LOCUS       sequence-173559-        8381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human full-length Akt1(D323A/D325A) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8381)
  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 8381)
  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(D323A/D325A)
FEATURES             Location/Qualifiers
     source          1..8381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(475..480,481..497,498..503))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    1011..1059
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1084..1103
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(2848..2957)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        2981..3072
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     2997..3014
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             3121..3150
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3157..3180
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3190..3210
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     CDS             3214..4653
                     /codon_start=1
                     /gene="AKT1"
                     /product="human serine/threonine protein kinase activated 
                     by growth factors"
                     /label=Akt1
                     /translation="MSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDV
                     DQREAPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQ
                     TVADGLKKQEEEEMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGK
                     VILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDR
                     LCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLM
                     LDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEANAYGRAVDWWGLGVVMY
                     EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDA
                     KEIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSM
                     ECVDSERRPHFPQFSYSASSTA"
     polyA_signal    4822..4956
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4859..4878)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4913..4932
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  4985..5150
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      5334..5788
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5421..5440)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5630..5651
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5814..5918
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5919..5987,5988..6779)
                     /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(6137..6156)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6950..7538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7439..7458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7692..7709
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  7843..8067
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(join(8134..8381,1..286))
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 gaacgtaagc cgcgagagcg ccaacaaccg cttcttggtc gaaggcagca agcgcgatga
       61 atgtcttact acggagcaag ttcccgaggt aatcggagtc cggctgatgt tgggagtagg
      121 tggctacgtc tccgaactca cgaccgaaaa gatcaagagc agcccgcatg gatttgactt
      181 ggtcagggcc gagcctacat gtgcgaatga tgcccatact tgagccacct aactttgttt
      241 tagggcgact gccctgctgc gtaacatcgt tgctgctgcg taacatcgtt gctgctccat
      301 aacatcaaac atcgacccac ggcgtaacgc gcttgctgct tggatgcccg aggcatagac
      361 tgtacaaaaa aacagtcata acaagccatg aaaaccgcca ctgcgccgtt accaccgctg
      421 cgttcggtca aggttctgga ccagttgcgt gagcgcatac gctacttgca ttacagttta
      481 cgaaccgaac aggcttatgt caactgggtt cgtgccttca tccgtttcca cggtgtgcgt
      541 cacccggcaa ccttgggcag cagcgaagtc gaggcatttc tgtcctggct ggcgaacgag
      601 cgcaaggttt cggtctccac gcatcgtcag gcattggcgg ccttgctgtt cttctacggc
      661 aaggtgctgt gcacggatct gcccttgctt caggagatcg gtagacctcg gccgtcgcgg
      721 cgcttgccgg tggtgctgac cccggatgaa gtggttcgca tcctcggttt tctggaaggc
      781 gagcatcgtt tgttcgccca ggactctagc tatagttcta gtggttggct acgtacccgt
      841 agtggctatg gcagggcttg ccgccccgac gttggctgcg agccctgggc cttcacccga
      901 acttgggggt tggggtgggg aaaaggaaga aacgcgggcg tattggtccc aatggggtct
      961 cggtggggta tcgacagagt gccagccctg ggaccgaacc ccgcgtttat gaacaaacga
