LOCUS       sequence-249098-        7172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Red-shifted fluorescent reporter for mitochondrial matrix Ca2+.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7172)
  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 7172)
  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-jRCaMP1b
FEATURES             Location/Qualifiers
     source          1..7172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             679..753
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             775..849
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             871..945
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             967..1041
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1147..1164
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1168..1200
                     /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             join(1204..1212,1213..1227)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(2529..2546)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2535..2759
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2805..3233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2892..2911)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3102..3123
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3242..3262)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3247..3576
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3427..3562
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3489..3508
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3643..4437
                     /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(3697..3716)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4307..4326
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4611..4732
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4648..4667)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4702..4721
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4781..4797)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4781..4797)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4794..4816)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4805..4821
                     /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(4829..4835,4836..4853,4854..4859))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4874..4895
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5012..5029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5183..5771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5263..5282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5942..6733,6734..6802))
                     /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     6565..6584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6803..6907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6982..7001)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcggat ccaccgccat gagcgtgctg acacctctgc tgctgagagg cctgacaggc
      721 tctgctagaa ggctgcctgt gcctagagcc aagatccact ctctgggcga ccctatgtct
      781 gtgctgactc cactgctcct gcggggactg actggatctg ctcgtagact gccagtgcca
      841 cgggctaaga ttcacagcct gggcgatcca atgagtgtgc ttaccccact cctgctcaga
      901 ggactgaccg gaagtgctag acgcctgcca gttccaaggg ccaaaatcca tagtctggga
      961 gatcccatgt ccgtgttgac acccctcttg ctgaggggac ttaccggctc agctcggaga
     1021 ctccctgttc caagagcaaa gattcattcc ctcggcgacc caagatctgg cagccgccac
     1081 atgctgcaga acgagcttgc tcttaagttg gctggacttg atattaacaa gactggagga
     1141 ggttctcatc atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt
     1201 cgggatctgt acgacgatga cgataaggat ctcgcaacaa tggtcgactc atcgcgacgt
     1261 aagtggaata agtggggtca cgcagtcaga gctataggtc ggctgagctc agcgaacaac
     1321 accgaaatga tgtacccagc ggatggtggt ctgcgtggtt acactcacat ggcgctgaaa
     1381 gttgatggcg gcggtcacct gtcctgttct ttcgtgacca cctaccgctc caaaaagact
     1441 gtcggcaaca ttaagatgcc tgccattcat tacgtcagcc accgtctgga gcgcctggag
     1501 gagagcgata acgaaatgtt tgtcgtacag cgtgaacacg cagttgccaa gtttgtgggc
     1561 ctgggtggtg gcggcggtac cggagggagc atgaactccc tgatcaagga gaacatgcgt
     1621 atgaaagtgg ttctggaagg ctccgtaaac ggccaccagt tcaaatgcac tggtgaaggc
     1681 gaaggcaacc cgtatatggg cacccagact atgcgtatca aagtgatcga gggtggtccg
     1741 ctgccgtttg cgttcgacat cctggcgacg tcctttatgt atggctcccg taccttcatc
     1801 aaatatccga aaggcatccc ggatttcttt aagcagtcct tcccggaagg ttttacctgg
     1861 gaacgtgtga cccgttacga agacggcggc gtaattaccg ttatgcaaga cacgtctctg
     1921 gaggatggct gcctggtgta tcacgtgcag gttcgcggtg tgaacttccc gagcaatggt
     1981 gctgtaatgc aaaagaaaac caaaggttgg gagcctacgg actcccaact gactgaagag
     2041 cagatcgcag aatttaaaga ggctttctcc ctatttgaca aggacgggga tgggacaata
     2101 acaaccaagg agatggggac ggtgatgcgg tctctggggc agaaccccac agaagcagag
     2161 ctgcaggaca tgatcaatga agtagatgcc gacggtgacg gcacaatcga cttccctgag
     2221 ttcctgatta tgatggcagg caaaatgaaa tacacagaca gtgaagaaga aattagagaa
     2281 gcgttcggcg tgtttgataa ggatggcaat ggctacatca gtgcagcaga gcttcgccac
     2341 gtgatgacaa accttggaga gaagttaaca gatgaagagg ttgatgaaat gatcagggaa
     2401 gcagacagcg atggggatgg tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg
     2461 aagtaggcgg ccgctcgagc atgcaaccgg tgtcgacgaa ttcgggcccg tttaaacccg
     2521 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2581 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2641 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2701 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2761 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2821 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2881 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2941 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3001 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3061 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3121 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3181 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3241 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3301 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3361 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3421 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3481 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3541 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3601 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3661 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3721 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3781 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3841 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3901 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3961 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     4021 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4081 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4141 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4201 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4261 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4321 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4381 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4441 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4501 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4561 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4621 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4681 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4741 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4801 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4861 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4921 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4981 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5041 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5101 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5161 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5221 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5281 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5341 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5401 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5461 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5521 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5581 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5641 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5701 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5761 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5821 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5881 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5941 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6001 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6061 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6121 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6181 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6241 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6301 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6361 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6421 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6481 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6541 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6601 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6661 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6721 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6781 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6841 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6901 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     6961 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     7021 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     7081 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     7141 gcttcgcgat gtacgggcca gatatacgcg tt
//
