LOCUS       sequence-109348-        7794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasma membrane-targeted FRET biosensor for monitoring calcineurin 
            (CaN) phosphatase activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7794)
  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 7794)
  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-PM-CaNAR2
FEATURES             Location/Qualifiers
     source          1..7794
                     /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      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(946..948,949..951,952..1662)
                     /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(991..1012)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1252..1271)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1599..1620
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2569..3279
                     /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(3346..3364)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3347..3364)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3384..3401)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3390..3614
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3660..4088
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3747..3766)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3957..3978
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4097..4117)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4102..4432
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4283..4418
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4345..4364
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4499..5293
                     /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(4553..4572)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5163..5182
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5467..5588
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5504..5523)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5558..5577
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5637..5653)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5637..5653)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5650..5672)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5661..5677
                     /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(5685..5691,5692..5709,5710..5715))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5730..5751
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5868..5885)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6039..6627)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6119..6138)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6798..7589,7590..7658))
                     /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     7421..7440
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7659..7763)
                     /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 gcttgcggcc
      901 gccaccatgg gctgcatcaa gagcaagcgc aaggacaagg atcccatggt gagcaagggc
      961 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1021 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1081 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1141 agctggggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     1201 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1261 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1321 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1381 gccatccacg gcaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac
     1441 ttcggcctca actgcaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1501 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1561 tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1621 accgccgccg ggatcactct cggcatggac gagctgtaca agcgcatgcc catgaacgcc
     1681 cccgagcggc agccccaacc cgacggcggg gacgccccag gccacgagcc tgggggcagc
     1741 ccccaagacg agcttgactt ctccatcctc ttcgactatg agtatttgaa tccgaacgaa
     1801 gaagagccga atgcacataa ggtcgccagc ccaccctccg gacccgcata ccccgatgat
     1861 gtaatggact atggcctcaa gccatacagc ccccttgcta gtctctctgg cgagcccccc
     1921 ggccgattcg gagagccgga tagggtaggg ccgcagaagt ttctgagcgc ggccaagcca
     1981 gcaggggcct cgggcctgag ccctcggatc gagatcactc cgtcccacga actgatccag
     2041 gcagtggggc ccctccgcat gagagacgcg ggcctcctgg tggagcagcc tcccctggcc
     2101 ggggtggccg ccagcccgag gttcaccctg cccgtgcccg gcttcgaggg ctaccgcgag
     2161 ccgctttgct tgagccccgc tagcagcggc tcctctgcca gcttcatttc tgacaccttc
     2221 tccccctaca cctcgccctg cgtctcgccc aataacggcg ggcccgacga cctgtgtccg
     2281 cagtttcaaa acatccctgc tcattattcc cccagaacct cgccaataat gtcacctcga
     2341 accagcctcg ccgaggacag ctgcctgggc cgccactcgc ccgtgccccg tccggcctcc
     2401 cgctcctcat cgcctggtgc caagcggagg cattcgtgcg ccgaggcctt ggttgccctg
     2461 ccgcccggag cctcacccca gcgctcccgg agcccctcgc cgcagccctc atctcacgtg
     2521 gcaccccagg accacggctc cccggctggg tacccccctg ttgagctctc taaaggtgaa
     2581 gaattattca ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac
     2641 aaattttctg tctccggtga aggtgaaggt gatgctacgt acggtaaatt gaccttaaaa
     2701 ttactctgta ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaggt
     2761 tatggtgttc aatgttttgc tagataccca gatcatatga aacaacatga ctttttcaag
     2821 tctgccatgc cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac
     2881 tacaagacca gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta
     2941 aaaggtattg attttaaaga agatggtaac attttaggtc acaaattgga atacaactat
     3001 aactctcaca atgtttacat cactgctgac aaacaaaaga atggtatcaa agctaacttc
     3061 aaaattagac acaacattga agatggtggt gttcaattag ctgaccatta tcaacaaaat
     3121 actccaattg gtgatggtcc agtcttgtta ccagacaacc attacttatc ctatcaatct
     3181 gccttattca aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttttgact
     3241 gctgctggta ttaccgaggg tatgaatgaa ttgtacaaat agtaagaatt ctgcagatat
     3301 ccatcacact ggcggccgct cgagcatgca tctagagggc cctattctat agtgtcacct
     3361 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3421 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3481 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3541 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3601 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3661 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3721 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3781 gttcgccggc tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag
     3841 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3901 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3961 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4021 agggattttg gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4081 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4141 ggcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     4201 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     4261 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4321 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     4381 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     4441 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     4501 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     4561 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     4621 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4681 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     4741 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     4801 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     4861 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4921 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4981 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     5041 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     5101 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     5161 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     5221 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     5281 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     5341 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5401 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5461 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5521 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5581 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5641 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5701 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5761 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5821 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5881 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5941 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6001 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6061 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6121 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6181 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     6241 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6301 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6361 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6421 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     6481 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6541 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6601 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6661 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6721 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6781 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6841 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6901 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6961 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7021 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7081 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7141 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7201 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7261 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7321 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7381 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7441 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7501 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7561 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7621 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7681 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7741 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
