LOCUS       sequence-243952-        7446 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of MARIO, a genetically-encoded fluorescent 
            Mg2+ sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7446)
  AUTHORS   Maeshima K, Matsuda T, Shindo Y, Imamura H, Tamura S, Imai R, 
            Kawakami S, Nagashima R, Soga T, Noji H, Oka K, Nagai T
  TITLE     A Transient Rise in Free Mg(2+) Ions Released from ATP-Mg Hydrolysis
            Contributes to Mitotic Chromosome Condensation.
  JOURNAL   Curr Biol. 2018 Feb 5;28(3):444-451.e6. doi: 
            10.1016/j.cub.2017.12.035. Epub 2018 Jan 18.
  PUBMED    29358072
REFERENCE   2  (bases 1 to 7446)
  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"; doi: 
            "10.1016/j.cub.2017.12.035"; journalName: "Curr Biol"; date: 
            "2018-02-5- 5"; volume: "28"; issue: "3"; pages: "444-451.e6"
COMMENT     Sequence Label: MARIO/ pcDNA3
FEATURES             Location/Qualifiers
     source          1..7446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        7..386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..590
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     541..561
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     635..654
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        635..653
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(686..688,689..691,692..1204)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     CDS             1973..2707
                     /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="MDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD
                     HMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTAVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     promoter        complement(2771..2789)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2772..2789)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2809..2826)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2815..3039
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3085..3513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3172..3191)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3382..3403
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3522..3542)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3527..3856
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3707..3842
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3769..3788
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3923..4717
                     /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(3977..3996)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4587..4606
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4891..5012
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4928..4947)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4982..5001
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5061..5077)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5061..5077)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5074..5096)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5085..5101
                     /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(5109..5115,5116..5133,5134..5139))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5154..5175
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5292..5309)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5463..6051)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5543..5562)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6222..7013,7014..7082))
                     /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     6845..6864
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7083..7187)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7262..7281)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
       61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      181 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      361 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      421 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      481 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      541 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      601 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      661 aagcttggta ccgagctcgg atcccatggt gagcaagggc gaggagctgt tcaccggggt
      721 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaggttca gcgtgtccgg
      781 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      841 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctggggcg tgcagtgctt
      901 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      961 ctacgtccag gagcgtacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1021 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1081 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacatcagcc acaacgtcta
     1141 tatcaccgcc gacaagcaga agaacggcat caaggcccac ttcaagatcc gccacaacat
     1201 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1261 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1321 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccc gcatgctgag
     1381 cctggcaacc cgcccggaac tggaagacat cgaagcatcg gcacgtttct ttgaagacga
     1441 cgacggcctg catgatcact ccttcttctt ctttgaagat gcggaagatc acgccggtaa
     1501 ctccactgtg gcatttacca tccgtgatgg tcgtctgttt actctgcgtg agcgtgaact
     1561 gcccgctttt cgtctgtatc gtatgcgtgc ccgtagccag tcgatggtag acggtaacgc
     1621 ctacgagttg ctgctggatc tgttcgaaac caaaatcgaa cagttggcag atgaaattga
     1681 aaatatctat agcgacctgg agcagttgag ccgggtgatt atggaagggc atcagggcga
     1741 tgagtacgac gaggcgctct ccactctggc ggaactggaa gatgacggct gggccgttgc
     1801 cctgtgtctg atggataccc agcgcgcgct caacttcctg gtgcgtaaag cgcgtttacc
     1861 gggtgggcaa ctggagcagg cgcgtgaaat cctgcgagat atcgaatccc tgctgccgca
     1921 taacgaatcc ctgttccaga aggtgaactt cctgatgcag gcggctgagc tcatggacgg
     1981 cggcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2041 gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa
     2101 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2161 cgagctgtac aagggtggca gcggtggcat ggtgagcaag ggcgaggagc tgttcaccgc
     2221 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2281 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagctga tctgcaccac
     2341 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgggctacg gcctgcagtg
     2401 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2461 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2521 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2581 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2641 ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa
     2701 catcgagtaa gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag
     2761 agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc
     2821 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     2881 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     2941 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3001 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     3061 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3121 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3181 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3241 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3301 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3361 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3421 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3481 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     3541 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3601 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3661 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3721 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3781 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3841 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     3901 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3961 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4021 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4081 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4141 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4201 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4261 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4321 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4381 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4441 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4501 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4561 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4621 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4681 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4741 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4801 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4861 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4921 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4981 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5041 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5101 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5161 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5221 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5281 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5341 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5401 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5461 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5521 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5581 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5641 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5701 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5761 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5821 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5881 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5941 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6001 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6061 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6121 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6181 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6241 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6301 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6361 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6421 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6481 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6541 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6601 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6661 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6721 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6781 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6841 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6901 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6961 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7021 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7081 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7141 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7201 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     7261 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     7321 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     7381 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     7441 gatata
//
