LOCUS       sequence-116204-        6687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of mCherry fused FRB to the mitochondrial 
            innermembrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6687)
  AUTHORS   Ichim G, Lopez J, Ahmed SU, Muthalagu N, Giampazolias E, Delgado ME,
            Haller M, Riley JS, Mason SM, Athineos D, Parsons MJ, van de Kooij 
            B, Bouchier-Hayes L, Chalmers AJ, Rooswinkel RW, Oberst A, Blyth K, 
            Rehm M, Murphy DJ, Tait SW
  TITLE     Limited mitochondrial permeabilization causes DNA damage and genomic
            instability in the absence of cell death.
  JOURNAL   Mol Cell. 2015 Mar 5;57(5):860-72. doi: 
            10.1016/j.molcel.2015.01.018. Epub 2015 Feb 19.
  PUBMED    25702873
REFERENCE   2  (bases 1 to 6687)
  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.molcel.2015.01.018"; journalName: "Mol Cell"; date: 
            "2015-03-5- 5"; volume: "57"; issue: "5"; pages: "860-72"
COMMENT     Sequence Label: pcDNA3 AIF 1-90 mCherry FRB
FEATURES             Location/Qualifiers
     source          1..6687
                     /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             1168..1197
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1204..1908
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1346..1364)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1614..1633
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1930..2208
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     promoter        complement(2239..2257)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2240..2257)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2277..2294)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2283..2507
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2553..2981
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2640..2659)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2850..2871
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2990..3010)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2995..3325
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3176..3311
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3238..3257
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3392..4186
                     /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(3446..3465)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4056..4075
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4360..4481
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4397..4416)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4451..4470
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4530..4546)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4530..4546)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4543..4565)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4554..4570
                     /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(4578..4584,4585..4602,4603..4608))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4623..4644
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4761..4778)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4932..5520)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5012..5031)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5691..6482,6483..6551))
                     /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     6314..6333
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6552..6656)
                     /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 gcttatgttc
      901 cggtgtggag gcctggcggc gggtgctttg aagcagaagc tggtgccctt ggtgcggacc
      961 gtgtgcgtcc gaagcccgag gcagaggaac cggctcccag gcaacttgtt ccagcgatgg
     1021 catgttcctc tagaactcca gatgacaaga caaatggcta gctctggtgc atcagggggc
     1081 aaaatcgata attctgtgtt agtccttatt gtgggcttat caacagtagg agctggtgcc
     1141 tatgcctaca agactatgaa agagatggaa caaaaactta tttctgaaga agatctgggt
     1201 accgtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     1261 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1321 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1381 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     1441 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     1501 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     1561 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     1621 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     1681 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     1741 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     1801 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     1861 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaaggc ggccgccgga
     1921 ggtggcggga tcctctggca tgagatgtgg catgaaggcc tggaagaggc atctcgtttg
     1981 tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc tggagccctt gcatgctatg
     2041 atggaacggg gcccccagac tctgaaggaa acatccttta atcaggccta tggtcgagat
     2101 ttaatggagg cccaagagtg gtgcaggaag tacatgaaat cagggaatgt caaggacctc
     2161 ctccaagcct gggacctcta ttatcatgtg ttccgacgaa tctcaaagtg actcgagcat
     2221 gcatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     2281 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2341 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2401 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2461 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2521 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2581 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2641 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2701 aaatcggggc atccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2761 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2821 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2881 caaccctatc tcggtctatt cttttgattt ataagggatt ttggggattt cggcctattg
     2941 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3001 cagttagggt gtggaaagtc cccaggctcc ccaggcaggc agaagtatgc aaagcatgca
     3061 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3121 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3181 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3241 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     3301 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     3361 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     3421 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3481 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3541 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     3601 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3661 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3721 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3781 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     3841 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     3901 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3961 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4021 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4081 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4141 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4201 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4261 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4321 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4381 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4441 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4501 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4561 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4621 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4681 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4741 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4801 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4861 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4921 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4981 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5041 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5101 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     5161 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5221 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5281 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5341 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5401 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5461 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5521 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5581 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5641 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5701 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5761 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5821 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5881 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5941 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6001 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6061 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6121 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6181 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6241 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6301 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6361 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6421 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6481 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6541 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6601 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6661 tttccccgaa aagtgccacc tgacgtc
//
