LOCUS       sequence-173564-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eGFP-tagged human Akt2(K14A/R25E) membrane-binding mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6126)
  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 6126)
  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-Akt2(K14A/R25E)
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      26..614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     515..534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        789..1092
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1093..1296
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1247..1267
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1335..1344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1341..1343,1344..1346,1347..2057)
                     /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(1386..1407)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1647..1666)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1994..2015
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3642..3763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3679..3698)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3733..3752
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3770..4225)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3907..3928)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4119..4138
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4252..4356
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4358..4715
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4358..4549
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4381..4401)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4566..4701
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4750..5544
                     /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             4750..5544
                     /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(4804..4823)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5414..5433
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5732..5751)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5776..5823
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
       61 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      121 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      181 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      241 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      301 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      361 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      421 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      481 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      541 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      601 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      661 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      721 ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      781 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      841 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      901 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      961 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     1021 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     1081 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     1141 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     1201 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     1261 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc
     1321 tagcgctacc ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1381 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1441 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1501 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1561 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1621 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1681 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1741 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     1801 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1861 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1921 tgctgctgcc cgacaaccac tacctgagca cccagtccaa gctgagcaaa gaccccaacg
     1981 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2041 tggacgagct gtacaagtcc ggactcagat ctcgagctat gaatgaggtg tctgtcatca
     2101 aagaaggctg gctccacgca cgtggtgaat acatcaagac ctggaggcca gagtacttcc
     2161 tgctgaagag cgacggctcc ttcattgggt acaaggagag gcccgaggcc cctgatcaga
     2221 ctctaccccc cttaaacaac ttctccgtag cagaatgcca gctgatgaag accgagaggc
     2281 cgcgacccaa cacctttgtc atacgctgcc tgcagtggac cacagtcatc gagaggacct
     2341 tccacgtgga ttctccagac gagagggagg agtggatgcg ggccatccag atggtcgcca
     2401 acagcctcaa gcagcgggcc ccaggcgagg accccatgga ctacaagtgt ggctccccca
     2461 gtgactcctc cacgactgag gagatggaag tggcggtcag caaggcacgg gctaaagtga
     2521 ccatgaatga cttcgactat ctcaaactcc ttggcaaggg aacctttggc aaagtcatcc
     2581 tggtgcggga gaaggccact ggccgctact acgccatgaa gatcctgcgg aaggaagtca
     2641 tcattgccaa ggatgaagtc gctcacacag tcaccgagag ccgggtcctc cagaacacca
     2701 ggcacccgtt cctcactgcg ctgaagtatg ccttccagac ccacgaccgc ctgtgctttg
     2761 tgatggagta tgccaacggg ggtgagctgt tcttccacct gtcccgggag cgtgtcttca
     2821 cagaggagcg ggcccggttt tatggtgcag agattgtctc ggctcttgag tacttgcact
     2881 cgcgggacgt ggtataccgc gacatcaagc tggaaaacct catgctggac aaagatggcc
     2941 acatcaagat cactgacttt ggcctctgca aagagggcat cagtgacggg gccaccatga
     3001 aaaccttctg tgggaccccg gagtacctgg cgcctgaggt gctggaggac aatgactatg
     3061 gccgggccgt ggactggtgg gggctgggtg tggtcatgta cgagatgatg tgcggccgcc
     3121 tgcccttcta caaccaggac cacgagcgcc tcttcgagct catcctcatg gaagagatcc
     3181 gcttcccgcg cacgctcagc cccgaggcca agtccctgct tgctgggctg cttaagaagg
     3241 accccaagca gaggcttggt ggggggccca gcgatgccaa ggaggtcatg gagcacaggt
     3301 tcttcctcag catcaactgg caggacgtgg tccagaagaa gctcctgcca cccttcaaac
     3361 ctcaggtcac gtccgaggtc gacacaaggt acttcgatga tgaatttacc gcccagtcca
     3421 tcacaatcac accccctgac cgctatgaca gcctgggctt actggagctg gaccagcgga
     3481 cccacttccc ccagttctcc tactcggcca gcatccgcga gtgatctaga taactgatca
     3541 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     3601 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     3661 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3721 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc
     3781 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3841 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3901 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3961 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     4021 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     4081 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     4141 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     4201 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4261 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4321 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     4381 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4441 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     4501 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4561 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4621 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4681 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     4741 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4801 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4861 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4921 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4981 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     5041 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     5101 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     5161 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     5221 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     5281 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5341 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     5401 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     5461 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5521 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5581 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5641 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5701 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     5761 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     5821 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     5881 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     5941 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     6001 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6061 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6121 cagacc
//