     1021 cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg gttccttccg
     1081 gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccggtacct cactgcacag
     1141 cggcgtccgg gtggctctgg tatcgctgcc tccaaacctc ctggatcttc ctgcaccact
     1201 tgtgtgcgtt cccgctgggg tccatcagat aatacgtcct gttaggcgtg tggacaaaga
     1261 aagttttaaa attcttggcc tctggtcgaa gttcttgtga ccaaggaatt tcacctttca
     1321 gaactttgtt gacaggatcc acataatata aatgtggtcc ttctgtgagc aacagctgtc
     1381 gtcgtcttgc aaataaaccc ttccgcttat ccactgggcc catctttagt attaaattat
     1441 tttctacaaa ctggtgccaa gggtttccgc cagcctgctt ctccaacaac aacctcttct
     1501 catcttcgga aaactgtaag tccagttcaa aggagttcga gtccagatcg tgaatgtact
     1561 gctctatgtt gctgcctgac ctctggggca ggcccgtgtc ggaggctgac agggagtgtg
     1621 aggaggagga cgaagacacc tgcatgcagc caaactggct caggagattg tcataattgc
     1681 catagcagtc ctcgtcgtct tccgacatag ccggcaggta agcggtgagc ttcggaggcg
     1741 tctgctggtg caggttctcc cacgtgacgg actcgaagaa cgggtgtgct ttaagaggtc
     1801 cgtatccttc catttcctca cagcctaacc gctttgtggc atctaaaacc aaaagtttct
     1861 ccacgaggtc tcttgcctta gggaagaatt tttctggaaa gtcatattcc aacttaatga
     1921 tcttctgaaa tataagatac tcgtttccag ctcggaatgg tgggagtcct gccacaagct
     1981 ggtatattat gcatccaaga gcccaaaggt ctgaactctt acaggcggac ttctccgtga
     2041 gcagctctgg agaaacgtac tgcgctgttc ccacgaatga gttggccctg gcttgtttgc
     2101 tctctgggga taagactttt gctgttccaa aatctgtgat ctggatgtgc atatcttcat
     2161 ttaacaaaat gttttccggt ttaaggtccc tgtgaatgat gcccttgccg tgcaagtact
     2221 ctaaagcaga cacaatctca gccgtgtaaa atcgggtaca ggtctcatcg aatgaaccga
     2281 ttttgcgaat atatttaagt agttctccat ttttggcata actaaggccg aaatacagct
     2341 tctcgtcgtc ctgaaatgtg aagtaaagct taacaaagaa ggggtgatcc aggcgcgaca
     2401 tgacatcccg ctctctggtt acatagggga ccttgttctc ttttatgata tgtcgcttct
     2461 ccagaatttt aatcgcatat tctctggagg ttgccagttc tcgagccagg acaaccgtgg
     2521 aaaaagagcc ttccccaagg attttcccaa acttgaagtc ctcaggccgc ttcttccgag
     2581 gctgcggcgg aggctgggca tgctgcaggg agccggcgcc gggccgaggc tcggctgcag
     2641 tgccgtccat ggcggggccc tgcctgctgc caccaggaat gccagggggc gtgctggact
     2701 cagtctgggt cctcaccatt gatggggatg ggcaggaaca taacaccacg ctggactgga
     2761 tgggcacggc gtcatacagc tggctggtgg tcctggccat gctagcacca tggctcgaga
     2821 tcccgggtga tcaagtcttc gtcgagtgat tgtaaataaa atgtaattta cagtatagta
     2881 ttttaattaa tatacaaatg atttgataat aattcttatt taactataat atattgtgtt
     2941 gggttgaatt aaaggtccgt atactccgga atattaatag atcatggaga taattaaaat
     3001 gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt tgtaataaaa
     3061 aaacctataa atattccgga ttattcatac cgtcccacca tcgggcgcgg atccatgtcg
     3121 caccatcacc atcaccatca ccatcaccat gcaagctgga gccacccgca gttcgaaaag
     3181 ggctccggtg aaaacctgta tttccaggcg cgcatgagcg acgtggctat tgtgaaggag
     3241 ggttggctgc acaaacgagg ggagtacatc aagacctggc ggccacgcta cttcctcctc
     3301 aagaatgatg gcaccttcat tggctacaag gagaggccgc aggatgtgga ccaacgtgag
     3361 gctcccctca acaacttctc tgtggcgcag tgccagctga tgaagacgga gcggccccgg
     3421 cccaacacct tcatcatccg ctgcctgcag tggaccactg tcatcgaacg caccttccat
     3481 gtggagactc ctgaggagcg ggaggagtgg acaaccgcca tccagactgt ggctgacggc
     3541 ctcaagaagc aggaggagga ggagatggac ttccggtcgg gctcacccag tgacaactca
     3601 ggggctgaag agatggaggt gtccctggcc aagcccaagc accgcgtgac catgaacgag
     3661 tttgagtacc tgaagctgct gggcaagggc actttcggca aggtgatcct ggtgaaggag
     3721 aaggccacag gccgctacta cgccatgaag atcctcaaga aggaagtcat cgtggccaag
     3781 gacgaggtgg cccacacact caccgagaac cgcgtcctgc agaactccag gcaccccttc
     3841 ctcacagccc tgaagtactc tttccagacc cacgaccgcc tctgctttgt catggagtac
     3901 gccaacgggg gcgagctgtt cttccacctg tcccgggagc gtgtgttctc cgaggaccgg
     3961 gcccgcttct atggcgctga gattgtgtca gccctggact acctgcactc ggagaagaac
     4021 gtggtgtacc gggacctcaa gctggagaac ctcatgctgg acaaggacgg gcacattaag
     4081 atcacagact tcgggctgtg caaggagggg atcaaggacg gtgccaccat gaagaccttt
     4141 tgcggcacac ctgagtacct ggcccccgag gtgctggagg ccaatgccta cggccgtgca
     4201 gtggactggt gggggctggg cgtggtcatg tacgagatga tgtgcggtcg cctgcccttc
     4261 tacaaccagg accatgagaa gctttttgag ctcatcctca tggaggagat ccgcttcccg
     4321 cgcacgcttg gtcccgaggc caagtccttg ctttcagggc tgctcaagaa ggaccccaag
     4381 cagaggcttg gcgggggctc cgaggacgcc aaggagatca tgcagcatcg cttctttgcc
