LOCUS       sequence-345234-        7226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Red/far-red FRET-based ERK activity reporter for multiplexed imaging
            applications..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7226)
  AUTHORS   Mo GCH, Posner C, Rodriguez EA, Sun T, Zhang J
  TITLE     A rationally enhanced red fluorescent protein expands the utility of
            FRET biosensors.
  JOURNAL   Nat Commun. 2020 Apr 15;11(1):1848. doi: 10.1038/s41467-020-15687-x.
  PUBMED    32296061
REFERENCE   2  (bases 1 to 7226)
  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 
            Commun."; date: "2020-04-15"; volume: "11(1):1848. doi"; pages: " 
            10.1038/s41467-020-15687-x"
COMMENT     Sequence Label: pcDNA3-smURFP-StagRFP-EKAR-EV
FEATURES             Location/Qualifiers
     source          1..7226
                     /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"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /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(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1009..1407
                     /codon_start=1
                     /product="dimeric small ultra-red fluorescent protein 
                     derived from a cyanobacterial phycobiliprotein (Rodriguez 
                     et al., 2016)"
                     /label=smURFP
                     /translation="MAKTSEQRVNIATLLTENKKKIVDKASQDLWRRHPDLIAPGGIAF
                     SQRDRALCLRDYGWFLHLITFCLLAGDKGPIESIGLISIREMYNSLGVPVPAMMESIRC
                     LKEASLSLLDEEDANETAPYFDYIIKAMS"
     CDS             1600..1947
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             2023..2718
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTVMALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHK"
     promoter        complement(2779..2797)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2780..2797)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2817..2834)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2823..3047
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3093..3521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3180..3199)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3390..3411
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3530..3550)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3535..3864
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3715..3850
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3777..3796
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3931..4725
                     /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(3985..4004)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4595..4614
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4899..5020
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4936..4955)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4990..5009
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5069..5085)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5069..5085)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5082..5104)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5093..5109
                     /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(5117..5123,5124..5141,5142..5147))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5162..5183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5300..5317)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5471..6059)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5551..5570)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6230..7021,7022..7090))
                     /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     6853..6872
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7091..7195)
                     /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 gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat ggctaagact
     1021 tccgaacaga gggtgaacat tgctacactg ctgacagaaa ataagaagaa aatcgtggat
     1081 aaggcttccc aggatctgtg gcggagacac ccagacctga tcgcaccagg aggaattgct
     1141 ttctctcaga gggaccgcgc tctgtgcctg cgagattacg gctggttcct gcatctgatc
     1201 accttttgtc tgctggccgg agataagggc cccatcgagt ctattgggct gatcagtatt
     1261 cgagaaatgt ataactcact gggagtgccc gtccctgcaa tgatggagag cattagatgc
     1321 ctgaaagaag ccagcctgtc cctgctggac gaagaggacg ccaacgagac cgcaccctac
     1381 tttgattaca ttattaaggc tatgagcgac gagctgtaca agcgcatgca catggcggac
     1441 gaggagaagc tgccgcccgg ctgggagaag cgcatgagcc gcagctcagg ccgagtgtac
     1501 tacttcaacc acatcactaa cgccagccag tgggagcggc ccagcggcaa cagcagcagt
     1561 ggtggcaaaa acgggcaggg ggagcctgcc aggggtacca gtgctggtgg tagtgctggt
     1621 ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt ccggcagtgc tggtggtagt
     1681 gctggtggta gtaccagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     1741 ggtagtgctg gtggttccgg cagtgctggt ggtagtgctg gtggtagtac cagtgctggt
     1801 ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg ttccggcagt
     1861 gctggtggta gtgctggtgg tagtaccagt gctggtggta gtgctggtgg tagtgctggt
     1921 ggtagtgctg gtggtagtgc tggtggttcc ggaccagatg tccctagaac tccagtggat
     1981 aaagcaaagc tgtcattcca attcccccgc gcacgggagc tcatgagcga gctgattaag
     2041 gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca cttcaagtgc
     2101 acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc
     2161 gagggcggcc ctctcccctt cgccttcgac atcctggcta ccagcttcat gtacggcagc
     2221 agaaccttca tcaaccacac ccagggcatc cccgatttct ttaagcagtc cttccctgag
     2281 ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag
     2341 gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc
     2401 ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggccaa caccgagatg
     2461 ctgtaccccg ctgacggcgg cctggaaggc agaaccgtca tggccctgaa gctcgtgggc
     2521 gggggccacc tgatctgcaa cttcaagacc acatacagat ccaagaaacc cgctaagaac
     2581 ctcaagatgc ccggcgtcta ctatgtggac cacagactgg aaagaatcaa ggaggccgac
     2641 aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga cctccctagc
     2701 aaactggggc acaagtaaga attctgcaga tatccatcac actggcggcc gctcgagcat
     2761 gcatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     2821 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2881 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2941 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3001 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3061 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3121 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3181 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3241 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3301 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3361 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3421 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3481 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3541 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3601 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3661 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3721 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3781 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3841 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     3901 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     3961 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4021 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4081 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4141 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4201 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4261 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4321 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4381 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4441 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4501 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4561 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4621 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4681 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4741 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4801 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4861 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4921 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4981 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5041 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5101 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5161 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5221 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5281 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5341 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5401 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5461 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5521 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5581 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5641 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5701 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5761 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5821 cagccactgg taacaggatt agcaaagcga ggtatgtagg cggtgctaca gagttcttga
     5881 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5941 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6001 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6061 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6121 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6181 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6241 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6301 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6361 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6421 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6481 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6541 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6601 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6661 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6721 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6781 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6841 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6901 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6961 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7021 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7081 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7141 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7201 ttccccgaaa agtgccacct gacgtc
//
