LOCUS       Exported                7740 bp ds-DNA     circular SYN 24-JAN-2017
DEFINITION  Ratiometric Pericam as described by Nagai et al 2001 (PMID 11248055)
            with an enhanced tandem mitochondrial localisation sequence from 
            human COX8A (2mt8-Pericam) as described by Fillipin et al 2005 (PMID
            15589993), cloned into the NheI / EcoRI sites of a modified pEGFP 
            backbone, with the original EGFP cut out at the EcoRI / NotI sites 
            (blunted and re-ligated). Ratiometric Pericam is a fluorescent 
            [Ca2+] probe consisting of a modified permuted EYFP (i.e. its C- and
            N-terminus were exchanged and connected by a Gly-rich linker, and 
            the mutations F46L, Q69K, H148D, V163A, S175G, and Y203F were 
            introduced) flanked by Calmodulin (CaM; from X. laevis, identical to
            UniProt ID P62155 except for D65Y) at the C-terminus and by an M13 
            peptide (the CaM binding site (K566-L591) of human MYLK2) at the 
            N-terminus. Increase in [Ca2+] leads to binding of CaM to M13 and 
            thus structural changes resulting in a shift of the excitation 
            maximum from ~410 nm to ~495 nm, while the emission peak remains at 
            ~515 nm. The fluorescence intensity ratio of Ex495/Ex410 increases 
            up to tenfold in a [Ca2+]-dependent manner, from 10% at 0.1 µM or 
            lower to 100% at 10 µM or higher.
            
            The two mitochondria transit peptide sequences (purple) are 
            identical to the pre-sequence (M1-L29 and M1-G33, respectively) of 
            human COX8A (UniProt ID P10176).
            M13 peptide sequence (red) is identical to the Calmodulin binding 
            site (K566-L591) of human MYLK2 (Myosin light chain kinase 2; 
            UniProt ID Q9H1R3).
            EYFP sequence (yellow) is identical with the SnapGene EYFP sequence 
            except for F46L, Q69K, H148D, V163A, S175G.
            Calmodulin protein sequence (blue) is identical to that of X. laevis
            Calmodulin (UniProt ID P62155) except for the mutation D65Y caused 
            by GAC-->TAC.
ACCESSION   .
VERSION     .
KEYWORDS    PB079C
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7740)
  AUTHORS   Nagai T, Sawano A, Park ES, Miyawaki A.
  TITLE     Circularly permuted green fluorescent proteins engineered to sense 
            Ca2+.
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 2001;98:3197-202.
  PUBMED    11248055
REFERENCE   2  (bases 1 to 7740)
  AUTHORS   Filippin L, Abad MC, Gastaldello S, MagalhA£es PJ, SandonA  D, 
            Pozzan T.
  TITLE     Improved strategies for the delivery of GFP-based Ca2+ sensors into 
            the mitochondrial matrix.
  JOURNAL   Cell Calcium 2005;37:129-36.
  PUBMED    15589993
REFERENCE   3  (bases 1 to 7740)
  AUTHORS   Philip Bohler
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jan 25, 2017 from SnapGene 3.3.1
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7740
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    579..920
                     /note="MESV Psi"
                     /note="Psi packaging element from MESV (murine embryonic 
                     stem cell virus)"
     CDS             987..1403
                     /note="gag (truncated)"
     CDS             1422..2903
                     /codon_start=1
                     /product="Ratiometric Pericam as described by Nagai et al 
                     2001 (PMID 11248055) with an enhanced tandem mitochondrial 
                     localisation sequence from human COX8A (2mt8-Pericam) as 
                     described by Fillipin et al 2005 (PMID 15589993), cloned 
                     into the NheI / EcoRI sites of a modified pEGFP backbone, 
                     with the original EGFP cut out at the EcoRI / NotI sites 
                     (blunted and re-ligated). Ratiometric Pericam is a 
                     fluorescent [Ca2+] probe consisting of a modified permuted 
                     EYFP (i.e. its C- and N-terminus were exchanged and 
                     connected by a Gly-rich linker, and the mutations F46L, 
                     Q69K, H148D, V163A, S175G, and Y203F were introduced) 
                     flanked by Calmodulin (CaM; from X. laevis, identical to 
                     UniProt ID P62155 except for D65Y) at the C-terminus and by
                     an M13 peptide (the CaM binding site (K566-L591) of human 
                     MYLK2) at the N-terminus. Increase in [Ca2+] leads to 
                     binding of CaM to M13 and thus structural changes resulting
                     in a shift of the excitation maximum from ~410 nm to ~495 
                     nm, while the emission peak remains at ~515 nm. The 
                     fluorescence intensity ratio of Ex495/Ex410 increases up to
                     tenfold in a [Ca2+]-dependent manner, from 10% at 0.1 µM or
                     lower to 100% at 10 µM or higher.
                     
                     The two mitochondria transit peptide sequences (purple) are
                     identical to the pre-sequence (M1-L29 and M1-G33, 
                     respectively) of human COX8A (UniProt ID P10176).
                     M13 peptide sequence (red) is identical to the Calmodulin 
                     binding site (K566-L591) of human MYLK2 (Myosin light chain
                     kinase 2; UniProt ID Q9H1R3).
