LOCUS       sequence-173555-        6728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human full-length Akt1(T308A/S473A) phosphomutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6728)
  AUTHORS   Ebner M, Lucic I, Leonard TA, Yudushkin I
  TITLE     PI(3,4,5)P3 Engagement Restricts Akt Activity to Cellular Membranes.
  JOURNAL   Mol Cell. 2017 Feb 2;65(3):416-431.e6. doi: 
            10.1016/j.molcel.2016.12.028.
  PUBMED    28157504
REFERENCE   2  (bases 1 to 6728)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2016.12"; journalName: "Mol Cell"; date: 
            "2017-02-2- 2"; volume: "65"; issue: "3"; pages: "416-431.e6"
COMMENT     Sequence Label: pFBD-Akt1(T308A/S473A)
FEATURES             Location/Qualifiers
     source          1..6728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      19..607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     508..527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     761..778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  912..1136
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(1203..1736)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1925..1930,1931..1947,1948..1953))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    2461..2509
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2534..2553
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(2645..2754)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        2778..2869
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     2794..2811
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             2918..2947
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2954..2977
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2987..3007
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     CDS             3011..4450
                     /codon_start=1
                     /gene="AKT1"
                     /product="human serine/threonine protein kinase activated 
                     by growth factors"
                     /label=Akt1
                     /translation="MSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDV
                     DQREAPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQ
                     TVADGLKKQEEEEMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGK
                     VILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDR
                     LCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLM
                     LDKDGHIKITDFGLCKEGIKDGATMKAFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMY
                     EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDA
                     KEIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSM
                     ECVDSERRPHFPQFAYSASSTA"
     polyA_signal    4619..4753
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4656..4675)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4710..4729
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  4782..4947
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      5131..5585
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5218..5237)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5427..5448
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5611..5715
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5716..5784,5785..6576)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5934..5953)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
       61 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
      121 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      181 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      241 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      301 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      361 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      421 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      481 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      541 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      601 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      661 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      721 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      781 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      841 catagaccag ccgcgtaacc tggcaaaatc ggttacggtt gagtaataaa tggatgccct
      901 gcgtaagcgg gtgtgggcgg acaataaagt cttaaactga acaaaataga tctaaactat
      961 gacaataaag tcttaaacta gacagaatag ttgtaaactg aaatcagtcc agttatgctg
     1021 tgaaaaagca tactggactt ttgttatggc taaagcaaac tcttcatttt ctgaagtgca
     1081 aattgcccgt cgtattaaag aggggcgtgg ccaagggcat ggtaaagact atattcgcgg
     1141 cgttgtgaca atttaccgaa caactccgcg gccgggaagc cgatctcggc ttgaacgaat
     1201 tgttaggtgg cggtacttgg gtcgatatca aagtgcatca cttcttcccg tatgcccaac
     1261 tttgtataga gagccactgc gggatcgtca ccgtaatctg cttgcacgta gatcacataa
     1321 gcaccaagcg cgttggcctc atgcttgagg agattgatga gcgcggtggc aatgccctgc
     1381 ctccggtgct cgccggagac tgcgagatca tagatataga tctcactacg cggctgctca
     1441 aacttgggca gaacgtaagc cgcgagagcg ccaacaaccg cttcttggtc gaaggcagca
     1501 agcgcgatga atgtcttact acggagcaag ttcccgaggt aatcggagtc cggctgatgt
     1561 tgggagtagg tggctacgtc tccgaactca cgaccgaaaa gatcaagagc agcccgcatg
     1621 gatttgactt ggtcagggcc gagcctacat gtgcgaatga tgcccatact tgagccacct
     1681 aactttgttt tagggcgact gccctgctgc gtaacatcgt tgctgctgcg taacatcgtt
     1741 gctgctccat aacatcaaac atcgacccac ggcgtaacgc gcttgctgct tggatgcccg
     1801 aggcatagac tgtacaaaaa aacagtcata acaagccatg aaaaccgcca ctgcgccgtt
     1861 accaccgctg cgttcggtca aggttctgga ccagttgcgt gagcgcatac gctacttgca
