LOCUS       sequence-235127-        7442 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the AKAR5-Delta sensor with N-terminal His 
            tag 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 7442)
  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 7442)
  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: pCDNA3_AKAR5-Delta
FEATURES             Location/Qualifiers
     source          1..7442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      12..21
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             33..50
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             54..86
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(90..98,99..113)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(120..122,123..125,126..638)
                     /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(1326..1328,1329..1331,1332..2042)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     ERVTAAGITLGMDELYK"
     promoter        complement(2106..2124)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2107..2124)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2144..2161)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2150..2374
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2420..2848
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2507..2526)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2717..2738
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2857..2877)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2862..3191
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3042..3177
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3104..3123
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3258..4052
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3312..3331)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3922..3941
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4226..4347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4263..4282)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4317..4336
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4396..4412)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4396..4412)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4409..4431)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4420..4436
                     /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)."
     promoter        complement(join(4444..4450,4451..4468,4469..4474))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4489..4510
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4627..4644)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4798..5386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4878..4897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5557..6348,6349..6417))
                     /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     6180..6199
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6418..6522)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6597..6616)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6788..7167
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7168..7371
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7322..7342
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7416..7435
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7416..7434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 agcttgcggc cgccaccatg gtgcggggtt ctcatcatca tcatcatcat ggtatggcta
       61 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatccca
      121 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
      181 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
      241 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
      301 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
      361 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
      421 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
      481 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
      541 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
      601 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
      661 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
      721 acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc
      781 tgctggagtt cgtgaccgcc gcccgcatgc ataagttttc tcaagaacag atcggcgaaa
      841 acattgtgtg cagggtcatt tgtaccacgg gtcaaattcc catccgagat ttgtcagctg
      901 atatttcaca agtgcttaag gaaaaacgat ccataaagaa agtttggaca tttggtagaa
      961 acccagcctg tgactatcat ttaggaaaca tttcaagact gtcaaataag catttccaaa
     1021 tactactagg agaagacggt aaccttttat tgaatgacat ttccactaat gggacctggt
     1081 taaatgggca aaaagtcgag aagaacagca atcagttact gtctcaaggt gatgaaataa
     1141 ccgttggtgt aggcgtggaa tcagatattt tatctctggt cattttcata aacgacaaat
     1201 ttaagcagtg cctcgagcag aacaaagttg atcgctctgc aggtaagcca ggcagcggcg
     1261 agggcagcac caagggcctg cgtcgcgcca ccctggtgga cggcggcacc ggcggcagcg
     1321 agctcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1381 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1441 cctacggcaa gctgaccctg aagctcatct gcaccaccgg caagctgccc gtgccctggc
     1501 ccaccctcgt gaccaccttc ggctacggcc tgatgtgctt cgcccgctac cccgaccaca
     1561 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1621 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1681 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1741 ggcacaagct ggagtacaac tggaacagcg tcaacgtcta tatcatggcc gacaagcaga
     1801 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1861 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1921 accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1981 tggtcctgct ggagagagtg accgccgccg ggatcactct cggcatggac gagctataca
     2041 agggtgaatt ctgcagatat ccatcacact ggcggccgct cgagcatgca tctagagggc
     2101 cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct
     2161 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2221 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2281 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2341 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2401 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2461 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2521 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2581 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2641 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2701 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2761 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2821 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     2881 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2941 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3001 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3061 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3121 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3181 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     3241 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3301 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3361 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3421 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3481 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3541 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3601 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3661 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     3721 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     3781 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     3841 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     3901 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     3961 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4021 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4081 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4141 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4201 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4261 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4321 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4381 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4441 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4501 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4561 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4621 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4681 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4741 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4801 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4861 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4921 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4981 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5041 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5101 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5161 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5221 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5281 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5341 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5401 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5461 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5521 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5581 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5641 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5701 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5761 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5821 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5881 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5941 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6001 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6061 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6121 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6181 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6241 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6301 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6361 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6421 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6481 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6541 gccacctgac gtcggcggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     6601 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     6661 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     6721 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
     6781 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     6841 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     6901 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     6961 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     7021 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     7081 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     7141 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     7201 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     7261 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     7321 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta
     7381 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc
     7441 ca
//
