LOCUS       sequence-109352-        7492 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endoplasmic reticulum (ER)-targeted FRET-based PKA activity 
            reporter; targeted to cytosolic face of ER membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7492)
  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 7492)
  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-AKAR4
FEATURES             Location/Qualifiers
     source          1..7492
                     /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="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2246..2980
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     promoter        complement(3044..3062)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3045..3062)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3082..3099)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3088..3312
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3358..3786
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3445..3464)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3655..3676
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3795..3815)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3800..4130
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3981..4116
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4043..4062
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4197..4991
                     /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(4251..4270)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4861..4880
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5165..5286
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5202..5221)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5256..5275
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5335..5351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5335..5351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5348..5370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5359..5375
                     /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(5383..5389,5390..5407,5408..5413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5428..5449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5566..5583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5737..6325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5817..5836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6496..7287,7288..7356))
                     /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     7119..7138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7357..7461)
                     /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 ccacaggttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1141 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1201 gacctggggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt
     1261 caagtccgcc atgcccgaag gctacgtcca ggagcgtacc atcttcttca aggacgacgg
     1321 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1381 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1441 cgccatcagt gacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccca
     1501 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1561 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1621 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1681 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagcgcatgc ataagttttc
     1741 tcaagaacag atcggcgaaa acattgtgtg cagggtcatt tgtaccacgg gtcaaattcc
     1801 catccgagat ttgtcagctg atatttcaca agtgcttaag gaaaaacgat ccataaagaa
     1861 agtttggaca tttggtagaa acccagcctg tgactatcat ttaggaaaca tttcaagact
     1921 gtcaaataag catttccaaa tactactagg agaagacggt aaccttttat tgaatgacat
     1981 ttccactaat gggacctggt taaatgggca aaaagtcgag aagaacagca atcagttact
     2041 gtctcaaggt gatgaaataa ccgttggtgt aggcgtggaa tcagatattt tatctctggt
     2101 cattttcata aacgacaaat ttaagcagtg cctcgagcag aacaaagttg atcgctctgc
     2161 aggtaagcca ggcagcggcg agggcagcac caagggcctg cgtcgcgcca ccctggtgga
     2221 cggcggcacc ggcggcagcg agctcatggg cggcgtgcag ctcgccgacc actaccagca
     2281 gaacaccccc atcggcgacg gccccgtgct gctgccngac aaccactacc tgagctacca
     2341 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     2401 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggaggta ccggtggatc
     2461 tatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2521 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2581 cggcaagctg accctgaagc tgatctgcac caccggcaag ctgcccgtgc cctggcccac
     2641 cctcgtgacc accctgggct acggccttca gtgcttcgcc cgctaccccg accacatgaa
     2701 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2761 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2821 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2881 caagctggag tacaactaca acagccacaa cgtctatatc accgccgaca agcagaagaa
     2941 cggcatcaag gccaacttca agatccgcca caacatcgag taagaattct gcagatatcc
     3001 atcacactgg cggccgctcg agcatgcatc tagagggccc tattctatag tgtcacctaa
     3061 atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     3121 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     3181 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     3241 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     3301 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     3361 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3421 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3481 tcgccggctt tccccgtcaa gctctaaatc ggggcatccc tttagggttc cgatttagtg
     3541 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3601 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3661 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3721 ggattttggg gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3781 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagg
     3841 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     3901 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3961 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4021 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     4081 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     4141 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     4201 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     4261 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4321 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     4381 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4441 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4501 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4561 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4621 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4681 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     4741 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4801 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4861 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4921 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4981 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     5041 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     5101 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5161 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5221 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5281 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5341 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5401 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5461 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5521 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5581 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5641 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5701 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5761 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5821 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5881 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     5941 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6001 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6061 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6121 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     6181 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6241 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6301 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6361 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6421 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6481 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6541 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6601 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6661 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6721 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6781 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6841 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6901 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6961 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7021 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7081 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7141 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7201 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7261 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7321 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7381 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7441 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
