LOCUS       Exported                5981 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    cytoplasmic EKAR (EGFP-mRFP)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5981)
  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 5981)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5981
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /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             2300..3016
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2300..3016
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     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    3095..3216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3106..3125)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3160..3179
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3397..3852
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3484..3503)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3694..3715
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3919..3938)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4038..4060
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4098..4116)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4184..4288
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4289..5149
                     /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(4507..4526)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5320..5908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5809..5828
                     /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 gcggaccggt 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 ccagtccgcc ctgagcaaag accccaacga
     2941 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     3001 ggacgagctg tacaaggatg ctcttcaaaa aaaacttgaa gaacttgaac ttgacgaatg
     3061 aactagtatc tgcggccgct tcgagcagac atgataagat acattgatga gtttggacaa
     3121 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     3181 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     3241 atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa
     3301 tgtggtaaaa tcgataagga tccgggctgg cgtaatagcg aagaggcccg caccgatcgc
     3361 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
     3421 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3481 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3541 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3601 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3661 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3721 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3781 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3841 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3901 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     3961 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4021 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4081 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4141 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4201 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4261 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4321 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4381 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4441 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4501 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4561 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4621 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4681 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4741 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4801 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     4861 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4921 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4981 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     5041 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5101 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5161 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5221 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5281 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5341 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5401 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5461 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5521 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5581 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5641 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5701 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5761 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5821 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5881 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5941 ggccttttgc tggccttttg ctcacatggc tcgacagatc t
//
