LOCUS       sequence-455037-        6546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For tagging mitochondria with Sigma FLAG epitopes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6546)
  AUTHORS   Chen WW, Freinkman E, Wang T, Birsoy K, Sabatini DM
  TITLE     Absolute Quantification of Matrix Metabolites Reveals the Dynamics 
            of Mitochondrial Metabolism.
  JOURNAL   Cell. 2016 Aug 25;166(5):1324-1337.e11. doi: 
            10.1016/j.cell.2016.07.040.
  PUBMED    27565352
REFERENCE   2  (bases 1 to 6546)
  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.cell.2016.07"; journalName: "Cell"; date: "2016-08-25- 
            25"; volume: "166"; issue: "5"; pages: "1324-1337.e11"
COMMENT     Sequence Label: pMXs-3XFLAG-EGFP-OMP25
FEATURES             Location/Qualifiers
     source          1..6546
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..417
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     347..369
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     385..401
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    427..801
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     739..755
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      866..875
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             875..940
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(941..943,944..1654)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(941..943,944..1654)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(983..1004)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1244..1263)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1591..1612
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1805..2379,2380..2382,2383..2391)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1972..1989)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2199..2218
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2392..2787
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             2958..3427
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3557..3574)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3728..4316)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3808..3827)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4480..5271,5272..5340))
                     /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     5103..5122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5341..5445)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5475..6068
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6131..6488
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
ORIGIN
        1 ggccagactg ttaccactcc cttaagtttg accttaggtc actggaaaga tgtcgagcgg
       61 atcgctcaca accagtcggt agatgtcaag aagagacgtt gggttacctt ctgctctgca
      121 gaatggccaa cctttaacgt cggatggccg cgagacggca cctttaaccg agacctcatc
      181 acccaggtta agatcaaggt cttttcacct ggcccgcatg gacacccaga ccaggtcccc
      241 tacatcgtga cctgggaagc cttggctttt gacccccctc cctgggtcaa gccctttgta
      301 caccctaagc ctccgcctcc tcttcctcca tccgccccgt ctctccccct tgaacctcct
      361 cgttcgaccc cgcctcgatc ctccctttat ccagccctca ctccttctct aggcgccccc
      421 atatggccat atgagatctt atatggggca cccccgcccc ttgtaaactt ccctgaccct
      481 gacatgacaa gagttactaa cagcccctct ctccaagctc acttacaggc tctctactta
      541 gtccagcacg aagtctggag acctctggcg gcagcctacc aagaacaact ggaccgaccg
      601 gtggtacctc acccttaccg agtcggcgac acagtgtggg tccgccgaca ccagactaag
      661 aacctagaac ctcgctggaa aggaccttac acagtcctgc tgaccacccc caccgccctc
      721 aaagtagacg gcatcgcagc ttggatacac gccgcccacg tgaaggctgc cgaccccggg
      781 ggtggaccat cctctagact gccggatcta gctagttaat taaggatccc agtgtggtgg
      841 tacgggaatt cctgcaggcc tcgaggccac catggactac aaagaccatg acggtgatta
      901 taaagatcat gacattgatt acaaggatga cgatgacaag gtgagcaagg gcgaggagct
      961 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1021 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1081 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1141 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1201 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1261 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1321 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1381 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1441 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1501 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     1561 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1621 cgggatcact ctcggcatgg acgagctgta caagccgcgg catcgaggcg acggagagcc
     1681 tagtggagtt cctgtagctg tggtgctgct gccagtgttt gcccttaccc tggtagcagt
     1741 ttgggccttc gtgagatacc gaaagcagct ctgagcggcc gctacgtaaa ttccgccccc
     1801 cccccccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     1861 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     1921 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     1981 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     2041 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     2101 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     2161 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     2221 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     2281 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     2341 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catggttcct
     2401 ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc
     2461 atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact
     2521 ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact
     2581 gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg
     2641 ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc
     2701 aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg
     2761 ccctctggtt atgtgtggga gggctaagtc gacgataaaa taaaagattt tatttagtct
     2821 ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac
     2881 gccattttgc aaggcatgga aaaatacata actgagaata gagaagttca gatcaaggtc
     2941 aggaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg
     3001 ccccggctca gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt
     3061 ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag
     3121 ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg
     3181 accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc
     3241 tgctccccga gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat
     3301 tgactgagtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt
     3361 gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg
     3421 tctttcacat gcagcatgta tcaaaattaa tttggttttt tttcttaagt atttacatta
     3481 aatggccata gttgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3541 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3601 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3661 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3721 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3781 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3841 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3901 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3961 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4021 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4081 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4141 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4201 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4261 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4321 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4381 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttgcgg ccggccgcaa
     4441 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4501 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4561 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4621 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4681 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4741 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4801 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4861 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4921 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4981 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5041 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5101 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5161 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5221 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5281 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5341 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5401 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5461 agtgccacct gcataatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg
     5521 ccattttgca aggcatggaa aaatacataa ctgagaatag aaaagttcag atcaaggtca
     5581 ggaacagatg gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     5641 cccggctcag ggccaagaac agatggaaca gctgaatatg ggccaaacag gatatctgtg
     5701 gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtccagc
     5761 cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga
     5821 ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct
     5881 gctccccgag ctcaataaaa gagcccacaa cccctcactc ggcgcgccag tcctccgatt
     5941 gactgagtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg
     6001 tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt
     6061 ctttcatttg ggggctcgtc cgggatcggg agacccctgc ccagggacca ccgacccacc
     6121 accgggaggt aagctggcca gcaacttatc tgtgtctgtc cgattgtcta gtgtctatga
     6181 ctgattttat gcgcctgcgt cggtactagt tagctaacta gctctgtatc tggcggaccc
     6241 gtggtggaac tgacgagttc ggaacacccg gccgcaaccc tgggagacgt cccagggact
     6301 tcgggggccg tttttgtggc ccgacctgag tccaaaaatc ccgatcgttt tggactcttt
     6361 ggtgcacccc cctaatagga gggatatgtg gttctggtag gagacgagaa cctaaaacag
     6421 ttcccgcctc cgtctgaatt tttgctttcg gtttgggacc gaagccgcgc cgcgcgtctt
     6481 gtctgctgca gcatcgttct gtgttgtctc tgtctgactg tgtttctgta tttgtctgaa
     6541 aattag
//