     4441 ggtatcgtgt ggcagcacgt gtacgagaag aagctcagcc cacccttcaa gccccaggtc
     4501 acgtcggaga ctgacaccag gtattttgat gaggagttca cggcccagat gatcaccatc
     4561 acaccacctg accaagatga cagcatggag tgtgtggaca gcgagcgcag gccccacttc
     4621 ccccagttct cctactcggc cagcagcacg gcctgagcgg ccgctttcga atctagagcc
     4681 tgcagtctcg acaagcttgt cgagaagtac tagaggatca taatcagcca taccacattt
     4741 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     4801 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     4861 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4921 tccaaactca tcaatgtatc ttatcatgtc tggatctgat cactgcttga gcctaggaga
     4981 tccgaaccag ataagtgaaa tctagttcca aactattttg tcatttttaa ttttcgtatt
     5041 agcttacgac gctacaccca gttcccatct attttgtcac tcttccctaa ataatcctta
     5101 aaaactccat ttccacccct cccagttccc aactattttg tccgcccaca gcggggcatt
     5161 tttcttcctg ttatgttttt aatcaaacat cctgccaact ccatgtgaca aaccgtcatc
     5221 ttcggctact ttttctctgt cacagaatga aaatttttct gtcatctctt cgttattaat
     5281 gtttgtaatt gactgaatat caacgcttat ttgcagcctg aatggcgaat gggacgcgcc
     5341 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     5401 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     5461 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     5521 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     5581 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttcttaatag tggactcttg
     5641 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     5701 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5761 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     5821 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5881 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5941 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6001 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6061 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6121 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6181 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6241 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6301 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6361 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6421 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6481 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6541 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6601 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6661 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6721 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6781 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6841 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6901 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6961 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7021 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7081 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7141 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7201 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7261 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7321 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7381 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7441 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7501 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7561 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     7621 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     7681 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     7741 tccttacgca tctgtgcggt atttcacacc gcatagacca gccgcgtaac ctggcaaaat
     7801 cggttacggt tgagtaataa atggatgccc tgcgtaagcg ggtgtgggcg gacaataaag
     7861 tcttaaactg aacaaaatag atctaaacta tgacaataaa gtcttaaact agacagaata
     7921 gttgtaaact gaaatcagtc cagttatgct gtgaaaaagc atactggact tttgttatgg
     7981 ctaaagcaaa ctcttcattt tctgaagtgc aaattgcccg tcgtattaaa gaggggcgtg
     8041 gccaagggca tggtaaagac tatattcgcg gcgttgtgac aatttaccga acaactccgc
     8101 ggccgggaag ccgatctcgg cttgaacgaa ttgttaggtg gcggtacttg ggtcgatatc
     8161 aaagtgcatc acttcttccc gtatgcccaa ctttgtatag agagccactg cgggatcgtc
     8221 accgtaatct gcttgcacgt agatcacata agcaccaagc gcgttggcct catgcttgag
     8281 gagattgatg agcgcggtgg caatgccctg cctccggtgc tcgccggaga ctgcgagatc
     8341 atagatatag atctcactac gcggctgctc aaacttgggc a
//
