LOCUS       sequence-235220-        7236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the tAKAR-alpha sensor with T391A mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7236)
  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 7236)
  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-alpha-T391A
FEATURES             Location/Qualifiers
     source          1..7236
                     /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      3536..3545
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3549..3683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3586..3605)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3640..3659
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3752..4109
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3752..3943
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3775..3795)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3960..4095
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4129..4146)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4129..4145)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4138..4160)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4358..4813
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4445..4464)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4655..4676
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4830..4849)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4949..4971
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5009..5027)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5095..5199
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5200..5268,5269..6060)
                     /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(5418..5437)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6231..6819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6720..6739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6973..6990
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7107..7128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7143..7148,7149..7166,7167..7173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7181..7197
                     /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     7186..7208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7205..7221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7205..7221
                     /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 gtcgcgccgc 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 ccggagtgta caagggtacc ccaggaaccc ccaagtctgc catcttggtg
     2941 ccgagtgaga agaaggtcgc catcatacgt actcctccaa aatctcctgc gactcccaag
     3001 cagcttcggc ttattaacca accactgcca gacctgaaga atgtcaaatc caaaatcgga
     3061 tcaacagaca acatcaaata ccagcctaaa ggggggcagg tacaaattgt taccaagaaa
     3121 atagacctaa gccatgtgac atccaaatgt ggctctctga agaacatccg ccacaggcca
     3181 ggtggcggac gtgtgaaaat tgagagtgta aaactagatt tcaaagaaaa ggcccaagct
     3241 aaagttggtt ctcttgataa tgctcatcat gtacctggag gtggtaatgt caagattgac
     3301 agccaaaagt tgaacttcag agagcatgct aaagcccgtg tggaccatgg ggctgagatc
     3361 attacacagt ccccaggcag atccagcgtg gcatcacccc gacgactcag caatgtctcc
     3421 tcgtctggaa gcatcaacct gctcgaatct cctcagcttg ccactttggc tgaggatgtc
     3481 actgctgcac tcgctaagca gggcttgtaa tctagagtcg acctgcagaa gcttggccgc
     3541 catggcccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3601 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3661 atgtatctta tcatgtctgg atcgggaatt aattcggcgc agcaccatgg cctgaaataa
     3721 cctctgaaag aggaacttgg ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg
     3781 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3841 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3901 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3961 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4021 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     4081 gcttttttgg aggcctaggc ttttgcaaaa agctgttaac agcttggcac tggccgtcgt
     4141 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     4201 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4261 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     4321 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4381 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4441 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4501 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4561 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4621 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4681 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     4741 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4801 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4861 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4921 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4981 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5041 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5101 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5161 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5221 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5281 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5341 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5401 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5461 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5521 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5581 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5641 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5701 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5761 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5821 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5881 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5941 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6001 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6061 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6121 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6181 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6241 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6301 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6361 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6421 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6481 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6541 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6601 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6661 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6721 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6781 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6841 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6901 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     6961 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     7021 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     7081 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     7141 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     7201 cacacaggaa acagctatga catgattacg aattaa
//
