LOCUS       Exported                7857 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  FRET-based biosensor for monitoring calcineurin (CaN) phosphatase 
            activity..
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3-CaNAR2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7857)
  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 7857)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7857
                     /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      901..910
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             979..1002
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1054..1075)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1315..1334)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1662..1683
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2632..3342
                     /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(3409..3427)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3410..3427)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3447..3464)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3453..3677
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3723..4151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3810..3829)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4020..4041
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4160..4180)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4165..4495
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4346..4481
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4408..4427
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4562..5356
                     /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(4616..4635)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5226..5245
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5530..5651
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5567..5586)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5621..5640
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5700..5716)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5700..5716)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5713..5735)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5724..5740
                     /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(5748..5778)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5793..5814
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5931..5948)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6102..6690)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6182..6201)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6861..7721)
                     /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     7484..7503
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7722..7826)
                     /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 tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat ggtgagcaag
     1021 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1081 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1141 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1201 ctgagctggg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1261 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1321 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1381 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1441 aacgccatcc acggcaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1501 aacttcggcc tcaactgcaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1561 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1621 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1681 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagcgcat gcccatgaac
     1741 gcccccgagc ggcagcccca acccgacggc ggggacgccc caggccacga gcctgggggc
     1801 agcccccaag acgagcttga cttctccatc ctcttcgact atgagtattt gaatccgaac
     1861 gaagaagagc cgaatgcaca taaggtcgcc agcccaccct ccggacccgc ataccccgat
     1921 gatgtaatgg actatggcct caagccatac agcccccttg ctagtctctc tggcgagccc
     1981 cccggccgat tcggagagcc ggatagggta gggccgcaga agtttctgag cgcggccaag
     2041 ccagcagggg cctcgggcct gagccctcgg atcgagatca ctccgtccca cgaactgatc
     2101 caggcagtgg ggcccctccg catgagagac gcgggcctcc tggtggagca gcctcccctg
     2161 gccggggtgg ccgccagccc gaggttcacc ctgcccgtgc ccggcttcga gggctaccgc
     2221 gagccgcttt gcttgagccc cgctagcagc ggctcctctg ccagcttcat ttctgacacc
     2281 ttctccccct acacctcgcc ctgcgtctcg cccaataacg gcgggcccga cgacctgtgt
     2341 ccgcagtttc aaaacatccc tgctcattat tcccccagaa cctcgccaat aatgtcacct
     2401 cgaaccagcc tcgccgagga cagctgcctg ggccgccact cgcccgtgcc ccgtccggcc
     2461 tcccgctcct catcgcctgg tgccaagcgg aggcattcgt gcgccgaggc cttggttgcc
     2521 ctgccgcccg gagcctcacc ccagcgctcc cggagcccct cgccgcagcc ctcatctcac
     2581 gtggcacccc aggaccacgg ctccccggct gggtaccccc ctgttgagct ctctaaaggt
     2641 gaagaattat tcactggtgt tgtcccaatt ttggttgaat tagatggtga tgttaatggt
     2701 cacaaatttt ctgtctccgg tgaaggtgaa ggtgatgcta cgtacggtaa attgacctta
     2761 aaattactct gtactactgg taaattgcca gttccatggc caaccttagt cactacttta
     2821 ggttatggtg ttcaatgttt tgctagatac ccagatcata tgaaacaaca tgactttttc
     2881 aagtctgcca tgccagaagg ttatgttcaa gaaagaacta tttttttcaa agatgacggt
     2941 aactacaaga ccagagctga agtcaagttt gaaggtgata ccttagttaa tagaatcgaa
     3001 ttaaaaggta ttgattttaa agaagatggt aacattttag gtcacaaatt ggaatacaac
     3061 tataactctc acaatgttta catcactgct gacaaacaaa agaatggtat caaagctaac
     3121 ttcaaaatta gacacaacat tgaagatggt ggtgttcaat tagctgacca ttatcaacaa
     3181 aatactccaa ttggtgatgg tccagtcttg ttaccagaca accattactt atcctatcaa
     3241 tctgccttat tcaaagatcc aaacgaaaag agagaccaca tggtcttgtt agaatttttg
     3301 actgctgctg gtattaccga gggtatgaat gaattgtaca aatagtaaga attctgcaga
     3361 tatccatcac actggcggcc gctcgagcat gcatctagag ggccctattc tatagtgtca
     3421 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3481 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3541 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3601 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     3661 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     3721 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3781 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3841 cacgttcgcc ggctttcccc gtcaagctct aaatcggggc atccctttag ggttccgatt
     3901 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3961 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4021 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4081 ataagggatt ttggggattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4141 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     4201 ccaggcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4261 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4321 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4381 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     4441 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4501 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     4561 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4621 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4681 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4741 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4801 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4861 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4921 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4981 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     5041 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     5101 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     5161 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     5221 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     5281 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     5341 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     5401 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5461 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5521 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5581 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5641 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5701 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5761 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5821 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5881 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5941 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6001 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6061 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6121 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6181 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6241 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc
     6301 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6361 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6421 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6481 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6541 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6601 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6661 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6721 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6781 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6841 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6901 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6961 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7021 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7081 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7141 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7201 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7261 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7321 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7381 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7441 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7501 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7561 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7621 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7681 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7741 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7801 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
