LOCUS       sequence-8821-ex        5929 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5929)
  AUTHORS   Harvey CD, Ehrhardt AG, Cellurale C, Zhong H, Yasuda R, Davis RJ, 
            Svoboda K.
  TITLE     A genetically encoded fluorescent sensor of ERK activity
  JOURNAL   PNAS 2008 Dec 9;105(49):19264-9
  PUBMED    19033456
REFERENCE   2  (bases 1 to 5929)
  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: "PNAS"; 
            date: "2008-12-9"; volume: "105(49)"; pages: "19264-"
COMMENT     Sequence Label: nuclear EKAR (EGFP-mRFP)
FEATURES             Location/Qualifiers
     source          1..5929
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..742
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1034..1052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1058..1732
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             1058..1732
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(1188..1206)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1456..1475
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1650..1673
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             join(2300..2302,2303..2305,2306..3019)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2345..2366)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2606..2625)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2953..2974
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3043..3164
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3054..3073)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3108..3127
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3345..3800
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3432..3451)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3642..3663
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3867..3886)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3986..4008
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4046..4064)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4132..4236
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4237..4305,4306..5097)
                     /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     complement(4455..4474)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5268..5856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5757..5776
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt acttaatacg actcactata ggctagcatg gcctcctccg aggacgtcat
     1081 caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc acgagttcga
     1141 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     1201 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tccagtacgg
     1261 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     1321 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     1381 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     1441 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga
     1501 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagatgaggc tgaagctgaa
     1561 ggacggcggc cactacgacg ccgaggtcaa gaccacctac atggccaaga agcccgtgca
     1621 gctgcccggc gcctacaaga ccgacatcaa gctggacatc acctcccaca acgaggacta
     1681 caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggcg ccgagctcaa
     1741 gcttatggcg gacgaggaga agctgccgcc cggctgggag aagcgcatga gccgcagctc
     1801 aggccgagtg tactacttca accacatcac taacgccagc cagtgggagc ggcccagcgg
     1861 caacagcagc agtggtggca aaaacgggca gggggagcct gccaggaagc ttcgaattct
     1921 gcagtcgact agggagcaag ggcttgtgtc tagaggcgga ggtggtggag gaggtggagg
     1981 tggtggaggt ggaggtggag gtggtggcgg tggtggagga ggaggagcta gaggcggagg
     2041 tggtggagga ggtggaggtg gtggaggtgg aggtggaggt ggtggcggtg gtggaggagg
     2101 aggagctagc ggcggaggtg gtggaggagg tggaggtggt ggaggtggag gtggaggtgg
     2161 tggcggtggt ggaggaggag gaatggctga ccaactgact gaagagtggt cgacggtacc
     2221 gcgggatatc cctgacgtac cacgtacacc agtaggaaag gcaaagctgt cattccaatt
     2281 cccgcgggcc cgggatccca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     2341 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2401 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2461 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2521 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2581 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2641 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2701 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2761 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2821 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2881 gctgctgccc gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga
     2941 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     3001 ggacgagctg tacaagtaag cggccgcttc gagcagacat gataagatac attgatgagt
     3061 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     3121 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     3181 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc
     3241 tctacaaatg tggtaaaatc gataaggatc cgggctggcg taatagcgaa gaggcccgca
     3301 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg
     3361 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3421 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3481 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3541 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3601 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3661 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3721 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3781 aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3841 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3901 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3961 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4021 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4081 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4141 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4201 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4261 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4321 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4381 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4441 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     4501 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4561 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4621 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4681 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4741 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4801 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4861 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4921 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4981 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5041 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5101 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5161 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5221 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5281 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5341 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5401 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5461 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5521 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5581 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5641 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5701 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5761 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5821 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5881 cggttcctgg ccttttgctg gccttttgct cacatggctc gacagatct
//
