LOCUS       sequence-313683-        6631 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 6631)
  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 6631)
  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..6631
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1202..1223
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1329..1350)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1590..1609)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2225..2242)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2231..2455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2501..2929
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2588..2607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2798..2819
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2938..2958)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2943..3272
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3123..3258
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3185..3204
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3339..4133
                     /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(3393..3412)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4003..4022
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4307..4428
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4344..4363)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4398..4417
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4477..4493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4477..4493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4490..4512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4501..4517
                     /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(4525..4531,4532..4549,4550..4555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4570..4591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4708..4725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4879..5464)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4959..4978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5635..6426,6427..6495))
                     /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     6258..6277
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6496..6600)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcgg ccgccaccgg atcccatgct gcgtcgcgcc accctggtgg
      961 acggcggcac cggcggcagc gagctcagct tctgcaacgt ctatatcaag gccgacaagc
     1021 agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac ggcggcgtgc
     1081 agctcgccta ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1141 acaaccacta cctgagcgtg cagtccaaac tttcgaaaga ccccaacgag aagcgcgatc
     1201 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     1261 acaagggcgg taccggaggg agcatggtga gcaagggcga ggagctgttc accggggtgg
     1321 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1381 agggtgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1441 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1501 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1561 acatccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1621 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1681 aggacggcaa catcctgggg cacaagctgg agtacaactt gctgcataag ttttctcaag
     1741 aacagatcgg cgaaaacatt gtgtgcaggg tcatttgtac cacgggtcaa attcccatcc
     1801 gagatttgtc agctgatatt tcacaagtgc ttaaggaaaa acgatccata aagaaagttt
     1861 ggacatttgg tagaaaccca gcctgtgact atcatttagg aaacatttca agactgtcaa
     1921 ataagcattt ccaaatacta ctaggagaag acggtaacct tttattgaat gacatttcca
     1981 ctaatgggac ctggttaaat gggcaaaaag tcgagaagaa cagcaatcag ttactgtctc
     2041 aaggtgatga aataaccgtt ggtgtaggcg tggaatcaga tattttatct ctggtcattt
     2101 tcataaacga caaatttaag cagtgcctcg agcagaacaa agttgatcgc taggaattcc
     2161 tgcagatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga
     2221 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2281 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2341 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2401 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2461 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2521 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2581 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2641 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2701 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2761 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2821 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2881 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     2941 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3001 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3061 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3121 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3181 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3241 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3301 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3361 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3421 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3481 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3541 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3601 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3661 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3721 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3781 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3841 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     3901 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3961 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4021 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4081 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4141 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4201 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4261 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4321 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4381 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4441 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4501 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4561 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4621 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4681 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4741 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4801 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4861 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4921 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4981 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5041 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5101 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5161 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5221 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5281 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5341 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5401 caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5461 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5521 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5581 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5641 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5701 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5761 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5821 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5881 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5941 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6001 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6061 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6121 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6181 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6241 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6301 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6361 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6421 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6481 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6541 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6601 cacatttccc cgaaaagtgc cacctgacgt c
//
