LOCUS       sequence-228229-        7324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the tAKAR4-alpha sensor for detecting PKA 
            activity using ratiometric imaging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7324)
  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 7324)
  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_tAKAR4-alpha
FEATURES             Location/Qualifiers
     source          1..7324
                     /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..1665)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2188..2922
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     regulatory      3569..3578
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3582..3716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3619..3638)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3673..3692
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3789..4146
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3789..3980
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3812..3832)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3997..4132
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4166..4183)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4166..4182)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4175..4197)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4395..4850
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4482..4501)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4692..4713
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4867..4886)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5021..5043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5096..5114)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5182..5286
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5287..5355,5356..6147)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRDDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5505..5524)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6318..6906
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6807..6826
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7060..7077
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(7230..7235,7236..7253,7254..7260)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7268..7284
                     /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     7273..7295
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7292..7308
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7292..7308
                     /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 ggccacaggt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1081 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1141 ctgacctggg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1201 ttcaagtccg ccatgcccga aggctacgtc caggagcgta ccatcttctt caaggacgac
     1261 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1321 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1381 aacgccatca gtgacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1441 cacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1501 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1561 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1621 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagcgcat gcataagttt
     1681 tctcaagaac agatcggcga aaacattgtg tgcagggtca tttgtaccac gggtcaaatt
     1741 cccatccgag atttgtcagc tgatatttca caagtgctta aggaaaaacg atccataaag
     1801 aaagtttgga catttggtag aaacccagcc tgtgactatc atttaggaaa catttcaaga
     1861 ctgtcaaata agcatttcca aatactacta ggagaagacg gtaacctttt attgaatgac
     1921 atttccacta atgggacctg gttaaatggg caaaaagtcg agaagaacag caatcagtta
     1981 ctgtctcaag gtgatgaaat aaccgttggt gtaggcgtgg aatcagatat tttatctctg
     2041 gtcattttca taaacgacaa atttaagcag tgcctcgagc agaacaaagt tgatcgctct
     2101 gcaggtaagc caggcagcgg cgagggcagc accaagggcc tgcgtcgcgc caccctggtg
     2161 gacggcggca ccggcggcag cgagctcatg ggcggcgtgc agctcgccga ccactaccag
     2221 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     2281 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2341 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggagg taccggtgga
     2401 tctatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2461 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2521 tacggcaagc tgaccctgaa gctgatctgc accaccggca agctgcccgt gccctggccc
     2581 accctcgtga ccaccctggg ctacggcctt cagtgcttcg cccgctaccc cgaccacatg
     2641 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2701 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2761 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2821 cacaagctgg agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag
     2881 aacggcatca aggccaactt caagatccgc cacaacatcg agtccggagt gtacaagggt
     2941 accccaggaa cccccaagtc tgccatcttg gtgccgagtg agaagaaggt cgccatcata
     3001 cgtactcctc caaaatctcc tgcgactccc aagcagcttc ggcttattaa ccaaccactg
     3061 ccagacctga agaatgtcaa atccaaaatc ggatcaacag acaacatcaa ataccagcct
     3121 aaaggggggc aggtacaaat tgttaccaag aaaatagacc taagccatgt gacatccaaa
     3181 tgtggctctc tgaagaacat ccgccacagg ccaggtggcg gacgtgtgaa aattgagagt
     3241 gtaaaactag atttcaaaga aaaggcccaa gctaaagttg gttctcttga taatgctcat
     3301 catgtacctg gaggtggtaa tgtcaagatt gacagccaaa agttgaactt cagagagcat
     3361 gctaaagccc gtgtggacca tggggctgag atcattacac agtccccagg cagatccagc
     3421 gtggcatcac cccgacgact cagcaatgtc tcctcgtctg gaagcatcaa cctgctcgaa
     3481 tctcctcagc ttgccacttt ggctgaggat gtcactgctg cactcgctaa gcagggcttg
     3541 taatctagag tcgacctgca gaagcttggc cgccatggcc caacttgttt attgcagctt
     3601 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3661 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcgatc
     3721 gggaattaat tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta
     3781 ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     3841 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3901 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3961 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4021 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4081 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4141 tgcaaaaagc tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     4201 accctggcgt tacccaactt aatcgccttg cagcacatcc ccccttcgcc agctggcgta
     4261 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgtagcctg aatggcgaat
     4321 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     4381 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4441 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4501 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4561 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4621 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4681 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     4741 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4801 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     4861 tctcagtaca atctgctctg atgccgcata gttaagccaa ctccgctatc gctacgtgac
     4921 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4981 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5041 aggttttcac cgtcatcacc gaaacgcgcg aggcagtatt cttgaagacg aaagggcctc
     5101 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5161 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5221 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5281 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5341 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5401 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5461 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5521 ttatcccgtg atgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5581 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5641 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5701 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5761 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5821 acgatgccag cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5881 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     5941 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6001 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6061 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6121 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6181 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6241 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6301 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6361 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6421 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     6481 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6541 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6601 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6661 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc
     6721 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6781 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6841 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6901 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6961 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     7021 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     7081 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatcc
     7141 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     7201 agttacctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     7261 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgaa
     7321 ttaa
//
