LOCUS       sequence-181806-        6477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses recruitable mCherry-tagged human Akt2(K14A) 
            membrane-binding mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6477)
  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 6477)
  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: pFKBPmCherry-Akt2(K14A)
FEATURES             Location/Qualifiers
     source          1..6477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      6..594
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     495..514
                     /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"
     CDS             join(1321..1323,1324..1644)
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             1669..2376
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1814..1832)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2082..2101
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3973..4094
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4010..4029)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4064..4083
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4101..4556)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4238..4259)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4450..4469
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4583..4687
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4689..5046
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4689..4880
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4712..4732)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4897..5032
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5081..5875
                     /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             5081..5875
                     /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(5135..5154)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5745..5764
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6063..6082)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6107..6154
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
       61 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
      121 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
      181 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
      241 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
      301 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      361 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      421 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      481 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      541 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      601 agcaacgcgg cctttttacg gttcctggcc ttttgctggc 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 ctagcccacc
     1321 atgggagtgc aggtggagac tatctcccca ggagacgggc gcaccttccc caagcgcggc
     1381 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaagt tgattcctcc
     1441 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg
     1501 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat
     1561 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc
     1621 gatgtggagc ttctaaaact ggaaggtgct acgcgtctgc ccggttctat ggtgagcaag
     1681 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     1741 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1801 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     1861 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     1921 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     1981 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     2041 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     2101 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     2161 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     2221 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     2281 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     2341 cgccactcca ccggcggcat ggacgagctg tacaagtccg gactcagatc tcgagctatg
     2401 aatgaggtgt ctgtcatcaa agaaggctgg ctccacgcac gtggtgaata catcaagacc
     2461 tggaggccac ggtacttcct gctgaagagc gacggctcct tcattgggta caaggagagg
     2521 cccgaggccc ctgatcagac tctacccccc ttaaacaact tctccgtagc agaatgccag
     2581 ctgatgaaga ccgagaggcc gcgacccaac acctttgtca tacgctgcct gcagtggacc
     2641 acagtcatcg agaggacctt ccacgtggat tctccagacg agagggagga gtggatgcgg
     2701 gccatccaga tggtcgccaa cagcctcaag cagcgggccc caggcgagga ccccatggac
     2761 tacaagtgtg gctcccccag tgactcctcc acgactgagg agatggaagt ggcggtcagc
     2821 aaggcacggg ctaaagtgac catgaatgac ttcgactatc tcaaactcct tggcaaggga
     2881 acctttggca aagtcatcct ggtgcgggag aaggccactg gccgctacta cgccatgaag
     2941 atcctgcgga aggaagtcat cattgccaag gatgaagtcg ctcacacagt caccgagagc
     3001 cgggtcctcc agaacaccag gcacccgttc ctcactgcgc tgaagtatgc cttccagacc
     3061 cacgaccgcc tgtgctttgt gatggagtat gccaacgggg gtgagctgtt cttccacctg
     3121 tcccgggagc gtgtcttcac agaggagcgg gcccggtttt atggtgcaga gattgtctcg
     3181 gctcttgagt acttgcactc gcgggacgtg gtataccgcg acatcaagct ggaaaacctc
     3241 atgctggaca aagatggcca catcaagatc actgactttg gcctctgcaa agagggcatc
     3301 agtgacgggg ccaccatgaa aaccttctgt gggaccccgg agtacctggc gcctgaggtg
     3361 ctggaggaca atgactatgg ccgggccgtg gactggtggg ggctgggtgt ggtcatgtac
     3421 gagatgatgt gcggccgcct gcccttctac aaccaggacc acgagcgcct cttcgagctc
     3481 atcctcatgg aagagatccg cttcccgcgc acgctcagcc ccgaggccaa gtccctgctt
     3541 gctgggctgc ttaagaagga ccccaagcag aggcttggtg gggggcccag cgatgccaag
     3601 gaggtcatgg agcacaggtt cttcctcagc atcaactggc aggacgtggt ccagaagaag
     3661 ctcctgccac ccttcaaacc tcaggtcacg tccgaggtcg acacaaggta cttcgatgat
     3721 gaatttaccg cccagtccat cacaatcaca ccccctgacc gctatgacag cctgggctta
     3781 ctggagctgg accagcggac ccacttcccc cagttctcct actcggccag catccgcgag
     3841 tgaggatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     3901 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     3961 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4021 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4081 atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     4141 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     4201 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     4261 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     4321 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     4381 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     4441 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     4501 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     4561 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4621 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4681 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     4741 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4801 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4861 tagtcagcaa ccatagcccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4921 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4981 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     5041 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     5101 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     5161 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     5221 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     5281 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     5341 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     5401 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     5461 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     5521 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     5581 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     5641 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     5701 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     5761 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     5821 cgctcccgat tcacagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     5881 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     5941 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     6001 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     6061 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     6121 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     6181 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     6241 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     6301 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     6361 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6421 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaagga
//
