LOCUS       sequence-235166-        6699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the tAKAR-delta 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 6699)
  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 6699)
  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-delta
FEATURES             Location/Qualifiers
     source          1..6699
                     /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      928..937
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             934..984
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     regulatory      1000..1009
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1006..1008,1009..1011,1012..1524)
                     /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(2428..2430,2431..2433,2434..2946)
                     /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      2999..3008
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3012..3146
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3049..3068)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3103..3122
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3215..3572
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3215..3406
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3238..3258)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3423..3558
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3592..3609)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3592..3608)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3601..3623)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3821..4276
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3908..3927)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4118..4139
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4293..4312)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4412..4434
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4472..4490)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4558..4662
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4663..4731,4732..5523)
                     /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(4881..4900)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5694..6282
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6183..6202
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6436..6453
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6570..6591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6606..6611,6612..6629,6630..6636)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6644..6660
                     /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     6649..6671
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6668..6684
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6668..6684
                     /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 attcgtcgcc accatgggcg tggccgactt gatcaagaag
      961 ttcgagtcca tctccaagga ggagggggat ccaccggtcg ccaccatggt gagcaagggc
     1021 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1081 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1141 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1201 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1261 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1321 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1381 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1441 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1501 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1561 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1621 tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1681 accgccgccc gcatgcataa gttttctcaa gaacagatcg gcgaaaacat tgtgtgcagg
     1741 gtcatttgta ccacgggtca aattcccatc cgagatttgt cagctgatat ttcacaagtg
     1801 cttaaggaaa aacgatccat aaagaaagtt tggacatttg gtagaaaccc agcctgtgac
     1861 tatcatttag gaaacatttc aagactgtca aataagcatt tccaaatact actaggagaa
     1921 gacggtaacc ttttattgaa tgacatttcc actaatggga cctggttaaa tgggcaaaaa
     1981 gtcgagaaga acagcaatca gttactgtct caaggtgatg aaataaccgt tggtgtaggc
     2041 gtggaatcag atattttatc tctggtcatt ttcataaacg acaaatttaa gcagtgcctc
     2101 gagcagaaca aagttgatcg ctctgcaggt aagccaggca gcggcgaggg cagcaccaag
     2161 ggcctgcgtc gcgccaccct ggtggacggc ggcaccggcg gcagcgagct catgggcagc
     2221 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     2281 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc
     2341 gatcacatgg tcctgctgga gagagtgacc gccgccggga tcactctcgg catggacgag
     2401 ctatacaagg gtggaactgg aggttcaatg gtgagcaagg gcgaggagct gttcaccggg
     2461 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     2521 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagctcat ctgcaccacc
     2581 ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cctgatgtgc
     2641 ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2701 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2761 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2821 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actggaacag cgtcaacgtc
     2881 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2941 atcgagtaag cggccgcaat tccccgggga tcctctagag tcgacctgca gaagcttggc
     3001 cgccatggcc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3061 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3121 tcaatgtatc ttatcatgtc tggatcggga attaattcgg cgcagcacca tggcctgaaa
     3181 taacctctga aagaggaact tggttaggta ccttctgagg cggaaagaac cagctgtgga
     3241 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3301 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3361 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3421 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3481 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3541 gaggcttttt tggaggccta ggcttttgca aaaagctgtt aacagcttgg cactggccgt
     3601 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     3661 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     3721 acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct
     3781 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     3841 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3901 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3961 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4021 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4081 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4141 acaacactca accctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     4201 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4261 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4321 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4381 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4441 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     4501 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     4561 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4621 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4681 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4741 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4801 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4861 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4921 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4981 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5041 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5101 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5161 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5221 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5281 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5341 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     5401 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     5461 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5521 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5581 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5641 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5701 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5761 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5821 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5881 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5941 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6001 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6061 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6121 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6181 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6241 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6301 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6361 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     6421 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     6481 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     6541 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     6601 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     6661 tttcacacag gaaacagcta tgacatgatt acgaattaa
//
