LOCUS       sequence-450136-        6632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Excitation-ratiometric biosensor for monitoring Protein Kinase Novel
            activity in living cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6632)
  AUTHORS   Bogomolovas J, Chen J
  TITLE     Illuminating understudied kinases: a generalizable biosensor 
            development method applied to protein kinase N.
  JOURNAL   Commun Biol. 2025 Jan 22;8(1):109. doi: 10.1038/s42003-025-07510-4.
  PUBMED    39843538
REFERENCE   2  (bases 1 to 6632)
  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: "Commun 
            Biol."; date: "2025-01-22"; volume: "8(1):109. doi"; pages: " 
            10.1038/s42003-025-07510-4"
COMMENT     Sequence Label: ExRai-PKNAR
FEATURES             Location/Qualifiers
     source          1..6632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1211..1230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1402..1781
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1782..1985
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1936..1956
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2030..2049
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2030..2048
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2389..2410
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2516..2537)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2777..2796)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(3411..3428)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3417..3641
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3687..4115
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3774..3793)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3984..4005
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4124..4144)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4289..4423
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4350..4369
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4504..5298
                     /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(4558..4577)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5168..5187
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5472..5593
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5509..5528)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5563..5582
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5642..5658)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5642..5658)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5655..5677)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5666..5682
                     /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(5690..5696,5697..5714,5715..5720))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5735..5756
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5873..5890)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6044..6632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6124..6143)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1381 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1441 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1501 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1561 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1621 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1681 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1741 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1801 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1861 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1921 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1981 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     2041 actataggga gacccaagct ggctagcgtt taaacttaag cttgcggccg ccaccggatc
     2101 catgaaggca cgactcaccg agggatgcac cttcagagac aagcagcatg gcggcaccgg
     2161 cggcagcgag ctcagcttct gcaacgtcta tatcaaggcc gacaagcaga agaacggcat
     2221 caaggcgaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgcctacca
     2281 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2341 gagcgtgcag tccaaacttt cgaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2401 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agggcggtac
     2461 cggagggagc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2521 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gtgagggcga
     2581 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2641 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2701 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctaca tccaggagcg
     2761 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     2821 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2881 cctggggcac aagctggagt acaacttgct gcataagttt tctcaagaac agatcggcga
     2941 aaacattgtg tgcagggtca tttgtaccac gggtcaaatt cccatccgag atttgtcagc
     3001 tgatatttca caagtgctta aggaaaaacg atccataaag aaagtttgga catttggtag
     3061 aaacccagcc tgtgactatc atttaggaaa catttcaaga ctgtcaaata agcatttcca
     3121 aatactacta ggagaagacg gtaacctttt attgaatgac atttccacta atgggacctg
     3181 gttaaatggg caaaaagtcg agaagaacag caatcagtta ctgtctcaag gtgatgaaat
     3241 aaccgttggt gtaggcgtgg aatcagatat tttatctctg gtcattttca taaacgacaa
     3301 atttaagcag tgcctcgagc agaacaaagt tgatcgctaa gaattctgca gatatccagc
     3361 acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     3421 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3481 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3541 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3601 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3661 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3721 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3781 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3841 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3901 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3961 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4021 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4081 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     4141 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4201 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4261 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccag ttccgcccat
     4321 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     4381 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     4441 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     4501 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     4561 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     4621 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     4681 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     4741 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4801 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4861 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4921 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4981 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     5041 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     5101 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     5161 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     5221 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     5281 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     5341 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     5401 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     5461 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     5521 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     5581 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     5641 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5701 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5761 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5821 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5881 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5941 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6001 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6061 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6121 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6181 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6241 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6301 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6361 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6421 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6481 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6541 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6601 agcagattac gcgcagaaaa aaaggatctc aa
//
