LOCUS       sequence-111768-        7851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mitochondria-targeted FRET biosensor for monitoring calcineurin 
            (CaN) phosphatase activity; targeted to cytosolic face of 
            mitochondrial outer membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7851)
  AUTHORS   Mehta S, Aye-Han NN, Ganesan A, Oldach L, Gorshkov K, Zhang J
  TITLE     Calmodulin-controlled spatial decoding of oscillatory Ca2+ signals 
            by calcineurin.
  JOURNAL   eLife. 2014 Jul 23;3:e03765. doi: 10.7554/eLife.03765.
  PUBMED    25056880
REFERENCE   2  (bases 1 to 7851)
  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: "eLife."; 
            date: "2014-07-23"; volume: "3:e03765. doi"; pages: " 
            10.7554/eLife.03765"
COMMENT     Sequence Label: pcDNA3-Mito-CaNAR2
FEATURES             Location/Qualifiers
     source          1..7851
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1000..1002,1003..1005,1006..1716)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     1653..1674
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(2623..2625,2626..2628,2629..3342)
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITEGMNELYK"
     promoter        complement(3403..3421)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3404..3421)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3441..3458)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3447..3671
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3717..4145
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3804..3823)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4014..4035
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4154..4174)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4159..4489
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4340..4475
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4402..4421
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4556..5350
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4610..4629)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5220..5239
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5524..5645
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5561..5580)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5615..5634
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5694..5710)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5694..5710)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5707..5729)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5718..5734
                     /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)."
     promoter        complement(join(5742..5748,5749..5766,5767..5772))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5787..5808
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5925..5942)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6096..6684)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6176..6195)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6855..7646,7647..7715))
                     /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     7478..7497
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7716..7820)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgccacc
      901 atggcaatcc agttgcgttc gctcttcccc ttggcgttgc ccggaatgct ggccctcctt
      961 ggctggtggt ggtttttctc tcgtaaaaaa gcggatccca tggtgagcaa gggcgaggag
     1021 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1081 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1141 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgagctgg
     1201 ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1261 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1321 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1381 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacgccatc
     1441 cacggcaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcggc
     1501 ctcaactgca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1561 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
     1621 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1681 gccgggatca ctctcggcat ggacgagctg tacaagcgca tgcccatgaa cgcccccgag
     1741 cggcagcccc aacccgacgg cggggacgcc ccaggccacg agcctggggg cagcccccaa
     1801 gacgagcttg acttctccat cctcttcgac tatgagtatt tgaatccgaa cgaagaagag
     1861 ccgaatgcac ataaggtcgc cagcccaccc tccggacccg cataccccga tgatgtaatg
     1921 gactatggcc tcaagccata cagccccctt gctagtctct ctggcgagcc ccccggccga
     1981 ttcggagagc cggatagggt agggccgcag aagtttctga gcgcggccaa gccagcaggg
     2041 gcctcgggcc tgagccctcg gatcgagatc actccgtccc acgaactgat ccaggcagtg
     2101 gggcccctcc gcatgagaga cgcgggcctc ctggtggagc agcctcccct ggccggggtg
     2161 gccgccagcc cgaggttcac cctgcccgtg cccggcttcg agggctaccg cgagccgctt
     2221 tgcttgagcc ccgctagcag cggctcctct gccagcttca tttctgacac cttctccccc
     2281 tacacctcgc cctgcgtctc gcccaataac ggcgggcccg acgacctgtg tccgcagttt
     2341 caaaacatcc ctgctcatta ttcccccaga acctcgccaa taatgtcacc tcgaaccagc
     2401 ctcgccgagg acagctgcct gggccgccac tcgcccgtgc cccgtccggc ctcccgctcc
     2461 tcatcgcctg gtgccaagcg gaggcattcg tgcgccgagg ccttggttgc cctgccgccc
     2521 ggagcctcac cccagcgctc ccggagcccc tcgccgcagc cctcatctca cgtggcaccc
     2581 caggaccacg gctccccggc tgggtacccc cctgttgagc tcatggtgag caaaggcgaa
     2641 gagctgttca ccggcgtggt gcccatcctg gtggagctgg acggcgacgt gaacggccac
     2701 aagttcagcg tgagcggcga gggcgagggc gacgccacct acggcaagct gaccctgaag
     2761 ctgctgtgca ccaccggcaa gctgcccgtg ccctggccca ccctggtgac caccctgggc
     2821 tacggcgtgc agtgcttcgc ccggtacccc gaccacatga agcagcacga cttcttcaag
     2881 agcgccatgc ccgagggcta cgtgcaggag cggaccatct tcttcaagga cgacggcaac
     2941 tacaagaccc gggccgaggt gaagttcgag ggcgacaccc tggtgaaccg gatcgagctg
     3001 aagggcatcg acttcaagga ggacggcaac atcctgggcc acaagctgga gtacaactac
     3061 aacagccaca acgtgtacat caccgccgac aagcagaaga acggcatcaa ggccaacttc
     3121 aagatccggc acaacatcga ggacggcggc gtgcagctgg ccgaccacta ccagcagaac
     3181 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagagc
     3241 gccctgttca aggaccccaa cgagaagcgg gaccacatgg tgctgctgga gttcctgacc
     3301 gccgccggca tcaccgaggg catgaacgag ctgtataagt aagaattctg cagatatcca
     3361 tcacactggc ggccgctcga gcatgcatct agagggccct attctatagt gtcacctaaa
     3421 tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3481 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3541 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3601 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3661 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3721 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3781 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3841 cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc
     3901 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3961 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4021 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4081 gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4141 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc
     4201 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     4261 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     4321 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4381 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     4441 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     4501 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
     4561 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4621 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4681 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     4741 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4801 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4861 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4921 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4981 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     5041 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     5101 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     5161 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     5221 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     5281 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     5341 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     5401 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     5461 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     5521 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5581 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5641 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     5701 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5761 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5821 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5881 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5941 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     6001 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     6061 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6121 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6181 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6241 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc
     6301 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6361 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6421 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6481 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     6541 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6601 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6661 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6721 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6781 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6841 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6901 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6961 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7021 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7081 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7141 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7201 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7261 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7321 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7381 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7441 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7501 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7561 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7621 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7681 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7741 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7801 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
