LOCUS       sequence-215467-        7903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the AKAR5 sensor tagged with nucleus export 
            signal (NES) for detecting PKA activity using two-photon 
            fluorescence lifetime imaging microscopy (2pFLIM).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7903)
  AUTHORS   Tillo SE, Xiong WH, Takahashi M, Miao S, Andrade AL, Fortin DA, Yang
            G, Qin M, Smoody BF, Stork PJS, Zhong H
  TITLE     Liberated PKA Catalytic Subunits Associate with the Membrane via 
            Myristoylation to Preferentially Phosphorylate Membrane Substrates.
  JOURNAL   Cell Rep. 2017 Apr 18;19(3):617-629. doi: 
            10.1016/j.celrep.2017.03.070.
  PUBMED    28423323
REFERENCE   2  (bases 1 to 7903)
  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.celrep.2017.03"; journalName: "Cell Rep"; date: 
            "2017-04-18- 18"; volume: "19"; issue: "3"; pages: "617-629"
COMMENT     Sequence Label: pCAGGS_cyt-AKAR5
FEATURES             Location/Qualifiers
     source          1..7903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1767..1776
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1773..1775,1776..1778,1779..2489)
                     /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"
     CDS             join(3012..3014,3015..3017,3018..3728)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     ERVTAAGITLGMDELYK"
     intron          3826..3959
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3942..3961
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3968..4556
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4021..4041)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4439..4450)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4583..4600)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4589..4700
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4927..4946)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4992..5047
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5046..5065)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5406..5422)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5419..5441)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5430..5446
                     /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(5454..5460,5461..5478,5479..5484))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5499..5520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5579..5774
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5625..5760
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5780..5914
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5817..5836)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5871..5890
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5982..5999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6153..6741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6233..6252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6912..7703,7704..7772))
                     /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     7535..7554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7773..7877)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg ggcgagggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcggtacc gcgggcccgg
     1741 gatccaccgg aagccacctg catgcggcca ccatggtgag caagggcgag gagctgttca
     1801 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1861 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1921 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1981 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2041 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2101 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2161 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2221 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     2281 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2341 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     2401 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2461 tcactctcgg catggacgag ctgtacaagc gcatgcataa gttttctcaa gaacagatcg
     2521 gcgaaaacat tgtgtgcagg gtcatttgta ccacgggtca aattcccatc cgagatttgt
     2581 cagctgatat ttcacaagtg cttaaggaaa aacgatccat aaagaaagtt tggacatttg
     2641 gtagaaaccc agcctgtgac tatcatttag gaaacatttc aagactgtca aataagcatt
     2701 tccaaatact actaggagaa gacggtaacc ttttattgaa tgacatttcc actaatggga
     2761 cctggttaaa tgggcaaaaa gtcgagaaga acagcaatca gttactgtct caaggtgatg
     2821 aaataaccgt tggtgtaggc gtggaatcag atattttatc tctggtcatt ttcataaacg
     2881 acaaatttaa gcagtgcctc gagcagaaca aagttgatcg ctctgcaggt aagccaggca
     2941 gcggcgaggg cagcaccaag ggcctgcgtc gcgccaccct ggtggacggc ggcaccggcg
     3001 gcagcgagct catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     3061 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     3121 atgccaccta cggcaagctg accctgaagc tcatctgcac caccggcaag ctgcccgtgc
     3181 cctggcccac cctcgtgacc accttcggct acggcctgat gtgcttcgcc cgctaccccg
     3241 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     3301 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     3361 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     3421 tcctggggca caagctggag tacaactgga acagcgtcaa cgtctatatc atggccgaca
     3481 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     3541 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     3601 ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg
     3661 atcacatggt cctgctggag agagtgaccg ccgccgggat cactctcggc atggacgagc
     3721 tatacaaggg tgaattcctt caaaaaaaac ttgaagaact tgaacttgac gaatgatcta
     3781 gagtccgagg taccgatgaa tagctaaggt cgaccgaggc cgcaggtaag tatcaaggtt
     3841 acaagacagg tttaaggaga ccacatagaa actgggcttg tcgagacaga gaagactctt
     3901 gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc tctccacagg
     3961 tgtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4021 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4081 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     4141 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     4201 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     4261 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     4321 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
     4381 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     4441 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     4501 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg
     4561 aattcgagct cggtacgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     4621 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     4681 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     4741 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggccagc ttttgttccc
     4801 tttagggtta attgcgcgct tggcgctaat catggtcata gctgtttcca ctcctcaggt
     4861 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     4921 ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct
     4981 gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg
     5041 tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt
     5101 ggtttagagt ttggcaacat atgccatatg ctggctgcca tgaacaaagg tggctataaa
     5161 gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct
     5221 tgacttgagg ttagattttt ttttatattt tgtttgtgtt atttttttct ttaacatccc
     5281 taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag
     5341 tcatagctgt ccctcttctc ttatgaagat ccctcgacct gcagcccaag cttggcgtaa
     5401 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5461 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5521 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
     5581 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     5641 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     5701 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     5761 taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa
     5821 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5881 caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc
     5941 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6001 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6061 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6121 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6181 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6241 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6301 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt
     6361 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6421 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6481 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6541 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     6601 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6661 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6721 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6781 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6841 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6901 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6961 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7021 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7081 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7141 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7201 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7261 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7321 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7381 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7441 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7501 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7561 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7621 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7681 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7741 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7801 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7861 caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
