LOCUS       sequence-109351-        7855 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endoplasmic reticulum (ER)-targeted FRET biosensor for monitoring 
            calcineurin (CaN) phosphatase activity; targeted to cytosolic face 
            of ER membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7855)
  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 7855)
  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-ER-CaNAR2
FEATURES             Location/Qualifiers
     source          1..7855
                     /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"
     CDS             join(1007..1009,1010..1012,1013..1723)
                     /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     complement(1052..1073)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1313..1332)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1660..1681
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2630..3340
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     LCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEF
                     LTAAGITEGMNELYK"
     promoter        complement(3407..3425)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3408..3425)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3445..3462)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3451..3675
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3721..4149
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3808..3827)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4018..4039
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4158..4178)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4163..4493
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4344..4479
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4406..4425
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4560..5354
                     /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(4614..4633)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5224..5243
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5528..5649
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5565..5584)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5619..5638
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5698..5714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5698..5714)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5711..5733)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5722..5738
                     /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(5746..5752,5753..5770,5771..5776))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5791..5812
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5929..5946)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6100..6688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6180..6199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6859..7650,7651..7719))
                     /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     7482..7501
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7720..7824)
                     /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 gcttcagttg
      901 tcaggaagag aaggctccca tggaccctgt ggtggtgctg gggctctgtc tctcctgttt
      961 gcttctcctt tcactctgga aacagagcta tgggggaggg gatcccatgg tgagcaaggg
     1021 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1081 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1141 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1201 gagctggggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt
     1261 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1321 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1381 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1441 cgccatccac ggcaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa
     1501 cttcggcctc aactgcaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1561 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1621 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1681 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagcgcatgc ccatgaacgc
     1741 ccccgagcgg cagccccaac ccgacggcgg ggacgcccca ggccacgagc ctgggggcag
     1801 cccccaagac gagcttgact tctccatcct cttcgactat gagtatttga atccgaacga
     1861 agaagagccg aatgcacata aggtcgccag cccaccctcc ggacccgcat accccgatga
     1921 tgtaatggac tatggcctca agccatacag cccccttgct agtctctctg gcgagccccc
     1981 cggccgattc ggagagccgg atagggtagg gccgcagaag tttctgagcg cggccaagcc
     2041 agcaggggcc tcgggcctga gccctcggat cgagatcact ccgtcccacg aactgatcca
     2101 ggcagtgggg cccctccgca tgagagacgc gggcctcctg gtggagcagc ctcccctggc
     2161 cggggtggcc gccagcccga ggttcaccct gcccgtgccc ggcttcgagg gctaccgcga
     2221 gccgctttgc ttgagccccg ctagcagcgg ctcctctgcc agcttcattt ctgacacctt
     2281 ctccccctac acctcgccct gcgtctcgcc caataacggc gggcccgacg acctgtgtcc
     2341 gcagtttcaa aacatccctg ctcattattc ccccagaacc tcgccaataa tgtcacctcg
     2401 aaccagcctc gccgaggaca gctgcctggg ccgccactcg cccgtgcccc gtccggcctc
     2461 ccgctcctca tcgcctggtg ccaagcggag gcattcgtgc gccgaggcct tggttgccct
     2521 gccgcccgga gcctcacccc agcgctcccg gagcccctcg ccgcagccct catctcacgt
     2581 ggcaccccag gaccacggct ccccggctgg gtacccccct gttgagctct ctaaaggtga
     2641 agaattattc actggtgttg tcccaatttt ggttgaatta gatggtgatg ttaatggtca
     2701 caaattttct gtctccggtg aaggtgaagg tgatgctacg tacggtaaat tgaccttaaa
     2761 attactctgt actactggta aattgccagt tccatggcca accttagtca ctactttagg
     2821 ttatggtgtt caatgttttg ctagataccc agatcatatg aaacaacatg actttttcaa
     2881 gtctgccatg ccagaaggtt atgttcaaga aagaactatt tttttcaaag atgacggtaa
     2941 ctacaagacc agagctgaag tcaagtttga aggtgatacc ttagttaata gaatcgaatt
     3001 aaaaggtatt gattttaaag aagatggtaa cattttaggt cacaaattgg aatacaacta
     3061 taactctcac aatgtttaca tcactgctga caaacaaaag aatggtatca aagctaactt
     3121 caaaattaga cacaacattg aagatggtgg tgttcaatta gctgaccatt atcaacaaaa
     3181 tactccaatt ggtgatggtc cagtcttgtt accagacaac cattacttat cctatcaatc
     3241 tgccttattc aaagatccaa acgaaaagag agaccacatg gtcttgttag aatttttgac
     3301 tgctgctggt attaccgagg gtatgaatga attgtacaaa tagtaagaat tctgcagata
     3361 tccatcacac tggcggccgc tcgagcatgc atctagaggg ccctattcta tagtgtcacc
     3421 taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     3481 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3541 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3601 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3661 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     3721 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3781 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3841 cgttcgccgg ctttccccgt caagctctaa atcggggcat ccctttaggg ttccgattta
     3901 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3961 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4021 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4081 aagggatttt ggggatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4141 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     4201 aggcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     4261 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4321 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4381 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     4441 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     4501 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     4561 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4621 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4681 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4741 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4801 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4861 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4921 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4981 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     5041 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     5101 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     5161 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     5221 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     5281 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5341 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     5401 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     5461 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     5521 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5581 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5641 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     5701 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5761 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5821 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5881 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5941 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     6001 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6061 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6121 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6181 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6241 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
     6301 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6361 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6421 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6481 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6541 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6601 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6661 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6721 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6781 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6841 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6901 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6961 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7021 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7081 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7141 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7201 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7261 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7321 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7381 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7441 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7501 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7561 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7621 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7681 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7741 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7801 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
