LOCUS       sequence-363201-        7300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded FRET-based sensor for monitoring JNK kinase 
            activity near a protein of interest. Must pair with GFP11-tagged POI
            to reconstitute donor fluorescence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7300)
  AUTHORS   Tenner B, Zhang JZ, Kwon Y, Pessino V, Feng S, Huang B, Mehta S, 
            Zhang J
  TITLE     FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized 
            signaling within endogenous microdomains.
  JOURNAL   Sci Adv. 2021 May 21;7(21). pii: 7/21/eabe4091. doi: 
            10.1126/sciadv.abe4091. Print 2021 May.
  PUBMED    34020947
REFERENCE   2  (bases 1 to 7300)
  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: "Sci Adv."; 
            date: "2021-05-21"; volume: "7(21). pii: 7/21/eabe4091. doi"; pages:
            " 10.1126/sciadv.abe4091. Print 2021 May"
COMMENT     Sequence Label: pcDNA3.1(+)-FluoSTEP-JNKAR
FEATURES             Location/Qualifiers
     source          1..7300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        105..484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        485..688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     639..659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     733..752
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        733..751
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             789..1499
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RITVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREVAV
                     AKFAGLGGGMDELYK"
     CDS             2064..2705
                     /codon_start=1
                     /product="fragment of superfolder GFP consisting of the 
                     first 10 beta-strands"
                     /label=GFP(1-10)
                     /note="associates with GFP11, the 11th beta-strand, to 
                     reconstitute fluorescent GFP (Cabantous et al., 2005; 
                     Kamiyama et al., 2016)"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     KYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
     primer_bind     complement(2761..2778)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2767..2991
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3037..3465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3124..3143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3334..3355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3474..3494)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3479..3808
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3659..3794
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3721..3740
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3875..4669
                     /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(3929..3948)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4539..4558
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4843..4964
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4880..4899)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4934..4953
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5013..5029)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5013..5029)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5026..5048)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5037..5053
                     /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(5061..5067,5068..5085,5086..5091))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5106..5127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5244..5261)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5415..6003)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5495..5514)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6174..6965,6966..7034))
                     /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     6797..6816
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7035..7139)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7214..7233)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
       61 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      661 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      721 gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt
      781 aagcttggat ggtgtctaag ggcgaagagc tgatcaagga aaatatgcgt atgaaggtgg
      841 tcatggaagg ttcggtcaac ggccaccaat tcaaatgcac aggtgaagga gaaggcaatc
      901 cgtacatggg aactcaaacc atgaggatca cagtcatcga gggaggaccc ctgccatttg
      961 cctttgacat tcttgccacg tcgttcatgt atggcagccg tacttttatc aagtacccga
     1021 aaggcattcc tgatttcttt aaacagtcct ttcctgaggg ttttacttgg gaaagagtta
     1081 cgagatacga agatggtgga gtcgtcaccg tcatgcagga caccagcctt gaggatggct
     1141 gtctcgttta ccacgtccaa gtcagagggg taaactttcc ctccaatggt cccgtgatgc
     1201 agaagaagac caagggttgg gagcctaata cagagatgat gtatccagca gatggtggtc
     1261 tgaggggata cactcatatg gcactgaaag ttgatggtgg tggccatctg tcttgctctt
     1321 tcgtaacaac ttacaggtca aaaaagaccg tcgggaacat caagatgccc ggtatccatg
     1381 ccgttgatca ccgcctggaa aggttagagg aaagtgacaa tgaaatgttc gtagtacaac
     1441 gcgaagtcgc agttgccaag ttcgccgggc ttggtggtgg gatggacgag ctgtacaagc
     1501 gcatgcataa gttttctcaa gaacagatcg gcgaaaacat tgtgtgcagg gtcatttgta
     1561 ccacgggtca aattcccatc cgagatttgt cagctgatat ttcacaagtg cttaaggaaa
     1621 aacgatccat aaagaaagtt tggacatttg gtagaaaccc agcctgtgac tatcatttag
     1681 gaaacatttc aagactgtca aataagcatt tccaaatact actaggagaa gacggtaacc
     1741 ttttattgaa tgacatttcc actaatggga cctggttaaa tgggcaaaaa gtcgagaaga
     1801 acagcaatca gttactgtct caaggtgatg aaataaccgt tggtgtaggc gtggaatcag
     1861 atattttatc tctggtcatt ttcataaacg acaaatttaa gcagtgcctc gagcagaaca
     1921 aagttgatcg ctctgcaggt aagccaggca gcggcgaggg cagcaccaag ggcctggtcg
     1981 acagtgtcaa gacccccgag gatgaaggca acccactgct cgagcagctc gagaagaagg
     2041 gaggtacagg aggttcagaa cttatgtcca aaggagaaga actgtttacc ggtgttgtgc
     2101 caattttggt tgaactcgat ggtgatgtca acggacataa gttctcagtg agaggcgaag
     2161 gagaaggtga cgccaccatt ggaaaattga ctcttaaatt catctgtact actggtaaac
     2221 ttcctgtacc atggccgact ctcgtaacaa cgcttacgta cggagttcag tgcttttcga
     2281 gatacccaga ccatatgaaa agacatgact tttttaagtc ggctatgcct gaaggttacg
     2341 tgcaagaaag aacaatttcg ttcaaagatg atggaaaata taaaactaga gcagttgtta
     2401 aatttgaagg agatactttg gttaaccgca ttgaactgaa aggaacagat tttaaagaag
     2461 atggtaatat tcttggacac aaactcgaat acaattttaa tagtcataac gtatacatca
     2521 ctgctgataa gcaaaagaac ggaattaaag cgaatttcac agtacgccat aatgtagaag
     2581 atggcagtgt tcaacttgcc gaccattacc aacaaaacac ccctattgga gacggtccgg
     2641 tacttcttcc tgataatcac tacctctcaa cacaaacagt cctgagcaaa gatccaaatg
     2701 aaaaaggaac aggtggcggc ggaagttagt ctagagggcc cgtttaaacc cgctgatcag
     2761 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2821 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2881 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2941 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3001 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3061 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3121 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3181 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3241 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3301 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3361 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3421 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     3481 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3541 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3601 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3661 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     3721 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     3781 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttatatat ccattttcgg
     3841 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3901 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3961 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     4021 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     4081 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4141 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4201 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4261 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4321 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4381 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4441 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4501 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4561 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4621 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4681 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4741 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4801 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     4861 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4921 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4981 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5041 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5101 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5161 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5221 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5281 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5341 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5401 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5461 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5521 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5581 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5641 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5701 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5761 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5821 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5881 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5941 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6001 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6061 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6121 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6181 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6241 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6301 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6361 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6421 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6481 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6541 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6601 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6661 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6721 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6781 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6841 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6901 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6961 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7021 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7081 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7141 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     7201 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     7261 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
//
