LOCUS       sequence-8820-ex        6701 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 6701)
  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 6701)
  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 (Cerulean-Venus)
FEATURES             Location/Qualifiers
     source          1..6701
                     /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"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      934..943
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(940..942,943..945,946..1656)
                     /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(2221..2223,2224..2226,2227..2940)
                     /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      2946..2955
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2959..3093
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2996..3015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3050..3069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3166..3523
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3166..3357
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3189..3209)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3374..3509
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3543..3560)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3543..3559)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3552..3574)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3772..4227
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3859..3878)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4069..4090
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4244..4263)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4398..4420
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4473..4491)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4559..4663
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4664..4732,4733..5524)
                     /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(4882..4901)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5695..6283
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6184..6203
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6437..6454
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(6607..6612,6613..6630,6631..6637)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6645..6661
                     /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     6650..6672
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6669..6685
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6669..6685
                     /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 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcgtaccg gtcgccacca tggtgagcaa gggcgaggag
      961 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1021 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1081 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctgg
     1141 ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1201 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1261 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1321 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacgccatc
     1381 agcgacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     1441 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1501 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
     1561 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1621 gccgggatca ctctcggcat ggacgagctg tacaagcgga tccatatggc ggacgaggag
     1681 aagctgccgc ccggctggga gaagcgcatg agccgcagct caggccgagt gtactacttc
     1741 aaccacatca ctaacgccag ccagtgggag cggcccagcg gcaacagcag cagtggtggc
     1801 aaaaacgggc agggggagcc tgccaggaag cttcgaattc tgcagtcgac tagggagcaa
     1861 gggcttgtgt ctagaggcgg aggtggtgga ggaggtggag gtggtggagg tggaggtgga
     1921 ggtggtggcg gtggtggagg aggaggagct agaggcggag gtggtggagg aggtggaggt
     1981 ggtggaggtg gaggtggagg tggtggcggt ggtggaggag gaggagctag cggcggaggt
     2041 ggtggaggag gtggaggtgg tggaggtgga ggtggaggtg gtggcggtgg tggaggagga
     2101 ggaatggctg accaactgac tgaagagtgg tcgacggtac cgcggaccgg tcctgacgta
     2161 ccacgtacac cagtaggaaa ggcaaagctg tcattccaat tcccgcgggc ccgggatatc
     2221 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2281 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2341 ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2401 ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
     2461 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2521 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2581 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2641 aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac
     2701 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc
     2761 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2821 tacctgagct accagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2881 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     2941 gcttggccgc catggcccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3001 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3061 aaactcatca atgtatctta tcatgtctgg atcgatcggg aattaattcg gcgcagcacc
     3121 atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa
     3181 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3241 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3301 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3361 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3421 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     3481 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctgt taacagcttg
     3541 gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     3601 cgccttgcag cacatccccc cttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     3661 cgcccttccc aacagttgcg tagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     3721 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3781 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3841 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3901 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3961 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     4021 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4081 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     4141 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4201 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     4261 ccgcatagtt aagccaactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4321 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4381 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4441 acgcgcgagg cagtattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt
     4501 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     4561 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4621 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4681 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4741 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4801 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4861 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgatg acgccgggca
     4921 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4981 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5041 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5101 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5161 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgccagcag caatggcaac
     5221 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5281 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5341 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5401 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5461 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5521 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5581 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5641 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5701 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5761 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5821 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     5881 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5941 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6001 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6061 cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg aagggagaaa
     6121 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6181 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6241 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6301 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     6361 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     6421 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     6481 accgcctctc cccgcgcgtt ggccgattca ttaatccagc tggcacgaca ggtttcccga
     6541 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tacctcactc attaggcacc
     6601 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     6661 atttcacaca ggaaacagct atgaccatga ttacgaatta a
//
