LOCUS       sequence-245097-        7243 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 7243)
  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 7243)
  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..7243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     CDS             913..987
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1009..1083
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1105..1179
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1201..1275
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1384..1401
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1405..1437
                     /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(1441..1449,1450..1464)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     promoter        complement(2795..2813)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2796..2813)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2833..2850)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2839..3063
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3109..3537
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3196..3215)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3406..3427
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3546..3566)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3551..3881
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3732..3867
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3794..3813
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3948..4742
                     /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(4002..4021)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4612..4631
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4916..5037
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4953..4972)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5007..5026
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5086..5102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5086..5102)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5099..5121)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5110..5126
                     /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(5134..5140,5141..5158,5159..5164))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5179..5200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5317..5334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5488..6076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5568..5587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6247..7038,7039..7107))
                     /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     6870..6889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7108..7212)
                     /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 gctggctagc
      901 ggatccaccg ccatgagcgt gctgacacct ctgctgctga gaggcctgac aggctctgct
      961 agaaggctgc ctgtgcctag agccaagatc cactctctgg gcgaccctat gtctgtgctg
     1021 actccactgc tcctgcgggg actgactgga tctgctcgta gactgccagt gccacgggct
     1081 aagattcaca gcctgggcga tccaatgagt gtgcttaccc cactcctgct cagaggactg
     1141 accggaagtg ctagacgcct gccagttcca agggccaaaa tccatagtct gggagatccc
     1201 atgtccgtgt tgacacccct cttgctgagg ggacttaccg gctcagctcg gagactccct
     1261 gttccaagag caaagattca ttccctcggc gacccaagat ctggcagcct cgccaccatg
     1321 ctgcagaacg agcttgctct taagttggct ggacttgata ttaacaagac tggaggaggt
     1381 tctcatcatc atcatcatca tggtatggct agcatgactg gtggacagca aatgggtcgg
     1441 gatctgtacg acgatgacga taaggatctc gcaacaatgg tcgactcatc gcgacgtaag
     1501 tggaataagt ggggtcacgc agtcagagct ataggtcggc tgagctcagc gaacaacacc
     1561 gaaatgatgt acccagcgga tggtggtctg cgtggttaca ctcacatggc gctgaaagtt
     1621 gatggcggcg gtcacctgtc ctgttctttc gtgaccacct accgctccaa aaagactgtc
     1681 ggcaacatta agatgcctgc cattcattac gtcagccacc gtctggagcg cctggaggag
     1741 agcgataacg aaatgtttgt cgtacagcgt gaacacgcag ttgccaagtt tgtgggcctg
     1801 ggtggtggcg gcggtaccgg agggagcatg aactccctga tcaaggagaa catgcgtatg
     1861 aaagtggttc tggaaggctc cgtaaacggc caccagttca aatgcactgg tgaaggcgaa
     1921 ggcaacccgt atatgggcac ccagactatg cgtatcaaag tgatcgaggg tggtccgctg
     1981 ccgtttgcgt tcgacatcct ggcgacgtcc tttatgtatg gctcccgtac cttcatcaaa
     2041 tatccgaaag gcatcccgga tttctttaag cagtccttcc cggaaggttt tacctgggaa
     2101 cgtgtgaccc gttacgaaga cggcggcgta attaccgtta tgcaagacac gtctctggag
     2161 gatggctgcc tggtgtatca cgtgcaggtt cgcggtgtga acttcccgag caatggtgct
     2221 gtaatgcaaa agaaaaccaa aggttgggag cctacggact cccaactgac tgaagagcag
     2281 atcgcagaat ttaaagaggc tttctcccta tttgacaagg acggggatgg gacaataaca
     2341 accaaggaga tggggacggt gatgcggtct ctggggcaga accccacaga agcagagctg
     2401 caggacatga tcaatgaagt agatgccgac ggtgacggca caatcgactt ccctgagttc
     2461 ctgattatga tggcaggcaa aatgaaatac acagacagtg aagaagaaat tagagaagcg
     2521 ttcggcgtgt ttgataagga tggcaatggc tacatcagtg cagcagagct tcgccacgtg
     2581 atgacaaacc ttggagagaa gttaacagat gaagaggttg atgaaatgat cagggaagca
     2641 gacagcgatg gggatggtca ggtaaactac gaagagtttg tacaaatgat gacagcgaag
     2701 taggcggccg ctcgagcatg caaccggtgt cgacgaattc tgcagatatc catcacactg
     2761 gcggccgctc gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag
     2821 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2881 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2941 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3001 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3061 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3121 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3181 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3241 ttccccgtca agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc
     3301 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3361 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3421 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg
     3481 ggatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3541 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag gcaggcagaa
     3601 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3661 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3721 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3781 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     3841 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     3901 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3961 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4021 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4081 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     4141 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4201 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4261 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4321 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4381 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4441 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4501 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4561 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4621 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4681 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4741 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4801 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4861 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4921 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4981 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5041 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5101 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5161 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5221 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5281 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5341 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5401 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5461 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5521 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5581 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5641 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta
     5701 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5761 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5821 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5881 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     5941 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6001 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6061 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6121 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6181 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6241 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6301 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6361 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6421 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6481 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6541 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6601 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6661 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6721 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6781 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6841 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6901 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6961 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7021 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7081 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7141 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7201 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
