LOCUS       sequence-181815-        6123 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eGFP-tagged human Akt1(D323A/D325A) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6123)
  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 6123)
  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: pEGFP-Akt1(D323A/D325A)
FEATURES             Location/Qualifiers
     source          1..6123
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     506..525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        780..1083
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1084..1287
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1238..1258
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1326..1335
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1332..1334,1335..1337,1338..2048)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1377..1398)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1638..1657)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1985..2006
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2070..3509
                     /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
                     ECVDSERRPHFPQFSYSASGTA"
     polyA_signal    3630..3751
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3667..3686)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3721..3740
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3758..4213)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3895..3916)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4107..4126
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4240..4344
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4346..4703
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4346..4537
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4369..4389)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4554..4689
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4738..5532
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4738..5532
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4792..4811)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5402..5421
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5720..5739)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5764..5811
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcatt
      721 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      781 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      841 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      901 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      961 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1021 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1081 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
     1141 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
     1201 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
     1261 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg ctagcgctac
     1321 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1381 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1441 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1501 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1561 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1621 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1681 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1741 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1801 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1861 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1921 ccgacaacca ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg
     1981 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2041 tgtacaagtc cggactcaga tctcgagcta tgagcgacgt ggctattgtg aaggagggtt
     2101 ggctgcacaa acgaggggag tacatcaaga cctggcggcc acgctacttc ctcctcaaga
     2161 atgatggcac cttcattggc tacaaggagc ggccgcagga tgtggaccaa cgtgaggctc
     2221 ccctcaacaa cttctctgtg gcgcagtgcc agctgatgaa gacggagcgg ccccggccca
     2281 acaccttcat catccgctgc ctgcagtgga ccactgtcat cgaacgcacc ttccatgtgg
     2341 agactcctga ggagcgggag gagtggacaa ccgccatcca gactgtggct gacggcctca
     2401 agaagcagga ggaggaggag atggacttcc ggtcgggctc acccagtgac aactcagggg
     2461 ctgaagagat ggaggtgtcc ctggccaagc ccaagcaccg cgtgaccatg aacgagtttg
     2521 agtacctgaa gctgctgggc aagggcactt tcggcaaggt gatcctggtg aaggagaagg
     2581 ccacaggccg ctactacgcc atgaagatcc tcaagaagga agtcatcgtg gccaaggacg
     2641 aggtggccca cacactcacc gagaaccgcg tcctgcagaa ctccaggcac cccttcctca
     2701 cagccctgaa gtactctttc cagacccacg accgcctctg ctttgtcatg gagtacgcca
     2761 acgggggcga gctgttcttc cacctgtccc gggagcgtgt gttctccgag gaccgggccc
     2821 gcttctatgg cgctgagatt gtgtcagccc tggactacct gcactcggag aagaacgtgg
     2881 tgtaccggga cctcaagctg gagaacctca tgctggacaa ggacgggcac attaagatca
     2941 cagacttcgg gctgtgcaag gaggggatca aggacggtgc caccatgaag accttttgcg
     3001 gcacacctga gtacctggcc cccgaggtgc tggaggccaa tgcctacggc cgtgcagtgg
     3061 actggtgggg gctgggcgtg gtcatgtacg agatgatgtg cggtcgcctg cccttctaca
     3121 accaggacca tgagaagctt tttgagctca tcctcatgga ggagatccgc ttcccgcgca
     3181 cgcttggtcc cgaggccaag tccttgcttt cagggctgct caagaaggac cccaagcaga
     3241 ggcttggcgg gggctccgag gacgccaagg agatcatgca gcatcgcttc tttgccggta
     3301 tcgtgtggca gcacgtgtac gagaagaagc tcagcccacc cttcaagccc caggtcacgt
     3361 cggagactga caccaggtat tttgatgagg agttcacggc ccagatgatc accatcacac
     3421 cacctgacca agatgacagc atggagtgtg tggacagcga gcgcaggccc cacttccccc
     3481 agttctccta ctcggccagc ggcacggcct gatctagata actgatcata atcagccata
     3541 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     3601 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     3661 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3721 gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg
     3781 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     3841 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     3901 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     3961 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4021 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4081 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4141 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4201 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4261 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4321 caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     4381 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4441 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4501 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4561 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4621 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4681 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     4741 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4801 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4861 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     4921 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4981 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     5041 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     5101 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     5161 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     5221 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     5281 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     5341 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     5401 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     5461 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5521 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     5581 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     5641 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     5701 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     5761 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     5821 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     5881 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     5941 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
     6001 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6061 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6121 aaa
//
