDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVEQRESPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGLKRQEEETMDFRSGSPSDNSGAEEMEVALAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMDILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDAKEIMQHRFFANIVWQDVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTAStop
In green: the HA tag sequence
In black: the rat Akt sequence
In red: the mutated aa: K179D.
4.Blast for sequences combined from all primer reads:
Nucleotide Blast:
ref|NM_033230.1| Rattus norvegicus v-akt murine thymoma viral oncogene homolog 1 (Akt1), mRNA
dbj|D30040.1|RATRPKAA Rattus norvegicus mRNA for RAC protein kinase alpha, complete cds; Length=1617
GENE ID: 24185 Akt1 | v-akt murine thymoma viral oncogene homolog 1[Rattus norvegicus]
Score = 2892 bits (1566), Expect = 0.0
Identities = 1570/1572 (99%), Gaps = 0/1572 (0%)
Strand=Plus/Plus
Query 657 AACGACGTAGCCATTGTGAAGGAGGGCTGGCTGCACAAACGAGGGGAATATATTAAAACC 716
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 46 AACGACGTAGCCATTGTGAAGGAGGGCTGGCTGCACAAACGAGGGGAATATATTAAAACC 105
Query 717 TGGCGGCCACGCTACTTCCTCCTCAAGAATGATGGCACCTTTATTGGCTACAAGGAACGG 776
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 106 TGGCGGCCACGCTACTTCCTCCTCAAGAATGATGGCACCTTTATTGGCTACAAGGAACGG 165
Query 777 CCTCAGGATGTGGAGCAGCGCGAGTCCCCACTCAACAACTTCTCAGTGGCACAATGTCAG 836
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 166 CCTCAGGATGTGGAGCAGCGCGAGTCCCCACTCAACAACTTCTCAGTGGCACAATGTCAG 225
Query 837 CTGATGAAGACAGAGCGGCCGAGGCCCAACACCTTCATCATCCGCTGCCTGCAGTGGACC 896
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 226 CTGATGAAGACAGAGCGGCCGAGGCCCAACACCTTCATCATCCGCTGCCTGCAGTGGACC 285
Query 897 ACGGTCATTGAGCGCACCTTCCATGTGGAAACGCCTGAGGAGCGGGAAGAGTGGACCACC 956
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 286 ACGGTCATTGAGCGCACCTTCCATGTGGAAACGCCTGAGGAGCGGGAAGAGTGGACCACC 345
Query 957 GCCATTCAGACTGTGGCTGATGGACTCAAACGGCAGGAGGAGGAGACGATGGACTTCCGG 1016
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 346 GCCATTCAGACTGTGGCTGATGGACTCAAACGGCAGGAGGAGGAGACGATGGACTTCCGG 405
Query 1017 TCAGGTTCACCCAGTGACAACTCAGGTGCTGAGGAGATGGAGGTGGCCCTGGCCAAGCCC 1076
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 406 TCAGGTTCACCCAGTGACAACTCAGGTGCTGAGGAGATGGAGGTGGCCCTGGCCAAGCCC 465
Query 1077 AAGCACCGTGTGACCATGAACGAGTTTGAGTACCTGAAGCTACTGGGCAAGGGCACCTTT 1136
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 466 AAGCACCGTGTGACCATGAACGAGTTTGAGTACCTGAAGCTACTGGGCAAGGGCACCTTT 525
Query 1137 GGGAAGGTGATCCTGGTGAAGGAGAAGGCCACAGGTCGCTACTATGCCATGGATATCCTC 1196
||||||||||||||||||||||||||||||||||||||||||||||||||| | ||||||
Sbjct 526 GGGAAGGTGATCCTGGTGAAGGAGAAGGCCACAGGTCGCTACTATGCCATGAAGATCCTC 585
Query 1197 AAGAAGGAGGTCATCGTTGCCAAGGATGAGGTTGCCCACACGCTTACTGAGAACCGTGTC 1256
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 586 AAGAAGGAGGTCATCGTTGCCAAGGATGAGGTTGCCCACACGCTTACTGAGAACCGTGTC 645
Query 1257 CTGCAGAACTCTAGGCATCCCTTCCTTACAGCCCTCAAGTACTCATTCCAGACCCACGAC 1316
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 646 CTGCAGAACTCTAGGCATCCCTTCCTTACAGCCCTCAAGTACTCATTCCAGACCCACGAC 705
Query 1317 CGCCTCTGCTTTGTCATGGAGTACGCCAATGGAGGCGAGCTCTTCTTCCACCTGTCTCGT 1376
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 706 CGCCTCTGCTTTGTCATGGAGTACGCCAATGGAGGCGAGCTCTTCTTCCACCTGTCTCGT 765
Query 1377 GAGCGCGTGTTTTCAGAGGACCGGGCCCGCTTCTACGGTGCGGAGATTGTGTCCGCCCTG 1436
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 766 GAGCGCGTGTTTTCAGAGGACCGGGCCCGCTTCTACGGTGCGGAGATTGTGTCCGCCCTG 825
Query 1437 GACTACTTGCACTCCGAGAAGAACGTGGTGTACCGGGACCTCAAGCTGGAGAACCTCATG 1496
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 826 GACTACTTGCACTCCGAGAAGAACGTGGTGTACCGGGACCTCAAGCTGGAGAACCTCATG 885
Query 1497 CTGGACAAGGACGGGCACATCAAGATAACGGACTTCGGGCTGTGCAAGGAGGGTATCAAG 1556
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 886 CTGGACAAGGACGGGCACATCAAGATAACGGACTTCGGGCTGTGCAAGGAGGGTATCAAG 945
Query 1557 GACGGTGCCACCATGAAGACGTTCTGCGGGACACCCGAGTACCTGGCCCCTGAGGTGCTG 1616
