LOCUS       sequence-173575-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eGFP-tagged human Akt2(T309A) phosphomutant.
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(T309A)
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     500..519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        774..1077
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1078..1281
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1232..1252
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1320..1329
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1326..1328,1329..1331,1332..2042)
                     /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(1371..1392)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1632..1651)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1979..2000
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3627..3748
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3664..3683)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3718..3737
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3755..4210)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3892..3913)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4104..4123
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4237..4341
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4343..4700
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4343..4534
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4366..4386)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4551..4686
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4735..5529
                     /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             4735..5529
                     /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(4789..4808)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5399..5418
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5717..5736)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5761..5808
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
       61 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
      121 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
      181 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
      241 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
      301 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
      361 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      421 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      481 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      541 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      601 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      661 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat
      721 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      781 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      841 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      901 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      961 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     1021 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     1081 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     1141 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     1201 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     1261 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg
     1321 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1381 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1441 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1501 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1561 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1621 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1681 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1741 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1801 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1861 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1921 accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     1981 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2041 agtccggact cagatctcga gctatgaatg aggtgtctgt catcaaagaa ggctggctcc
     2101 acaagcgtgg tgaatacatc aagacctgga ggccacggta cttcctgctg aagagcgacg
     2161 gctccttcat tgggtacaag gagaggcccg aggcccctga tcagactcta ccccccttaa
     2221 acaacttctc cgtagcagaa tgccagctga tgaagaccga gaggccgcga cccaacacct
     2281 ttgtcatacg ctgcctgcag tggaccacag tcatcgagag gaccttccac gtggattctc
     2341 cagacgagag ggaggagtgg atgcgggcca tccagatggt cgccaacagc ctcaagcagc
     2401 gggccccagg cgaggacccc atggactaca agtgtggctc ccccagtgac tcctccacga
     2461 ctgaggagat ggaagtggcg gtcagcaagg cacgggctaa agtgaccatg aatgacttcg
     2521 actatctcaa actccttggc aagggaacct ttggcaaagt catcctggtg cgggagaagg
     2581 ccactggccg ctactacgcc atgaagatcc tgcggaagga agtcatcatt gccaaggatg
     2641 aagtcgctca cacagtcacc gagagccggg tcctccagaa caccaggcac ccgttcctca
     2701 ctgcgctgaa gtatgccttc cagacccacg accgcctgtg ctttgtgatg gagtatgcca
     2761 acgggggtga gctgttcttc cacctgtccc gggagcgtgt cttcacagag gagcgggccc
     2821 ggttttatgg tgcagagatt gtctcggctc ttgagtactt gcactcgcgg gacgtggtat
     2881 accgcgacat caagctggaa aacctcatgc tggacaaaga tggccacatc aagatcactg
     2941 actttggcct ctgcaaagag ggcatcagtg acggggccac catgaaagcc ttctgtggga
     3001 ccccggagta cctggcgcct gaggtgctgg aggacaatga ctatggccgg gccgtggact
     3061 ggtgggggct gggtgtggtc atgtacgaga tgatgtgcgg ccgcctgccc ttctacaacc
     3121 aggaccacga gcgcctcttc gagctcatcc tcatggaaga gatccgcttc ccgcgcacgc
     3181 tcagccccga ggccaagtcc ctgcttgctg ggctgcttaa gaaggacccc aagcagaggc
     3241 ttggtggggg gcccagcgat gccaaggagg tcatggagca caggttcttc ctcagcatca
     3301 actggcagga cgtggtccag aagaagctcc tgccaccctt caaacctcag gtcacgtccg
     3361 aggtcgacac aaggtacttc gatgatgaat ttaccgccca gtccatcaca atcacacccc
     3421 ctgaccgcta tgacagcctg ggcttactgg agctggacca gcggacccac ttcccccagt
     3481 tctcctactc ggccagcatc cgcgagtgat ctagataact gatcataatc agccatacca
     3541 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     3601 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3661 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3721 gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta
     3781 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     3841 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     3901 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     3961 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4021 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4081 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4141 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4201 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4261 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4321 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     4381 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4441 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4501 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4561 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4621 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4681 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4741 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4801 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4861 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     4921 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4981 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     5041 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     5101 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5161 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5221 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     5281 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5341 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5401 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5461 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5521 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5581 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5641 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5701 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     5761 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     5821 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     5881 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     5941 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     6001 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6061 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6121 agatca
//
