LOCUS       sequence-235137-        6693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the tAKAR-beta 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 6693)
  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 6693)
  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-beta
FEATURES             Location/Qualifiers
     source          1..6693
                     /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      994..1003
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1000..1002,1003..1005,1006..1518)
                     /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(2422..2424,2425..2427,2428..2940)
                     /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      2993..3002
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3006..3140
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3043..3062)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3097..3116
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3209..3566
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3209..3400
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3232..3252)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3417..3552
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3586..3603)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3586..3602)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3595..3617)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3815..4270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3902..3921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4112..4133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4287..4306)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4406..4428
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4466..4484)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4552..4656
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4657..4725,4726..5517)
                     /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(4875..4894)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5688..6276
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6177..6196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6430..6447
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6564..6585
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6600..6605,6606..6623,6624..6630)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6638..6654
                     /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     6643..6665
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6662..6678
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6662..6678
                     /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 attcgccacc atgctgcaga acgagcttgc tcttaagttg
      961 gctggacttg atattaacaa gactggaccg gtcgccacca tggtgagcaa gggcgaggag
     1021 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1081 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1141 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1201 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1261 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1321 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1381 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1441 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1501 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1561 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
     1621 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1681 gcccgcatgc ataagttttc tcaagaacag atcggcgaaa acattgtgtg cagggtcatt
     1741 tgtaccacgg gtcaaattcc catccgagat ttgtcagctg atatttcaca agtgcttaag
     1801 gaaaaacgat ccataaagaa agtttggaca tttggtagaa acccagcctg tgactatcat
     1861 ttaggaaaca tttcaagact gtcaaataag catttccaaa tactactagg agaagacggt
     1921 aaccttttat tgaatgacat ttccactaat gggacctggt taaatgggca aaaagtcgag
     1981 aagaacagca atcagttact gtctcaaggt gatgaaataa ccgttggtgt aggcgtggaa
     2041 tcagatattt tatctctggt cattttcata aacgacaaat ttaagcagtg cctcgagcag
     2101 aacaaagttg atcgctctgc aggtaagcca ggcagcggcg agggcagcac caagggcctg
     2161 cgtcgcgcca ccctggtgga cggcggcacc ggcggcagcg agctcatggg cagcgtgcag
     2221 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     2281 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     2341 atggtcctgc tggagagagt gaccgccgcc gggatcactc tcggcatgga cgagctatac
     2401 aagggtggaa ctggaggttc aatggtgagc aagggcgagg agctgttcac cggggtggtg
     2461 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     2521 ggcgagggcg atgccaccta cggcaagctg accctgaagc tcatctgcac caccggcaag
     2581 ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgat gtgcttcgcc
     2641 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2701 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2761 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2821 gacggcaaca tcctggggca caagctggag tacaactgga acagcgtcaa cgtctatatc
     2881 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     2941 taagcggccg caattccccg gggatcctct agagtcgacc tgcagaagct tggccgccat
     3001 ggcccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3061 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3121 tatcttatca tgtctggatc gggaattaat tcggcgcagc accatggcct gaaataacct
     3181 ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt
     3241 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3301 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3361 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3421 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3481 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3541 tttttggagg cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg ccgtcgtttt
     3601 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     3661 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     3721 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3781 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     3841 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3901 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3961 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4021 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4081 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4141 ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4201 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4261 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4321 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4381 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4441 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4501 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4561 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4621 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4681 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4741 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4801 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4861 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     4921 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4981 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5041 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5101 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5161 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5221 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5281 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5341 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5401 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5461 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5521 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5581 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5641 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5701 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5761 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5821 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5881 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5941 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6001 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6061 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6121 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6181 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6241 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6301 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6361 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6421 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     6481 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     6541 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     6601 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     6661 acaggaaaca gctatgacat gattacgaat taa
//
