LOCUS       sequence-307467-        7565 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET-based biosensor for monitoring Akt activity in non-raft plasma 
            membrane regions..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7565)
  AUTHORS   Gao X, Zhang J
  TITLE     Spatiotemporal analysis of differential Akt regulation in plasma 
            membrane microdomains.
  JOURNAL   Mol Biol Cell. 2008 Oct;19(10):4366-73. doi: 
            10.1091/mbc.E08-05-0449. Epub 2008 Aug 13.
  PUBMED    18701703
REFERENCE   2  (bases 1 to 7565)
  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: 
            "10.1091/mbc.E08-05-0449"; journalName: "Mol Biol Cell"; date: 
            "2008-10"; volume: "19"; issue: "10"; pages: "4366-73"
COMMENT     Sequence Label: pcDNA3-AktAR-Kras
FEATURES             Location/Qualifiers
     source          1..7565
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        117..496
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        497..700
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     651..671
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     745..764
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        745..763
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      783..792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             804..821
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             825..857
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(861..869,870..884)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(891..893,894..896,897..1607)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2160..2894
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     promoter        complement(3000..3018)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3001..3018)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3038..3055)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3044..3268
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3314..3742
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3401..3420)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3611..3632
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3751..3771)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3756..4085
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3936..4071
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3998..4017
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4152..4946
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4206..4225)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4816..4835
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5120..5241
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5157..5176)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5211..5230
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5290..5306)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5290..5306)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5303..5325)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5314..5330
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5338..5344,5345..5362,5363..5368))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5383..5404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5521..5538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5692..6280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5772..5791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6451..7242,7243..7311))
                     /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     7074..7093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7312..7416)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7491..7510)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
       61 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      121 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      181 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      241 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      301 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      361 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      421 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      481 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      541 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      601 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      661 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      721 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagcttgcgg
      781 ccgccaccat ggtgcggggt tctcatcatc atcatcatca tggtatggct agcatgactg
      841 gtggacagca aatgggtcgg gatctgtacg acgatgacga taaggatccc atggtgagca
      901 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      961 acggccacag gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1021 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1081 ccctgacctg gggcgtgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact
     1141 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg taccatcttc ttcaaggacg
     1201 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1261 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1321 acaacgccat cagtgacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg
     1381 cccacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     1441 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     1501 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1561 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagcgc atgcataagt
     1621 tttctcaaga acagatcggc gaaaacattg tgtgcagggt catttgtacc acgggtcaaa
     1681 ttcccatccg agatttgtca gctgatattt cacaagtgct taaggaaaaa cgatccataa
     1741 agaaagtttg gacatttggt agaaacccag cctgtgacta tcatttagga aacatttcaa
     1801 gactgtcaaa taagcatttc caaatactac taggagaaga cggtaacctt ttattgaatg
     1861 acatttccac taatgggacc tggttaaatg ggcaaaaagt cgagaagaac agcaatcagt
     1921 tactgtctca aggtgatgaa ataaccgttg gtgtaggcgt ggaatcagat attttatctc
     1981 tggtcatttt cataaacgac aaatttaagc agtgcctcga gcagaacaaa gttgatcgct
     2041 ctgcaggtaa gccaggcagc ggcgagggca gcaccaaggg cctggtcgac cctcgtccgc
     2101 gctcgtgcac ctggccggac cccaggccgg agtttggagg taccggcggc agcgagctca
     2161 tgggcggcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     2221 tgctgctgcc cgacaaccac tacctgagct accagtccaa gctgagcaaa gaccccaacg
     2281 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2341 tggacgagct gtacaaggga ggtaccggtg gatctatggt gagcaagggc gaggagctgt
     2401 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     2461 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagctgatct
     2521 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg ggctacggcc
     2581 ttcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     2641 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     2701 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2761 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     2821 acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc
     2881 gccacaacat cgagaagaag aagaagaaga agagcaagac caagtgcgtg atcatgtaag
     2941 aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga gggccctatt
     3001 ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     3061 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3121 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3181 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3241 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3301 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3361 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3421 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3481 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3541 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3601 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3661 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3721 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3781 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3841 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3901 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3961 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4021 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4081 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4141 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4201 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4261 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4321 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4381 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4441 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4501 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4561 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4621 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4681 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4741 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4801 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4861 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4921 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4981 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5041 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5101 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5161 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5221 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5281 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5341 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5401 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5461 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5521 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5581 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5641 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5701 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5761 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5821 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5881 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5941 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6001 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6061 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6121 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6181 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6241 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6301 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6361 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6421 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6481 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6541 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6601 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6661 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6721 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6781 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6841 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6901 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6961 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     7021 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7081 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7141 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7201 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7261 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7321 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7381 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7441 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     7501 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     7561 tagtg
//
