LOCUS       sequence-140025-        6758 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 6758)
  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 6758)
  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: cytoplasmic EKAR (Cerulean-Venus)
FEATURES             Location/Qualifiers
     source          1..6758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        827..845
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     827..844
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     858..877
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      933..942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(939..941,942..944,945..1655)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2220..2222,2223..2225,2226..2936)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      3007..3016
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3020..3154
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3057..3076)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3111..3130
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3223..3580
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3223..3414
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3246..3266)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3431..3566
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3600..3617)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3600..3616)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3609..3631)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3829..4284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3916..3935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4126..4147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4301..4320)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4455..4477
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4530..4548)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4616..4720
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4721..4789,4790..5581)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRDDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4939..4958)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5752..6340
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6241..6260
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6494..6511
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(6664..6669,6670..6687,6688..6694)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6702..6718
                     /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)."
     primer_bind     6707..6729
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6726..6742
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6726..6742
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac
      661 accgggaccg atccagcctc cgcggccggg aacggtgcat tggaacgcgg attccccgtg
      721 ccaagagtga cgtaagtacc gcctatagag tctataggcc cacccccttg gcttcgttag
      781 aacgcggcta caattaatac ataaccttat gtatcataca catacgattt aggtgacact
      841 atagaataac atccactttg cctttctctc cacaggtgtc cactcccagg tccaactgca
      901 cctcggttct atcgattgaa ttcgtaccgg tcgccaccat ggtgagcaag ggcgaggagc
      961 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1021 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1081 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctggg
     1141 gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1201 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1261 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1321 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacgccatca
     1381 gcgacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     1441 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1501 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc
     1561 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1621 ccgggatcac tctcggcatg gacgagctgt acaagcggat ccatatggcg gacgaggaga
     1681 agctgccgcc cggctgggag aagcgcatga gccgcagctc aggccgagtg tactacttca
     1741 accacatcac taacgccagc cagtgggagc ggcccagcgg caacagcagc agtggtggca
     1801 aaaacgggca gggggagcct gccaggaagc ttcgaattct gcagtcgact agggagcaag
     1861 ggcttgtgtc tagaggcgga ggtggtggag gaggtggagg tggtggaggt ggaggtggag
     1921 gtggtggcgg tggtggagga ggaggagcta gaggcggagg tggtggagga ggtggaggtg
     1981 gtggaggtgg aggtggaggt ggtggcggtg gtggaggagg aggagctagc ggcggaggtg
     2041 gtggaggagg tggaggtggt ggaggtggag gtggaggtgg tggcggtggt ggaggaggag
     2101 gaatggctga ccaactgact gaagagtggt cgacggtacc gcggaccggt cctgacgtac
     2161 cacgtacacc agtaggaaag gcaaagctgt cattccaatt cccgcgggcc cgggatatca
     2221 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     2281 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     2341 gcaagctgac cctgaagctg atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     2401 tcgtgaccac cctgggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc
     2461 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2521 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2581 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2641 agctggagta caactacaac agccacaacg tctatatcac cgccgacaag cagaagaacg
     2701 gcatcaaggc caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg
     2761 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2821 acctgagcta ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2881 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggatg
     2941 ctcttcaaaa aaaacttgaa gaacttgaac ttgacgaatg atctagagtc gacctgcaga
     3001 agcttggccg ccatggccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3061 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3121 caaactcatc aatgtatctt atcatgtctg gatcgggaat taattcggcg cagcaccatg
     3181 gcctgaaata acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca
     3241 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3301 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3361 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3421 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3481 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3541 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctgttaa cagcttggca
     3601 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     3661 cttgcagcac atcccccctt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     3721 ccttcccaac agttgcgtag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
     3781 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     3841 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3901 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3961 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4021 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4081 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4141 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     4201 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4261 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     4321 catagttaag ccaactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     4381 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4441 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4501 cgcgaggcag tattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4561 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     4621 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4681 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4741 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4801 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4861 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4921 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgatgacg ccgggcaaga
     4981 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5041 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5101 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5161 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5221 gaatgaagcc ataccaaacg acgagcgtga caccacgatg ccagcagcaa tggcaacaac
     5281 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5341 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5401 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     5461 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5521 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5581 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5641 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5701 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5761 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5821 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5881 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5941 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6001 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6061 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6121 actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc
     6181 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6241 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6301 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6361 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6421 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6481 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     6541 gcctctcccc gcgcgttggc cgattcatta atccagctgg cacgacaggt ttcccgactg
     6601 gaaagcgggc agtgagcgca acgcaattaa tgtgagttac ctcactcatt aggcacccca
     6661 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     6721 tcacacagga aacagctatg accatgatta cgaattaa
//