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 946 GACGGTGCCACCATGAAGACGTTCTGCGGGACACCCGAGTACCTGGCCCCTGAGGTGCTG 1005
Query 1617 GAGGACAACGACTATGGCCGTGCAGTGGACTGGTGGGGGCTGGGCGTGGTCATGTACGAG 1676
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1006 GAGGACAACGACTATGGCCGTGCAGTGGACTGGTGGGGGCTGGGCGTGGTCATGTACGAG 1065
Query 1677 ATGATGTGCGGCCGCCTGCCCTTCTACAACCAGGACCATGAGAAGCTGTTCGAGCTCATC 1736
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1066 ATGATGTGCGGCCGCCTGCCCTTCTACAACCAGGACCATGAGAAGCTGTTCGAGCTCATC 1125
Query 1737 CTAATGGAGGAGATCCGCTTCCCACGCACACTCGGGCCGGAGGCCAAGTCCCTGCTCTCG 1796
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1126 CTAATGGAGGAGATCCGCTTCCCACGCACACTCGGGCCGGAGGCCAAGTCCCTGCTCTCG 1185
Query 1797 GGGCTGCTCAAGAAGGACCCTACACAGAGGCTCGGTGGGGGCTCCGAGGACGCCAAGGAG 1856
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1186 GGGCTGCTCAAGAAGGACCCTACACAGAGGCTCGGTGGGGGCTCCGAGGACGCCAAGGAG 1245
Query 1857 ATCATGCAGCACCGCTTCTTTGCCAACATCGTGTGGCAAGATGTGTATGAGAAGAAGCTG 1916
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1246 ATCATGCAGCACCGCTTCTTTGCCAACATCGTGTGGCAAGATGTGTATGAGAAGAAGCTG 1305
Query 1917 AGCCCACCTTTCAAGCCCCAGGTCACCTCTGAGACCGACACCAGGTATTTTGATGAGGAG 1976
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1306 AGCCCACCTTTCAAGCCCCAGGTCACCTCTGAGACCGACACCAGGTATTTTGATGAGGAG 1365
Query 1977 TTCACAGCTCAGATGATCACCATCACACCGCCTGATCAAGATGACAGCATGGAGTGTGTG 2036
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1366 TTCACAGCTCAGATGATCACCATCACACCGCCTGATCAAGATGACAGCATGGAGTGTGTG 1425
Query 2037 GACAGTGAACGGAGGCCGCACTTTCCCCAGTTCTCCTACTCAGCCAGTGGCACAGCCTGA 2096
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1426 GACAGTGAACGGAGGCCGCACTTTCCCCAGTTCTCCTACTCAGCCAGTGGCACAGCCTGA 1485
Query 2097 GGCCTGGGGTGGTGGCTGGCAGCCCTGCACCCCTCTGCATTGCCGAGTCCAGAAGCCCCG 2156
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1486 GGCCTGGGGTGGTGGCTGGCAGCCCTGCACCCCTCTGCATTGCCGAGTCCAGAAGCCCCG 1545
Query 2157 CATGGATCATCTGTACCTGATGGTTTTGTTTCTCGGATGTGCTGGGGAGGAACCTCGCCA 2216
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1546 CATGGATCATCTGTACCTGATGGTTTTGTTTCTCGGATGTGCTGGGGAGGAACCTCGCCA 1605
Query 2217 GCCTCGGAATTC 2228
||||||||||||
Sbjct 1606 GCCTCGGAATTC 1617
Protein Blast:
Query= combined sequence from the 4 primer reads (481R, 462R, 1191R, 1172F)
Subject= ref|NP_150233.1| sp|P47196.1|AKT1_RAT dbj|BAA06279.1| gb|EDL97422.1| GENE ID: 24185 Akt1 | RAC-alpha serine/threonine-protein kinase [Rattus norvegicus]; AltName: Full=Protein kinase B;PKB; thymoma viral proto-oncogene 1; v-akt murine thymoma viral oncogene homolog 1
Length=480
Score = 1000 bits (2585), Identities = 478/479 (99%), Positives = 478/479 (99%), Gaps = 0/479 (0%)
Query 1 NDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVEQRESPLNNFSVAQCQ 60
Sbjct 2 NDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVEQRESPLNNFSVAQCQ 61
Query 61 LMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGLKRQEEETMDFR 120
Sbjct 62 LMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGLKRQEEETMDFR 121
Query 121 SGSPSDNSGAEEMEVALAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMDIL 180
Sbjct 122 SGSPSDNSGAEEMEVALAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKIL 181
Query 181 KKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSR 240
Sbjct 182 KKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSR 241
Query 241 ERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIK 300
Sbjct 242 ERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIK 301
Query 301 DGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELI 360
Sbjct 302 DGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELI 361
Query 361 LMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDAKEIMQHRFFANIVWQDVYEKKL 420
Sbjct 362 LMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDAKEIMQHRFFANIVWQDVYEKKL 421
Query 421 SPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA 479
Sbjct 422 SPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA 480