LOCUS       sequence-307195-        7460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET-based biosensor for monitoring Akt activity in lipid raft 
            plasma membrane regions..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7460)
  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 7460)
  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-Lyn-AktAR
FEATURES             Location/Qualifiers
     source          1..7460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(946..948,949..951,952..1662)
                     /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             2215..2949
                     /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(3013..3031)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3014..3031)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3051..3068)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3057..3281
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3327..3755
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3414..3433)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3624..3645
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3764..3784)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3769..4098
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3949..4084
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4011..4030
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4165..4959
                     /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(4219..4238)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4829..4848
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5133..5254
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5170..5189)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5224..5243
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5303..5319)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5303..5319)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5316..5338)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5327..5343
                     /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(5351..5357,5358..5375,5376..5381))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5396..5417
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5534..5551)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5705..6293)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5785..5804)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6464..7255,7256..7324))
                     /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     7087..7106
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7325..7429)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg gctgcatcaa gagcaagcgc aaggacaagg atcccatggt gagcaagggc
      961 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1021 cacaggttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1081 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1141 acctggggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     1201 aagtccgcca tgcccgaagg ctacgtccag gagcgtacca tcttcttcaa ggacgacggc
     1261 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1321 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1381 gccatcagtg acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggcccac
     1441 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1501 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1561 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1621 accgccgccg ggatcactct cggcatggac gagctgtaca agcgcatgca taagttttct
     1681 caagaacaga tcggcgaaaa cattgtgtgc agggtcattt gtaccacggg tcaaattccc
     1741 atccgagatt tgtcagctga tatttcacaa gtgcttaagg aaaaacgatc cataaagaaa
     1801 gtttggacat ttggtagaaa cccagcctgt gactatcatt taggaaacat ttcaagactg
     1861 tcaaataagc atttccaaat actactagga gaagacggta accttttatt gaatgacatt
     1921 tccactaatg ggacctggtt aaatgggcaa aaagtcgaga agaacagcaa tcagttactg
     1981 tctcaaggtg atgaaataac cgttggtgta ggcgtggaat cagatatttt atctctggtc
     2041 attttcataa acgacaaatt taagcagtgc ctcgagcaga acaaagttga tcgctctgca
     2101 ggtaagccag gcagcggcga gggcagcacc aagggcctgg tcgaccctcg tccgcgctcg
     2161 tgcacctggc cggaccccag gccggagttt ggaggtaccg gcggcagcga gctcatgggc
     2221 ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2281 ctgcccgaca accactacct gagctaccag tccaagctga gcaaagaccc caacgagaag
     2341 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     2401 gagctgtaca agggaggtac cggtggatct atggtgagca agggcgagga gctgttcacc
     2461 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2521 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagct gatctgcacc
     2581 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgggcta cggccttcag
     2641 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2701 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2761 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2821 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2881 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccgccac
     2941 aacatcgagt aagaattctg cagatatcca tcacactggc ggccgctcga gcatgcatct
     3001 agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt
     3061 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3121 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3181 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3241 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3301 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3361 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3421 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3481 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3541 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3601 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3661 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3721 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3781 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3841 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3901 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3961 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4021 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4081 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     4141 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4201 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4261 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4321 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4381 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4441 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4501 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4561 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4621 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4681 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4741 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4801 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4861 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4921 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4981 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     5041 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5101 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5161 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5221 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5281 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5341 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5401 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5461 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5521 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5581 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5641 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5701 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5761 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5821 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5881 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5941 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6001 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6061 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6121 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6181 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6241 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6301 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6361 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6421 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6481 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6541 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6601 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6661 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6721 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6781 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6841 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6901 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6961 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7021 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7081 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7141 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7201 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7261 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7321 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7381 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7441 gaaaagtgcc acctgacgtc
//
