LOCUS       sequence-250067-        7129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for mitochondrial matrix Ca2+.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7129)
  AUTHORS   Ashrafi G, de Juan-Sanz J, Farrell RJ, Ryan TA
  TITLE     Molecular Tuning of the Axonal Mitochondrial Ca(2+) Uniporter 
            Ensures Metabolic Flexibility of Neurotransmission.
  JOURNAL   Neuron. 2019 Dec 10. pii: S0896-6273(19)30984-5. doi: 
            10.1016/j.neuron.2019.11.020.
  PUBMED    31862210
REFERENCE   2  (bases 1 to 7129)
  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"; journalName: "Neuron. 
            2019 Dec 10. pii: S0896-6273(19)30984-5. doi: 
            10.1016/j.neuron.2019.11.020."
COMMENT     Sequence Label: CMV-Mito4x-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..7129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             75..869
                     /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(129..148)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     739..758
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1043..1164
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1080..1099)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1134..1153
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1213..1229)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1213..1229)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1226..1248)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1237..1253
                     /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(1261..1267,1268..1285,1286..1291))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1306..1327
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1444..1461)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1615..2203)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1695..1714)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2374..3165,3166..3234))
                     /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     2997..3016
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3235..3339)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3414..3433)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3605..3984
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3985..4188
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4139..4159
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4233..4252
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4233..4251
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4283..4357
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             4379..4453
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             4475..4549
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             4571..4645
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             4703..4720
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(4760..4768,4769..4783)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             4811..4867
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     complement(5468..5487)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(6090..6107)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6096..6320
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6366..6794
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6453..6472)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6663..6684
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6803..6823)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6808..7129,1..8)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6988..7123
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7050..7069
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
       61 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
      121 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
      181 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
      241 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
      301 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
      361 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
      421 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
      481 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
      541 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
      601 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
      661 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
      721 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
      781 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
      841 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
      901 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
      961 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     1021 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     1081 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     1141 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     1201 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     1261 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     1321 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     1381 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     1441 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     1501 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     1561 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1621 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1681 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1741 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1801 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1861 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1921 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1981 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2041 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2101 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2161 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2221 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2281 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2341 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2401 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2461 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2521 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2581 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2641 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2701 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2761 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2821 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2881 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2941 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3001 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3061 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3121 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3181 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3241 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3301 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3361 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     3421 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     3481 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     3541 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     3601 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     3661 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     3721 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     3781 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     3841 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     3901 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     3961 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     4021 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     4081 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     4141 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
     4201 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
     4261 gctggctagc ggatccaccg ccatgagcgt gctgacacct ctgctgctga gaggcctgac
     4321 aggctctgct agaaggctgc ctgtgcctag agccaagatc cactctctgg gcgaccctat
     4381 gtctgtgctg actccactgc tcctgcgggg actgactgga tctgctcgta gactgccagt
     4441 gccacgggct aagattcaca gcctgggcga tccaatgagt gtgcttaccc cactcctgct
     4501 cagaggactg accggaagtg ctagacgcct gccagttcca agggccaaaa tccatagtct
     4561 gggagatccc atgtccgtgt tgacacccct cttgctgagg ggacttaccg gctcagctcg
     4621 gagactccct gttccaagag caaagattca ttccctcggc gacccaagat ctggcagcgc
     4681 taaagaccct accatgggta gccaccacca ccatcaccac ggcatggctt ctatgacagg
     4741 cggccagcag atgggccgcg acctgtacga tgacgacgat aaggacctgg ccacaatggt
     4801 ggacagcagc aggcggaagt ggaacaagac aggccacgcc gttagagcca tcggcagact
     4861 gtctagcctg gaaaacgtgt acatcaaggc cgacaagcag aagaacggca tcaaggctaa
     4921 cttcaagatc cggcacaaca tcgaggacgg cggagtgcag ctggcttacc actaccagca
     4981 gaacacccct atcggcgacg gaccagttct gctgcctgac aaccactacc tgagcgtgca
     5041 gagcaagctg agcaaggacc ccaacgagaa gagggaccac atggtgctgc tggaatttgt
     5101 gaccgccgct ggcatcacac tcggcatgga cgaactgtac aaaggcggca caggcggctc
     5161 tatggtgtcc aagggcgaag aactgttcac cggcgtggtg cccattctgg tggaactgga
     5221 tggggatgtg aacggccaca agttcagcgt cagcggagaa ggcgaaggcg acgccacata
     5281 cggaaagctg accctgaagt tcatctgcac caccggcaaa ctgcccgtgc cttggcctac
     5341 actggtcacc acactgacat acggcgtgca gtgcttcagc agataccccg accatatgaa
     5401 gcagcacgac ttcttcaaga gcgccatgcc tgagggctac atccaagaga gaaccatctt
     5461 ctttaaggac gacggcaact acaagaccag ggccgaagtg aagttcgagg gcgacaccct
     5521 ggtcaacaga atcgagctga agggcatcga cttcaaagag gacggcaaca tcctgggcca
     5581 caagctcgag tacaacctgc ctgaccagct gaccgaggaa cagatcgccg agttcaaaga
     5641 agagttcagc ctgttcgaca aggacggcga cggcacaatc accaccaaag aactgggcac
     5701 cgtgatgaga agcctgggac agaaccctac agaggccgag ctgcaggaca tgatcaacga
     5761 ggttgacgct gatggcgacg gaactatcga cttccccgag ttcctgacca tgatggccag
     5821 aaagatgaag tacagggaca cagaggaaga gatccgcgag gccttcggcg tgttcgataa
     5881 ggatggaaac ggctacatct ctgccgctga gctgagacac gtgatgacca acctgggcga
     5941 gaagctgaca gacgaagagg tggacgagat gatcagagag gccgacattg acggcgacgg
     6001 gcaagtgaac tacgaagagt ttgtccagat gatgaccgcc aagtgaaccg gtgtcgacga
     6061 attcgggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     6121 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     6181 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     6241 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     6301 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     6361 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     6421 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     6481 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     6541 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     6601 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     6661 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     6721 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     6781 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     6841 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     6901 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     6961 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     7021 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     7081 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggct
//
