LOCUS       Exported                8375 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  FRET-based calcineurin (CaN) activation biosensor..
ACCESSION   .
VERSION     .
KEYWORDS    pcDN3.1(+)-CaNARi
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8375)
  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 8375)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8375
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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      920..929
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             926..1642
                     /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             3218..3937
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3969..3986)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3975..4199
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4245..4673
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4332..4351)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4542..4563
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4682..4702)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4687..5016
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4867..5002
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4929..4948
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5083..5877
                     /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(5137..5156)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5747..5766
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6051..6172
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6088..6107)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6142..6161
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6221..6237)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6221..6237)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6234..6256)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6245..6261
                     /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(6269..6299)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6314..6335
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6452..6469)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6623..7208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6703..6722)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7379..8239)
                     /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     8002..8021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8240..8344)
                     /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 gtttaaactt aagcttgccg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
      961 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaggttca gcgtgtccgg
     1021 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     1081 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctggggcg tgcagtgctt
     1141 cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     1201 ctacgtccag gagcgtacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1261 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1321 ggaggacggc aacatcctgg ggcacaagct ggagtacaac gccatcagtg acaacgtcta
     1381 tatcaccgcc gacaagcaga agaacggcat caaggcccac ttcaagatcc gccacaacat
     1441 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1501 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1561 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1621 cggcatggac gagctgtaca agggatccat gtccgagccc aaggcaattg atcccaagtt
     1681 gtcgacgacc gacagggtgg tgaaagctgt tccatttcct ccaagtcacc ggcttacagc
     1741 aaaagaagtg tttgataatg atggaaaacc tcgtgtggat atcttaaagg cgcatcttat
     1801 gaaggaggga aggctggaag agagtgttgc attgagaata ataacagagg gtgcatcaat
     1861 tcttcgacag gaaaaaaatt tgctggatat tgatgcgcca gtcactgttt gtggggacat
     1921 tcatggacaa ttctttgatt tgatgaagct ctttgaagtc gggggatctc ctgccaacac
     1981 tcgctacctc ttcttagggg actatgttga cagagggtac ttcagtattg aatgtgtgct
     2041 gtatttgtgg gccttgaaaa ttctctaccc caaaacactg tttttacttc gtggaaatca
     2101 tgaatgtaga catctaacag agtatttcac atttaaacaa gaatgtaaaa taaagtattc
     2161 agaacgagta tatgatgcct gtatggatgc ctttgactgc cttcccctgg ctgccctgat
     2221 gaaccaacag ttcctgtgtg tgcatggtgg tttgtctcca gagattaaca ctttagatga
     2281 tatcagaaaa ttagaccgat tcaaagaacc acctgcatat ggacctatgt gtgatatcct
     2341 gtggtcagac cccctggaag attttggaaa tgagaagact caggaacatt tcactcacaa
     2401 cacagtcagg gggtgttcat acttctacag ttacccggct gtatgtgaat tcttacagca
     2461 caataacttg ttatctatac tccgagccca cgaagcccaa gatgcagggt accgcatgta
     2521 caggaaaagc caaacaacag gcttcccttc tctaattaca attttttcag caccaaatta
     2581 cttagatgta tacaataaca aagctgcagt attgaagtat gagaacaatg ttatgaatat
     2641 caggcaattc aactgttctc ctcatccata ctggcttcca aatttcatgg atgtttttac
     2701 ttggtccctt ccatttgttg gggaaaaagt gactgagatg ctggtaaatg tcctcaacat
     2761 ctgctcagat gatgaactag ggtcagaaga agatggattt gatggtgcaa cagctgcagc
     2821 ccggaaagag gtgataagga acaagatccg agcaataggc aaaatggcca gagtgttctc
     2881 agtgctcaga gaagagagtg agagtgtgct gacgctgaaa ggcttgaccc caactggcat
     2941 gctccccagc ggagtacttt ctggagggaa gcaaaccctg caaagcgcta ctgttgaggc
     3001 tattgaggct gatgaagcta tcaaaggatt ttcaccacaa cataagatca ctagcttcga
     3061 ggaagccaag ggcttagacc gaattaatga gaggatgccg cctcgcagag atgccatgcc
     3121 ctctgacgcc aaccttaact ccatcaacaa ggctctcacc tcagagacta acggcacgga
     3181 cagcaatggc agtaatagca gcaatattca gctcgagatg gtgagcaagg gcgaggagct
     3241 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     3301 cagcgtgtcc ggtgagggcg agggcgatgc cacctacggc aagctgaccc tgaagctgat
     3361 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg
     3421 cctgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     3481 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     3541 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     3601 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     3661 ccacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca actttaagat
     3721 ccgccacaac atcgaggacg gcggcgtgca gctcgccgac cactaccagc agaacacccc
     3781 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccaagct
     3841 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     3901 cgggatcact ctcggcatgg acgagctgta caagtaatct agagggcccg tttaaacccg
     3961 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4021 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4081 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4141 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     4201 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     4261 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4321 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4381 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4441 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4501 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4561 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4621 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     4681 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4741 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     4801 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4861 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4921 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     4981 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     5041 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     5101 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     5161 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     5221 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     5281 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     5341 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     5401 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     5461 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     5521 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     5581 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     5641 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     5701 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     5761 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     5821 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     5881 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     5941 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     6001 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     6061 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     6121 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     6181 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     6241 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     6301 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     6361 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     6421 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     6481 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     6541 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6601 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6661 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6721 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6781 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6841 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6901 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6961 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     7021 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     7081 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     7141 aaaccaccgc tggtagcggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     7201 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     7261 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     7321 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7381 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7441 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7501 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7561 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7621 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7681 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7741 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7801 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7861 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7921 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7981 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     8041 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     8101 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     8161 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     8221 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     8281 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     8341 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
