LOCUS       sequence-250063-        6903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for mitochondrial outer membrane Ca2+.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6903)
  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 6903)
  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-OMM-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..6903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             107..901
                     /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(161..180)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     771..790
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1075..1196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1112..1131)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1166..1185
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1245..1261)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1245..1261)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1258..1280)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1269..1285
                     /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(1293..1299,1300..1317,1318..1323))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1338..1359
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1476..1493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1647..2235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1727..1746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2406..3197,3198..3266))
                     /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     3029..3048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3267..3371)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3446..3465)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3637..4016
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4017..4220
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4171..4191
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4265..4284
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4265..4283
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4337..4346
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4478..4486,4487..4504,4505..4507,4508..4540,4541..4543,
                     4544..4567,4568..4594,4595..4651,4652..4657,4658..4927,
                     4928..4945,4946..5380,5381..5386,5387..5830)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      4574..4583
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     4864..4885
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4991..5012)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5252..5271)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(5896..5913)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5902..6126
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6172..6600
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6259..6278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6469..6490
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6609..6629)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6614..6903,1..40)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6794..6903,1..26)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6856..6875
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
       61 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
      121 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
      181 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
      241 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
      301 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
      361 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
      421 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
      481 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
      541 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
      601 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
      661 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
      721 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
      781 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
      841 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
      901 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
      961 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     1021 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     1081 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     1141 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     1201 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     1261 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     1321 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     1381 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1441 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     1501 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1561 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1621 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2281 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2341 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2401 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2461 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2521 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     2581 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     2641 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     2701 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     2761 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     2821 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     2881 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     2941 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3001 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3061 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3121 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3181 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3241 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3301 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3361 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     3421 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3481 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     3541 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     3601 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     3661 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     3721 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     3781 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     3841 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     3901 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     3961 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     4021 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     4081 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     4141 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     4201 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg
     4261 aaattaatac gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttgg
     4321 taccgagctc ggatccgcca ccatggtggg aagaaactct gctatcgccg ctggcgtttg
     4381 tggcgctctg ttcatcggct actgcatcta cttcgacaga aagagaagaa gcgaccccaa
     4441 caccggtaag acctactgct actacagcag caagcttatg ggttctcatc atcatcatca
     4501 tcatggtatg gctagcatga ctggtggaca gcaaatgggt cgggatctgt acgacgatga
     4561 cgataaggat ctcgccacca tggtcgactc atcacgtcgt aagtggaata agacaggtca
     4621 cgcagtcaga gctataggtc ggctgagctc actcgagaac gtctatatca aggccgacaa
     4681 gcagaagaac ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcggcgt
     4741 gcagctcgcc taccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     4801 cgacaaccac tacctgagcg tgcagtccaa actttcgaaa gaccccaacg agaagcgcga
     4861 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     4921 gtacaagggc ggtaccggag ggagcatggt gagcaagggc gaggagctgt tcaccggggt
     4981 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     5041 cgagggtgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     5101 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     5161 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     5221 ctacatccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     5281 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     5341 ggaggacggc aacatcctgg ggcacaagct ggagtacaac ctgccggacc aactgactga
     5401 agagcagatc gcagaattta aagaggaatt ctccctattt gacaaggacg gggatgggac
     5461 aataacaacc aaggagctgg ggacggtgat gcggtctctg gggcagaacc ccacagaagc
     5521 agagctgcag gacatgatca atgaagtaga tgccgacggt gacggcacaa tcgacttccc
     5581 tgagttcctg acaatgatgg caagaaaaat gaaatacagg gacacggaag aagaaattag
     5641 agaagcgttc ggtgtgtttg ataaggatgg caatggctac atcagtgcag cagagcttcg
     5701 ccacgtgatg acaaaccttg gagagaagtt aacagatgaa gaggttgatg aaatgatcag
     5761 ggaagcagac atcgatgggg atggtcaggt aaactacgaa gagtttgtac aaatgatgac
     5821 agcgaagtga aagctgatat ccagcacagt ggcggccgct cgagtctaga gggcccgttt
     5881 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     5941 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     6001 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     6061 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     6121 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     6181 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     6241 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     6301 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     6361 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     6421 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     6481 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     6541 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     6601 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     6661 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     6721 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     6781 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     6841 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     6901 gaa
//
