LOCUS       Exported                4759 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses a mitochondrial matrix-targeted photoactivable GFP that 
            enables detection and quantification of organelle fusion in living 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    mito-PAGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4759)
  AUTHORS   Karbowski M, Arnoult D, Chen H, Chan DC, Smith CL, Youle RJ
  TITLE     Quantitation of mitochondrial dynamics by photolabeling of 
            individual organelles shows that mitochondrial fusion is blocked 
            during the Bax activation phase of apoptosis.
  JOURNAL   J Cell Biol. 2004 Feb 16. 164(4):493-9.
  PUBMED    14769861
REFERENCE   2  (bases 1 to 4759)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4759
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        115..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        419..622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     573..593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             651..725
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      753..762
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             759..1475
                     /codon_start=1
                     /product="photoactivatable GFP variant (Patterson et al., 
                     2002)"
                     /label=PA-GFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(804..825)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1065..1084)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1412..1433
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1601..1722
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1638..1657)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1692..1711
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1729..2184)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1866..1887)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2078..2097
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2211..2315
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2317..2674
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(2340..2360)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2525..2660
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2587..2606
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2709..3503
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2763..2782)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3373..3392
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3691..3710)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3735..3782
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4111..4699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4600..4619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
       61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      241 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      421 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      481 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      541 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      601 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc atgtccgtcc
      661 tgacgccgct gctgctgcgg ggcttgacag gctcggcccg gcggctccca gtgccgcgcg
      721 ccaagatcca ttcgttgggg gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc
      781 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
      841 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
      901 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcagctacg
      961 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1021 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1081 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1141 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     1201 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     1261 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1321 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagccac cagtccgccc
     1381 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1441 ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgcgac tctagatcat
     1501 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     1561 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     1621 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     1681 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg
     1741 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     1801 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     1861 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     1921 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     1981 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     2041 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     2101 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     2161 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2221 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2281 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     2341 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2401 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     2461 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     2521 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2581 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     2641 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     2701 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     2761 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     2821 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     2881 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     2941 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     3001 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     3061 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     3121 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     3181 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     3241 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     3301 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     3361 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     3421 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     3481 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     3541 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     3601 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     3661 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     3721 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     3781 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     3841 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     3901 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     3961 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4021 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4081 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4141 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4201 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4261 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4321 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4381 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4441 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4501 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4561 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4621 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4681 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4741 ctggcctttt gctcacatg
//
