LOCUS       sequence-317683-        6633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Enhanced excitation-ratiometric biosensor for monitoring Protein 
            Kinase A activity in living cells and in vivo..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6633)
  AUTHORS   Zhang JF, Liu B, Hong I, Mo A, Roth RH, Tenner B, Lin W, Zhang JZ, 
            Molina RS, Drobizhev M, Hughes TE, Tian L, Huganir RL, Mehta S, 
            Zhang J
  TITLE     An ultrasensitive biosensor for high-resolution kinase activity 
            imaging in awake mice.
  JOURNAL   Nat Chem Biol. 2020 Sep 28. pii: 10.1038/s41589-020-00660-y. doi: 
            10.1038/s41589-020-00660-y.
  PUBMED    32989297
REFERENCE   2  (bases 1 to 6633)
  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"; journalName: "Nat Chem 
            Biol. 2020 Sep 28. pii: 10.1038/s41589-020-00660-y. doi: 
            10.1038/s41589-020-00660-y."
COMMENT     Sequence Label: pcDNA3.1(+)-ExRai-AKAR2
FEATURES             Location/Qualifiers
     source          1..6633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(52..71)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        243..622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        623..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     777..797
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     871..890
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        871..889
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1210..1231
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1337..1358)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1598..1617)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2232..2249)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2238..2462
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2508..2936
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2595..2614)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2805..2826
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2945..2965)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2950..3279
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3130..3265
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3192..3211
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3346..4140
                     /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(3400..3419)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4010..4029
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4314..4435
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4351..4370)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4405..4424
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4484..4500)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4484..4500)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4497..4519)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4508..4524
                     /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(4532..4538,4539..4556,4557..4562))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4577..4598
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4715..4732)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4886..5474)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4966..4985)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5645..6436,6437..6505))
                     /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     6268..6287
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6506..6610)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
       61 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
      121 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
      181 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      241 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      301 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      361 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      421 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      481 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      541 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      601 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      661 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      721 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      781 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
      841 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
      901 tggctagcgt ttaaacttaa gcttgcggcc gccaccggat cccatgctgc gtcgcgccac
      961 cctggtggac ggcggcaccg gcggcagcga gctcagcttc tgcaacgtct atatcaaggc
     1021 cgacaagcag aagaacggca tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg
     1081 cggcgtgcag ctcgcctacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1141 gctgcccgac aaccactacc tgagcgtgca gtccaaactt tcgaaagacc ccaacgagaa
     1201 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1261 cgagctgtac aagggcggta ccggagggag catggtgagc aagggcgagg agctgttcac
     1321 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1381 gtccggcgag ggtgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1441 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1501 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1561 cgaaggctac atccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1621 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1681 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttgc tgcataagtt
     1741 ttctcaagaa cagatcggcg aaaacattgt gtgcagggtc atttgtacca cgggtcaaat
     1801 tcccatccga gatttgtcag ctgatatttc acaagtgctt aaggaaaaac gatccataaa
     1861 gaaagtttgg acatttggta gaaacccagc ctgtgactat catttaggaa acatttcaag
     1921 actgtcaaat aagcatttcc aaatactact aggagaagac ggtaaccttt tattgaatga
     1981 catttccact aatgggacct ggttaaatgg gcaaaaagtc gagaagaaca gcaatcagtt
     2041 actgtctcaa ggtgatgaaa taaccgttgg tgtaggcgtg gaatcagata ttttatctct
     2101 ggtcattttc ataaacgaca aatttaagca gtgcctcgag cagaacaaag ttgatcgcta
     2161 agaattctgc agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac
     2221 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2281 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2341 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2401 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2461 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2521 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2581 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2641 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2701 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2761 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2821 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2881 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2941 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3001 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3061 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3121 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3181 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3241 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     3301 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3361 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3421 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3481 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     3541 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3601 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3661 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3721 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3781 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3841 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     3901 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3961 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4021 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4081 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4141 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4201 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4261 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4321 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4381 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4441 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4501 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4561 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4621 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4681 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4741 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4801 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4861 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4921 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4981 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5041 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5101 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5161 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5221 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5281 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5341 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5401 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5461 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5521 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5581 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5641 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5701 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5761 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5821 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5881 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5941 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6001 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6061 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6121 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6181 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6241 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6301 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6361 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6421 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6481 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6541 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6601 gggttccgcg cacatttccc cgaaaagtgc cac
//
