LOCUS       sequence-212242-        7740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral transfection of cells to express ratiometric Pericam as 
            described by Nagai et al 2001 (PMID 11248055) localising to the 
            mitochondrial matrix via the transit peptide from human COX8A..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7740)
  AUTHORS   Bohler P, Stuhldreier F, Anand R, Kondadi AK, Schlutermann D, 
            Berleth N, Deitersen J, Wallot-Hieke N, Wu W, Frank M, Niemann H, 
            Wesbuer E, Barbian A, Luyten T, Parys JB, Weidtkamp-Peters S, 
            Borchardt A, Reichert AS, Pena-Blanco A, Garcia-Saez AJ, Itskanov S,
            van der Bliek AM, Proksch P, Wesselborg S, Stork B
  TITLE     The mycotoxin phomoxanthone A disturbs the form and function of the 
            inner mitochondrial membrane.
  JOURNAL   Cell Death Dis. 2018 Feb 19;9(3):286. doi: 
            10.1038/s41419-018-0312-8.
  PUBMED    29459714
REFERENCE   2  (bases 1 to 7740)
  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: "Cell Death 
            Dis."; date: "2018-02-19"; volume: "9(3):286. doi"; pages: " 
            10.1038/s41419-018-0312-8"
COMMENT     Sequence Label: pMSCVpuro-Mito-Pericam
FEATURES             Location/Qualifiers
     source          1..7740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1424..1498
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1520..1594
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1625..1702
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             1745..1996
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSFQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     1933..1954
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2066..2087)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2327..2346)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        2916..3415
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2951..2974)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3348..3367
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3436..4035
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3436..3455)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3932..3952
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4135..4649
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4811..4827
                     /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(4835..4841,4842..4859,4860..4865))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4880..4901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5018..5035)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5189..5777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5269..5288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5948..6739,6740..6808))
                     /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     6571..6590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6809..6913)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6981..6999
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7037..7059)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7159..7178
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7372..7394
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7522..7541
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccggaat tagatccgct agcatgtccg tcctgacgcc
     1441 gctgctgctg cggggcttga caggctcggc ccggcggctc ccagtgccgc gcgccaagat
     1501 ccattcgttg ggggacccca tgtccgtcct gacgccgctg ctgctgcggg gcttgacagg
     1561 ctcggcccgg cggctcccag tgccgcgcgc caagatccat tcgttgccgc cggagggaga
     1621 tctcaagagg cgctggaaga aaaacttcat tgccgtcagc gctgccaacc ggttcaagaa
     1681 gatctccagc tccggggcac tggggtctgc aggctacaac agcgacaacg tctatatcat
     1741 ggccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagga
     1801 cggcggcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1861 gctgctgccc gacaaccact acctgagctt ccagtccgcc ctgagcaaag accccaacga
     1921 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1981 ggacgagctg tacaaggtcg acggtggcag cggtggcacc ggtgtgagca agggcgagga
     2041 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2101 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagct
     2161 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta
     2221 cggcctgaag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2281 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2341 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2401 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacggtac
     2461 cgaccaactg acagaagagc agattgcaga gttcaaagaa gccttctcat tattcgacaa
     2521 ggatggggac ggcaccatca ccacaaagga acttggcacc gttatgaggt cgcttggaca
     2581 aaacccaacg gaagcagaat tgcaggatat gatcaatgaa gtcgatgctg atggcaatgg
     2641 aacgatttac tttcctgaat ttcttactat gatggctaga aaaatgaagg acacagacag
     2701 cgaagaggaa atccgagaag cattccgtgt ttttgacaag gatgggaacg gctacatcag
     2761 cgctgctcag ttacgtcacg tcatgacaaa cctcggggag aagttaacag atgaagaagt
     2821 tgatgaaatg ataagggaag cagatatcga tggtgatggc caagtaaact atgaagagtt
     2881 tgtacaaatg atgacagcaa agtaagaatt ctaccgggta ggggaggcgc ttttcccaag
     2941 gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct
     3001 ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg
     3061 ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct
     3121 cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga
     3181 cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg
     3241 ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg
     3301 cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct
     3361 tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctg
     3421 cagcccaagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     3481 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     3541 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     3601 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     3661 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     3721 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     3781 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     3841 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     3901 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     3961 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     4021 aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga
     4081 ccccatgcat cgataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa
     4141 gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa
     4201 atacataact gagaatagag aagttcagat caaggttagg aacagagaga cagcagaata
     4261 tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga
     4321 tggtccccag atgcggtccc gccctcagca gtttctagag aaccatcaga tgtttccagg
     4381 gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc
     4441 tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac
     4501 tcggcgcgcc agtcctccga tagactgcgt cgcccgggta cccgtgtatc caataaaccc
     4561 tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga
     4621 ttgactaccc gtcagcgggg gtctttcatg ggtaacagtt tcttgaagtt ggagaacaac
     4681 attctgaggg taggagtcga atattaagta atcctgactc aattagccac tgttttgaat
     4741 ccacatactc caatactcct gaaatagttc attatggaca gcgcagaaag agctggggag
     4801 aattgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4861 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4921 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4981 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5041 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5101 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5161 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5221 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5281 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5341 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5401 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5461 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5521 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5581 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5641 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5701 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5761 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5821 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5881 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5941 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6001 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6061 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6121 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6181 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6241 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6301 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6361 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6421 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6481 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6541 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6601 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6661 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6721 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6781 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6841 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6901 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     6961 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     7021 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     7081 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     7141 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     7201 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
     7261 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     7321 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     7381 gacgttgtaa aacgacggcg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     7441 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     7501 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     7561 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg cgattagtcc aatttgttaa
     7621 agacaggata tcagtggtcc aggctctagt tttgactcaa caatatcacc agctgaagcc
     7681 tatagagtac gagccataga taaaataaaa gattttattt agtctccaga aaaagggggg
//
