LOCUS       sequence-235174-        7770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the tAKAR-epsilon sensor 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 7770)
  AUTHORS   Ma L, Jongbloets BC, Xiong WH, Melander JB, Qin M, Lameyer TJ, 
            Harrison MF, Zemelman BV, Mao T, Zhong H
  TITLE     A Highly Sensitive A-Kinase Activity Reporter for Imaging 
            Neuromodulatory Events in Awake Mice.
  JOURNAL   Neuron. 2018 Aug 22;99(4):665-679.e5. doi: 
            10.1016/j.neuron.2018.07.020. Epub 2018 Aug 9.
  PUBMED    30100256
REFERENCE   2  (bases 1 to 7770)
  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.neuron.2018.07.020"; journalName: "Neuron"; date: 
            "2018-08-22- 22"; volume: "99"; issue: "4"; pages: "665-679.e5"
COMMENT     Sequence Label: pRK5_tAKAR-epsilon
FEATURES             Location/Qualifiers
     source          1..7770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      943..952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(949..951,952..954,955..1467)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     CDS             join(2371..2373,2374..2376,2377..2889)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIE"
     regulatory      4070..4079
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4083..4217
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4120..4139)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4174..4193
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4286..4643
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4286..4477
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4309..4329)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4494..4629
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4663..4680)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4663..4679)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4672..4694)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4892..5347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4979..4998)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5189..5210
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5364..5383)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5483..5505
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5543..5561)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5629..5733
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5734..5802,5803..6594)
                     /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     complement(5952..5971)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6765..7353
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7254..7273
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7507..7524
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7641..7662
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7677..7682,7683..7700,7701..7707)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7715..7731
                     /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)."
     primer_bind     7720..7742
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7739..7755
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7739..7755
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcccgggc tcgagaccgg tcgccaccat ggtgagcaag
      961 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1021 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1081 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1141 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1201 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1261 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1321 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1381 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1441 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1501 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1561 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1621 gtgaccgccg cccgcatgca taagttttct caagaacaga tcggcgaaaa cattgtgtgc
     1681 agggtcattt gtaccacggg tcaaattccc atccgagatt tgtcagctga tatttcacaa
     1741 gtgcttaagg aaaaacgatc cataaagaaa gtttggacat ttggtagaaa cccagcctgt
     1801 gactatcatt taggaaacat ttcaagactg tcaaataagc atttccaaat actactagga
     1861 gaagacggta accttttatt gaatgacatt tccactaatg ggacctggtt aaatgggcaa
     1921 aaagtcgaga agaacagcaa tcagttactg tctcaaggtg atgaaataac cgttggtgta
     1981 ggcgtggaat cagatatttt atctctggtc attttcataa acgacaaatt taagcagtgc
     2041 ctcgagcaga acaaagttga tcgctctgca ggtaagccag gcagcggcga gggcagcacc
     2101 aagggcctgc gtcgcgccac cctggtggac ggcggcaccg gcggcagcga gctcatgggc
     2161 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2221 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
     2281 cgcgatcaca tggtcctgct ggagagagtg accgccgccg ggatcactct cggcatggac
     2341 gagctataca agggtggaac tggaggttca atggtgagca agggcgagga gctgttcacc
     2401 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2461 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagct catctgcacc
     2521 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcctgatg
     2581 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2641 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2701 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2761 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactggaa cagcgtcaac
     2821 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2881 aacatcgagt ccggactcag atctcgagct atggatgatg atatcgccgc gctcgtcgtc
     2941 gacaacggct ccggcatgtg caaggccggc ttcgcgggcg acgatgcccc ccgggccgtc
     3001 ttcccctcca tcgtggggcg ccccaggcac cagggcgtga tggtgggcat gggtcagaag
     3061 gattcctatg tgggcgacga ggcccagagc aagagaggca tcctcaccct gaagtacccc
     3121 atcgagcacg gcatcgtcac caactgggac gacatggaga aaatctggca ccacaccttc
     3181 tacaatgagc tgcgtgtggc tcccgaggag caccccgtgc tgctgaccga ggcccccctg
     3241 aaccccaagg ccaaccgcga gaagatgacc cagatcatgt ttgagacctt caacacccca
     3301 gccatgtacg ttgctatcca ggctgtgcta tccctgtacg cctctggccg taccactggc
     3361 atcgtgatgg actccggtga cggggtcacc cacactgtgc ccatctacga ggggtatgcc
     3421 ctcccccatg ccatcctgcg tctggacctg gctggccggg acctgactga ctacctcatg
     3481 aagatcctca ccgagcgcgg ctacagcttc accaccacgg ccgagcggga aatcgtgcgt
     3541 gacattaagg agaagctgtg ctacgtcgcc ctggacttcg agcaagagat ggccacggct
     3601 gcttccagct cctccctgga gaagagctac gagctgcctg acggccaggt catcaccatt
     3661 ggcaatgagc ggttccgctg ccctgaggca ctcttccagc cttccttcct gggcatggag
     3721 tcctgtggca tccacgaaac taccttcaac tccatcatga agtgtgacgt ggacatccgc
     3781 aaagacctgt acgccaacac agtgctgtct ggcggcacca ccatgtaccc tggcattgcc
     3841 gacaggatgc agaaggagat cactgccctg gcacccagca caatgaagat caagatcatt
     3901 gctcctcctg agcgcaagta ctccgtgtgg atcggcggct ccatcctggc ctcgctgtcc
     3961 accttccagc agatgtggat cagcaagcag gagtatgacg agtccggccc ctccatcgtc
     4021 caccgcaaat gcttctaggg atcctctaga gtcgacctgc agaagcttgg ccgccatggc
     4081 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4141 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4201 cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa ataacctctg
     4261 aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     4321 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4381 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4441 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4501 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4561 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4621 ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg tcgttttaca
     4681 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     4741 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     4801 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4861 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4921 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4981 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5041 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5101 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5161 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5221 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5281 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     5341 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     5401 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5461 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5521 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5581 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5641 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5701 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5761 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5821 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5881 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5941 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6001 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6061 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6121 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6181 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6241 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6301 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6361 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6421 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6481 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6541 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6601 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6661 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6721 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6781 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6841 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6901 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6961 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7021 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7081 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7141 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7201 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7261 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7321 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7381 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     7441 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     7501 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     7561 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     7621 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     7681 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     7741 ggaaacagct atgacatgat tacgaattaa
//