     1921 ttacagttta cgaaccgaac aggcttatgt caactgggtt cgtgccttca tccgtttcca
     1981 cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc gaggcatttc tgtcctggct
     2041 ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag gcattggcgg ccttgctgtt
     2101 cttctacggc aaggtgctgt gcacggatct gcccttgctt caggagatcg gtagacctcg
     2161 gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa gtggttcgca tcctcggttt
     2221 tctggaaggc gagcatcgtt tgttcgccca ggactctagc tatagttcta gtggttggct
     2281 acgtacccgt agtggctatg gcagggcttg ccgccccgac gttggctgcg agccctgggc
     2341 cttcacccga acttgggggt tggggtgggg aaaaggaaga aacgcgggcg tattggtccc
     2401 aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc ccgcgtttat
     2461 gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg
     2521 gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccggtaccg
     2581 catgctatgc atcagctgct agcaccatgg ctcgagatcc cgggtgatca agtcttcgtc
     2641 gagtgattgt aaataaaatg taatttacag tatagtattt taattaatat acaaatgatt
     2701 tgataataat tcttatttaa ctataatata ttgtgttggg ttgaattaaa ggtccgtata
     2761 ctccggaata ttaatagatc atggagataa ttaaaatgat aaccatctcg caaataaata
     2821 agtattttac tgttttcgta acagttttgt aataaaaaaa cctataaata ttccggatta
     2881 ttcataccgt cccaccatcg ggcgcggatc catgtcgcac catcaccatc accatcacca
     2941 tcaccatgca agctggagcc acccgcagtt cgaaaagggc tccggtgaaa acctgtattt
     3001 ccaggcgcgc atgagcgacg tggctattgt gaaggagggt tggctgcaca aacgagggga
     3061 gtacatcaag acctggcggc cacgctactt cctcctcaag aatgatggca ccttcattgg
     3121 ctacaaggag aggccgcagg atgtggacca acgtgaggct cccctcaaca acttctctgt
     3181 ggcgcagtgc cagctgatga agacggagcg gccccggccc aacaccttca tcatccgctg
     3241 cctgcagtgg accactgtca tcgaacgcac cttccatgtg gagactcctg aggagcggga
     3301 ggagtggaca accgccatcc agactgtggc tgacggcctc aagaagcagg aggaggagga
     3361 gatggacttc cggtcgggct cacccagtga caactcaggg gctgaagaga tggaggtgtc
     3421 cctggccaag cccaagcacc gcgtgaccat gaacgagttt gagtacctga agctgctggg
     3481 caagggcact ttcggcaagg tgatcctggt gaaggagaag gccacaggcc gctactacgc
     3541 catgaagatc ctcaagaagg aagtcatcgt ggccaaggac gaggtggccc acacactcac
     3601 cgagaaccgc gtcctgcaga actccaggca ccccttcctc acagccctga agtactcttt
     3661 ccagacccac gaccgcctct gctttgtcat ggagtacgcc aacgggggcg agctgttctt
     3721 ccacctgtcc cgggagcgtg tgttctccga ggaccgggcc cgcttctatg gcgctgagat
     3781 tgtgtcagcc ctggactacc tgcactcgga gaagaacgtg gtgtaccggg acctcaagct
     3841 ggagaacctc atgctggaca aggacgggca cattaagatc acagacttcg ggctgtgcaa
     3901 ggaggggatc aaggacggtg ccaccatgaa ggccttttgc ggcacacctg agtacctggc
     3961 ccccgaggtg ctggaggaca atgactacgg ccgtgcagtg gactggtggg ggctgggcgt
     4021 ggtcatgtac gagatgatgt gcggtcgcct gcccttctac aaccaggacc atgagaagct
     4081 ttttgagctc atcctcatgg aggagatccg cttcccgcgc acgcttggtc ccgaggccaa
     4141 gtccttgctt tcagggctgc tcaagaagga ccccaagcag aggcttggcg ggggctccga
     4201 ggacgccaag gagatcatgc agcatcgctt ctttgccggt atcgtgtggc agcacgtgta
     4261 cgagaagaag ctcagcccac ccttcaagcc ccaggtcacg tcggagactg acaccaggta
     4321 ttttgatgag gagttcacgg cccagatgat caccatcaca ccacctgacc aagatgacag
     4381 catggagtgt gtggacagcg agcgcaggcc ccacttcccc cagttcgcct actcggccag
     4441 cagcacggcc tgagcggccg ctttcgaatc tagagcctgc agtctcgaca agcttgtcga
     4501 gaagtactag aggatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     4561 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     4621 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4681 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4741 tcatgtctgg atctgatcac tgcttgagcc taggagatcc gaaccagata agtgaaatct
     4801 agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct acacccagtt
     4861 cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc cacccctccc
     4921 agttcccaac tattttgtcc gcccacagcg gggcattttt cttcctgtta tgtttttaat
     4981 caaacatcct gccaactcca tgtgacaaac cgtcatcttc ggctactttt tctctgtcac
     5041 agaatgaaaa tttttctgtc atctcttcgt tattaatgtt tgtaattgac tgaatatcaa
     5101 cgcttatttg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     5161 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5221 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5281 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5341 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5401 tgacgttgga gtccacgttc ttaatagtgg actcttgttc caaactggaa caacactcaa
     5461 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5521 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5581 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5641 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5701 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5761 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5821 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5881 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5941 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     6001 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     6061 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6121 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6181 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6241 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6301 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6361 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6421 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6481 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6541 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6601 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6661 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6721 cccgtaga
//
