LOCUS       sequence-76084-e        5677 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses MagFRET-1 in the nucleus of mammalian cells. FRET sensor 
            based on Cerulean/Citrine for Mg2+ imaging. Sensor does not respond 
            to normal Ca2+ levels..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5677)
  AUTHORS   Lindenburg LH, Vinkenborg JL, Oortwijn J, Aper SJ, Merkx M
  TITLE     MagFRET: The First Genetically Encoded Fluorescent Mg(2+) Sensor.
  JOURNAL   PLoS One. 2013 Dec 2;8(12):e82009. doi: 
            10.1371/journal.pone.0082009.
  PUBMED    24312622
REFERENCE   2  (bases 1 to 5677)
  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: "PLoS One.";
            date: "2013-12-2"; volume: "8(12):e82009. doi"; pages: " 
            10.1371/journal.pone.0082009"
COMMENT     Sequence Label: pCMVMagFRET-1-NLS
FEATURES             Location/Qualifiers
     source          1..5677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      600..609
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(615..617,618..620,621..1133)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIE"
     CDS             join(1539..1541,1542..1544,1545..2255)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2265..2285
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2289..2309
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2313..2333
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2465..2586
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2502..2521)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2556..2575
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2593..3048)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2730..2751)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2942..2961
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3075..3179
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3181..3538
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3181..3372
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3204..3224)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3389..3524
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3573..4367
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3573..4367
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3627..3646)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4237..4256
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4555..4574)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4599..4646
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4975..5563
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5464..5483
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgccg
      601 ccaccatggg ccatatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      661 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      721 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggt aagctgcccg
      781 tgccctggcc caccctcgtg accaccctga cctggggcgt gcagtgcttc gcccgctacc
      841 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      901 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      961 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1021 acatcctggg gcacaagctg gagtacaacg ccatcagcga caacgtctat atcaccgccg
     1081 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggca
     1141 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1201 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1261 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactagc gaggaacaga
     1321 aacaagaaat taaagatgct tttgaactgt ttgatacaga caaagatgaa gcaatagatt
     1381 atcatgaact gaaggtggca atgagagcct tggggtttga tgtaaaaaaa gctgatgtac
     1441 tgaagattct taaagattat gacagagaag ccacagggaa aatcaccttt gaagatttta
     1501 atgaagttgt gacagactgg atattggaaa gagatgccat ggtgagcaag ggcgaggagc
     1561 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1621 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1681 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg
     1741 gcctgatgtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1801 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1861 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1921 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gcttgagtac aactacaaca
     1981 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2041 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2101 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc
     2161 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2221 ccgggatcac tctcggcatg gacgagctgt acaagggcgg atctcccaag aaaaaacgca
     2281 aggtggatcc aaagaaaaag cgtaaagttg atccaaaaaa gaagagaaag gtagatctat
     2341 aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     2401 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     2461 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2521 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2581 atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     2641 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2701 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2761 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     2821 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     2881 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     2941 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3001 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     3061 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3121 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     3181 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3241 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3301 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3361 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3421 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     3481 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     3541 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3601 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3661 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3721 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     3781 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3841 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3901 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3961 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4021 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4081 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4141 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4201 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4261 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4321 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4381 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4441 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4501 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     4561 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     4621 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     4681 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     4741 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     4801 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     4861 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4921 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4981 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5041 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5101 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     5161 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5221 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5281 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5341 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5401 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5461 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5521 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5581 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5641 ccctgattct gtggataacc gtattaccgc catgcat
//
