LOCUS       sequence-215468-        7911 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the AKAR5 sensor C-terminally fused to the 
            C-terminus of KRas (membrane targeting) 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 7911)
  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 7911)
  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_m-AKAR5
FEATURES             Location/Qualifiers
     source          1..7911
                     /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          3834..3967
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3950..3969
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3976..4564
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4029..4049)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4447..4458)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4591..4608)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4597..4708
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4935..4954)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5000..5055
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5054..5073)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5414..5430)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5427..5449)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5438..5454
                     /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(5462..5468,5469..5486,5487..5492))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5507..5528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5587..5782
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5633..5768
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5788..5922
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5825..5844)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5879..5898
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5990..6007)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6161..6749)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6241..6260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6920..7711,7712..7780))
                     /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     7543..7562
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7781..7885)
                     /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 tgaattcggt aaaaagaaga aaaagaagtc aaagacaaag tgtgtaatta
     3781 tgtaatctag agtcgaggta ccgatgaata gctaaggtcg accgaggccg caggtaagta
     3841 tcaaggttac aagacaggtt taaggagacc acatagaaac tgggcttgtc gagacagaga
     3901 agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt tgcctttctc
     3961 tccacaggtg tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     4021 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     4081 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     4141 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     4201 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     4261 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     4321 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc
     4381 tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     4441 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     4501 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     4561 ccgcctggaa ttcgagctcg gtacgatcag cctcgactgt gccttctagt tgccagccat
     4621 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     4681 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     4741 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggccagctt
     4801 ttgttccctt tagggttaat tgcgcgcttg gcgctaatca tggtcatagc tgtttccact
     4861 cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac
     4921 aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt
     4981 gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat
     5041 tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat
     5101 gagtatttgg tttagagttt ggcaacatat gccatatgct ggctgccatg aacaaaggtg
     5161 gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag
     5221 aaaagccttg acttgaggtt agattttttt ttatattttg tttgtgttat ttttttcttt
     5281 aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact
     5341 actcccagtc atagctgtcc ctcttctctt atgaagatcc ctcgacctgc agcccaagct
     5401 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5461 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5521 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5581 ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     5641 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     5701 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     5761 tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa
     5821 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5881 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg
     5941 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     6001 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6061 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6121 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6181 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6241 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6301 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     6361 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6421 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6481 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6541 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6601 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6661 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6721 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6781 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6841 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6901 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6961 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     7021 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     7081 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7141 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7201 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7261 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7321 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7381 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7441 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7501 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7561 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7621 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7681 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7741 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7801 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7861 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
