LOCUS       sequence-109681-        6967 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Endoplasmic reticulum (ER)-targeted version of a high-affinity 
            FRET-based biosensor for monitoring Ca2+/CaM levels; targeted to 
            cytosolic face of ER membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6967)
  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 6967)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pcDNA3.1(+)-ER-BSCaM2
FEATURES             Location/Qualifiers
     source          1..6967
                     /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(1012..1014,1015..1017,1018..1728)
                     /codon_start=1
                     /product="enhanced yellow variant of GFP"
                     /label=Topaz YFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1804..2517
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHRFSVSGKGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAH
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(2561..2578)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2567..2791
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2837..3265
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2924..2943)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3134..3155
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3274..3294)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3279..3608
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3459..3594
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3521..3540
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3675..4469
                     /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(3729..3748)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4339..4358
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4643..4764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4680..4699)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4734..4753
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4813..4829)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4813..4829)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4826..4848)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4837..4853
                     /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(4861..4867,4868..4885,4886..4891))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4906..4927
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5044..5061)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5215..5800)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5295..5314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5971..6762,6763..6831))
                     /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     6594..6613
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6832..6936)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 gctggctagc
      901 gccaccatgg accctgtggt ggtgctgggg ctctgtctct cctgtttgct tctcctttca
      961 ctctggaaac agagctatgg gggagggaag cttggtaccg agctcggatc catggtgagc
     1021 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1081 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1141 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1201 accttcggct acggcgtgca gtgcttcgcc cgctaccccg accacatgcg ccagcacgac
     1261 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1321 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1381 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1441 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1501 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1561 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1621 taccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1681 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg taccagcagc
     1741 cgtcagaaat ggcagaaaac cggacatgcg gtgcgtgcga ttggccgtct ggctgctacc
     1801 ggtagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1861 gacgtaaacg gccacaggtt cagcgtgtcc ggcaagggcg agggcgatgc cacctacggc
     1921 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1981 gtgaccaccc tgacctgggg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     2041 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgtac catcttcttc
     2101 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2161 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2221 ctggagtaca actacatcag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc
     2281 atcaaggccc acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2341 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2401 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2461 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtgactc
     2521 gagcatgcat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     2581 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2641 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2701 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2761 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2821 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2881 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2941 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3001 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3061 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3121 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3181 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3241 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     3301 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3361 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3421 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3481 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3541 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3601 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     3661 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3721 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3781 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3841 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3901 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3961 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     4021 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     4081 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     4141 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4201 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4261 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4321 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4381 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4441 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     4501 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     4561 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4621 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     4681 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4741 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     4801 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4861 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4921 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4981 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5041 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5101 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5161 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5221 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5281 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5341 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5401 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5461 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5521 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5581 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5641 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5701 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtttttttgt
     5761 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5821 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5881 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5941 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6001 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6061 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6121 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6181 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6241 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6301 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6361 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6421 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6481 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6541 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6601 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6661 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6721 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6781 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6841 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6901 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6961 tgacgtc
//
