LOCUS       sequence-495713-        7440 bp    DNA     circular SYN 18-DEC-2025
DEFINITION  For detection of ER-mitochondrial contact.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7440)
  AUTHORS   Chen C, Rafael KA, Cho G, Lim Y
  TITLE     Split-Luciferase Reassembly Assay to Measure Endoplasmic 
            Reticulum-Mitochondria Contacts in Live Cells.
  JOURNAL   J Vis Exp. 2024 Oct 11;(212):10.3791/66862. doi: 10.3791/66862.
  PUBMED    39465944
REFERENCE   2  (bases 1 to 7440)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Vis 
            Exp."; date: "2024-10-11"; volume: "(212):10.3791/66862. doi"; 
            pages: " 10.3791/66862"
COMMENT     Sequence Label: pCAG-MitoRlucN-T2A-RlucCER-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..7440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1891..1914
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2206..2259
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2926..2955
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    join(3077..3652,3653..3655,3656..3664)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3245..3262)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3472..3491
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3665..4375
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3810..3828)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4078..4097
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4461..4480)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4526..4581
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4580..4599)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4942..4958)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4942..4958)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4955..4977)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4966..4982
                     /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(4990..4996,4997..5014,5015..5020))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5035..5056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5115..5310
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5161..5296
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5316..5450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5353..5372)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5407..5426
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5518..5535)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5689..6277)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5769..5788)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6448..7239,7240..7308))
                     /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     7071..7090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7309..7413)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagcac catggccatc
     1741 cagctgcggt ctctctttcc actggccttg ccaggtttgc tggctctcct gggatggtgg
     1801 tggtttttca gccgcaagaa ggacctggaa cttaagctgc gaattctgca gtcaactgtg
     1861 ccccgcgcaa gagaccctcc cgtcgcaact gattacaagg atgacgacga taaggaattc
     1921 atggcttcca aggtgtacga ccccgagcaa cgcaaacgca tgatcactgg gcctcagtgg
     1981 tgggctcgct gcaagcaaat gaacgtgctg gactccttca tcaactacta tgattccgag
     2041 aagcacgccg agaacgccgt gatttttctg catggtaacg ctacctccag ctacctgtgg
     2101 aggcacgtcg tgcctcacat cgagcccgtg gctagatgca tcatccctga tctgatcgga
     2161 atgggtaagt ccggcaagag cgggaatggc tcagcttcga gtggagaggg cagaggaagt
     2221 ctgctaacat gcggtgacgt cgaggagaat cccggccctt atcgcctcct ggatcactac
     2281 aagtacctca ccgcttggtt cgagctgctg aaccttccaa agaaaatcat ctttgtgggc
     2341 cacgactggg gggctgctct ggcctttcac tacgcctacg agcaccaaga caggatcaag
     2401 gccatcgtcc atatggagag tgtcgtggac gtgatcgagt cctgggacga gtggcctgac
     2461 atcgaggagg atatcgccct gatcaagagc gaagagggcg agaaaatggt gcttgagaat
     2521 aacttcttcg tcgagaccgt gctcccaagc aagatcatgc ggaaactgga gcctgaggag
     2581 ttcgctgcct acctggagcc attcaaggag aagggcgagg ttagacggcc taccctctcc
     2641 tggcctcgcg agatccctct cgttaaggga ggcaagcccg acgtcgtcca gattgtccgc
     2701 aactacaacg cctaccttcg ggccagcgac gatctgccta agctgttcat cgagtccgac
     2761 cctgggttct tttccaacgc tattgtcgag ggagctaaga agttccctaa caccgagttc
     2821 gtgaaggtga agggcctcca cttcctccag gaggacgctc cagatgaaat gggtaagtac
     2881 atcaagagct tcgtggagcg cgtgctgaag aacgagcagg ctagcgagca gaagctgatc
     2941 tcagaggagg acctgagcgg tctgaggtct agggcccaag ccagcaattc tcgagtcatg
     3001 gtctatatcg gaatcgcaat cttcctgttc gttggcttgt tcatgaaatg atctgcagaa
     3061 gcttctaggg gatccgcccc tctccctccc ccccccccta acgttactgg ccgaagccgc
     3121 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     3181 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     3241 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     3301 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     3361 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     3421 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     3481 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     3541 gatctggggc ctcggtacac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     3601 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac
     3661 aaccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     3721 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     3781 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     3841 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     3901 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     3961 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     4021 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     4081 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     4141 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     4201 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     4261 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     4321 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagcggc
     4381 cgcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     4441 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     4501 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     4561 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     4621 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     4681 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     4741 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     4801 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     4861 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag
     4921 cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4981 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5041 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5101 gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     5161 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     5221 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     5281 ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg
     5341 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5401 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta
     5461 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5521 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5581 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5641 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5701 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5761 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5821 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5881 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5941 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6001 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6061 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6121 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6181 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6241 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6301 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6361 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6421 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6481 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6541 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6601 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6661 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6721 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6781 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6841 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6901 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6961 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7021 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7081 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7141 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7201 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7261 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7321 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7381 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgg
//