                     EYFP sequence (yellow) is identical with the SnapGene EYFP 
                     sequence except for F46L, Q69K, H148D, V163A, S175G.
                     Calmodulin protein sequence (blue) is identical to that of 
                     X. laevis Calmodulin (UniProt ID P62155) except for the 
                     mutation D65Y caused by GAC-->TAC."
                     /note="Pericam"
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAKIHSLGDPMSVLTPLLLRGLT
                     GSARRLPVPRAKIHSLPPEGDLKRRWKKNFIAVSAANRFKKISSSGALGSAGYNSDNVY
                     IMADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSFQSALSKD
                     PNEKRDHMVLLEFVTAAGITLGMDELYKVDGGSGGTGVSKGEELFTGVVPILVELDGDV
                     NGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQH
                     DFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK
                     LEYNGTDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINE
                     VDADGNGTIYFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAQLRHVMTNLG
                     EKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     promoter        2914..3413
                     /note="PGK promoter"
     CDS             3434..4033
                     /codon_start=1
                     /note="PuroR"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     LTR             4133..4647
                     /note="3' LTR"
     protein_bind    4809..4825
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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(4833..4863)
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     rep_origin      complement(5187..5775)
                     /direction=LEFT
                     /note="ori"
     CDS             complement(5946..6806)
                     /codon_start=1
                     /note="AmpR"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6807..6911)
                     /note="AmpR promoter"
     primer_bind     complement(7036..7054)
                     /note="pGEX-3 rvs (GATC)"
     primer_bind     7581..7596
                     /note="pET upstream primer (Novagen)"
                     /note="ATGCGTCCGGCGTAGA"
     LTR             7739..515
                     /note="5' LTR"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatccgctag catgtccgtc ctgacgccgc
     1441 tgctgctgcg gggcttgaca ggctcggccc ggcggctccc agtgccgcgc gccaagatcc
     1501 attcgttggg ggaccccatg tccgtcctga cgccgctgct gctgcggggc ttgacaggct
     1561 cggcccggcg gctcccagtg ccgcgcgcca agatccattc gttgccgccg gagggagatc
     1621 tcaagaggcg ctggaagaaa aacttcattg ccgtcagcgc tgccaaccgg ttcaagaaga
     1681 tctccagctc cggggcactg gggtctgcag gctacaacag cgacaacgtc tatatcatgg
     1741 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg
     1801 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1861 tgctgcccga caaccactac ctgagcttcc agtccgccct gagcaaagac cccaacgaga
     1921 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1981 acgagctgta caaggtcgac ggtggcagcg gtggcaccgg tgtgagcaag ggcgaggagc
     2041 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2101 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagctca
     2161 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg
     2221 gcctgaagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2281 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2341 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2401 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacggtaccg
     2461 accaactgac agaagagcag attgcagagt tcaaagaagc cttctcatta ttcgacaagg
     2521 atggggacgg caccatcacc acaaaggaac ttggcaccgt tatgaggtcg cttggacaaa
     2581 acccaacgga agcagaattg caggatatga tcaatgaagt cgatgctgat ggcaatggaa
     2641 cgatttactt tcctgaattt cttactatga tggctagaaa aatgaaggac acagacagcg
     2701 aagaggaaat ccgagaagca ttccgtgttt ttgacaagga tgggaacggc tacatcagcg
     2761 ctgctcagtt acgtcacgtc atgacaaacc tcggggagaa gttaacagat gaagaagttg
     2821 atgaaatgat aagggaagca gatatcgatg gtgatggcca agtaaactat gaagagtttg
     2881 tacaaatgat gacagcaaag taagaattct accgggtagg ggaggcgctt ttcccaaggc
     2941 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     3001 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     3061 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     3121 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
     3181 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     3241 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     3301 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     3361 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
     3421 gcccaagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     3481 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     3541 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     3601 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     3661 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     3721 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     3781 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     3841 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     3901 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     3961 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     4021 gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc
     4081 ccatgcatcg ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga
     4141 ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaat
     4201 acataactga gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg
     4261 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     4321 gtccccagat gcggtcccgc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     4381 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     4441 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     4501 ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc
     4561 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     4621 gactacccgt cagcgggggt ctttcatggg taacagtttc ttgaagttgg agaacaacat
     4681 tctgagggta ggagtcgaat attaagtaat cctgactcaa ttagccactg ttttgaatcc
     4741 acatactcca atactcctga aatagttcat tatggacagc gcagaaagag ctggggagaa
     4801 ttgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4861 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4921 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4981 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5041 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5101 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5161 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5221 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5281 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5341 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5401 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5461 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5521 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5581 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5641 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5701 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5761 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5821 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5881 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5941 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6001 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6061 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6121 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6181 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6241 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6301 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6361 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6421 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6481 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6541 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6601 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6661 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6721 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6781 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6841 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6901 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6961 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     7021 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     7081 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     7141 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     7201 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     7261 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     7321 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     7381 cgttgtaaaa cgacggcgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     7441 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     7501 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     7561 cgccggtgat gccggccacg atgcgtccgg cgtagaggcg attagtccaa tttgttaaag
     7621 acaggatatc agtggtccag gctctagttt tgactcaaca atatcaccag ctgaagccta
     7681 tagagtacga gccatagata aaataaaaga ttttatttag tctccagaaa aaggggggaa
//
