LOCUS       Exported                7137 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Mammalian expression plasmid to target the roGFP1-mApple sensor to 
            the mitochondrial matrix..
ACCESSION   .
VERSION     .
KEYWORDS    GW1-mito-roGFP1-mApple
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7137)
  AUTHORS   Norcross S, Trull KJ, Snaider J, Doan S, Tat K, Huang L, Tantama M
  TITLE     Extending roGFP Emission via Forster-Type Resonance Energy Transfer 
            Relay Enables Simultaneous Dual Compartment Ratiometric Redox 
            Imaging in Live Cells.
  JOURNAL   ACS Sens. 2017 Nov 22;2(11):1721-1729. doi: 
            10.1021/acssensors.7b00689. Epub 2017 Nov 7.
  PUBMED    29072071
REFERENCE   2  (bases 1 to 7137)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7137
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             189..263
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             285..359
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             381..455
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             477..551
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             606..1322
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(651..672)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(912..931)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1259..1280
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1344..2051
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="VSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAF
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNFE
                     DGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALKS
                     EIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     1754..1773
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2177..2311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2214..2233)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2268..2287
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2335..2351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2335..2351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2348..2370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2359..2375
                     /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(2383..2413)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2428..2449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2566..2583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2737..3325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2817..2836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3496..4356)
                     /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     4119..4138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     4566..4584
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4622..4644)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4744..4763
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4831..4850)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     4995..5017
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5009..5026
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5010..5026
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        5575..5954
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5955..6158
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6109..6129
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6155..6179
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 aagcttcccg gccacagcaa tcgatccatg acacaaccag tgacaagcag tcccccagtg
       61 aaaccgcatc cgcatccgag tccgataccg ataaagattc tgaatcggag tgagtgccgc
      121 gtccgagagc gttccctgtc cacgtccacc atcggcggag caggtgtgcc tgaggcctct
      181 agaccaccat gagcgtgctg acaccattgt tgctcagagg cctcactggt tcagccaggc
      241 ggctgccagt cccaagagct aagatccata gtctgggaga tccaatgtca gttctcactc
      301 cccttttgct taggggactg accggaagcg ctcggcggtt gcctgtccca cgcgccaaga
      361 ttcactccct gggcgaccct atgagtgtcc ttactcctct tttgctgaga ggcttgacag
      421 ggagcgctcg gaggctccca gtgcctcgcg caaagatcca ttctctcgga gatcccatgt
      481 ccgtcctcac tcctctgctg ctgcgcgggc ttacaggttc cgctcgccgc ctgcctgttc
      541 ctcgggcaaa gatacacagt ctgggcgatc ctcaccggcg tggaggagca tctggtggag
      601 ctagtatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      661 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
      721 cctacggcaa gctgaccctg aagttcatct ccaccaccgg caagctgccc gtgccctggc
      781 ccaccctcgt gaccactctg tcttacggcg tgcagtgctt cagccgctac cccgaccaca
      841 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
      901 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
      961 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1021 ggcacaagct ggagtacaac tacaactgcc acaacgtcta tatcatggcc gacaagcaga
     1081 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1141 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1201 accactacct gagcacctgc tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1261 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtata
     1321 agggaggttc aggaggtcga tccgtgagca agggcgagga gaataacatg gccatcatca
     1381 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     1441 tcgagggcga gggcgagggc cgcccctacg aggcctttca gaccgctaag ctgaaggtga
     1501 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     1561 ccaaggtcta cattaagcac ccagccgaca tccccgacta cttcaagctg tccttccccg
     1621 agggcttcag gtgggagcgc gtgatgaact tcgaggacgg cggcattatt cacgttaacc
     1681 aggactcctc cctgcaggac ggcgtgttca tctacaaggt gaagctgcgc ggcaccaact
     1741 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tccgaggagc
     1801 ggatgtaccc cgaggacggc gccctgaaga gcgagatcaa gaagaggctg aagctgaagg
     1861 acggcggcca ctacgccgcc gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     1921 tgcccggcgc ctacatcgtc gacatcaagt tggacatcgt gtcccacaac gaggactaca
     1981 ccatcgtgga acagtacgaa cgcgccgagg gtcgtcactc taccggcggc atggacgagc
     2041 tgtacaagta ataaactcga cgaattcatt gatcataatc agccatacca catttgtaga
     2101 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     2161 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2221 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2281 actcatcaat gtatcttatc atgtctggat ccgtaccgag ctcgcgtaat catggtcata
     2341 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2401 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2461 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2521 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2581 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2641 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2701 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2761 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2821 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2881 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     2941 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3001 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3061 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3121 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     3181 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3241 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3301 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3361 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3421 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3481 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3541 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3601 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3661 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3721 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3781 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3841 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3901 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3961 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4021 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4081 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4141 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4201 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4261 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4321 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aataagcggc
     4381 cgcggccatg ccggccacta gtctcgagtt attattgaag catttatcag ggttattgtc
     4441 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4501 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     4561 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     4621 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     4681 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     4741 atgcggcatc agagcagatt gtactgagag tgcaccatat gaagacgtcg cctcctcact
     4801 acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata aaaaaaatta
     4861 gtcagcgcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     4921 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     4981 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca tctgcagtga
     5041 ataataaaat gtgtgtttgt ccgaaatacg cgtttgagat ttctgtcccg actaaattca
     5101 tgtcgcgcga tagtggtgtt tatcgccgat agagatggcg atattggaaa aatcgatatt
     5161 tgaaaatatg gcatattgaa aatgtcgccg atgtgagttt ctgtgtaact gatatcgcca
     5221 tttttccaaa agttgatttt tgggcatacg cgatatctgg cgatacgctt atatcgttta
     5281 cgggggatgg cgatagacgc ctttggtgac ttgggcgatt ctgtgtgtcg caaatatcgc
     5341 agtttcgata taggtgacag acgatatgag gctatatcgc cgatagaggc gacatcaagc
     5401 tggcacatgg ccaatgcata tcgatctata cattgaatca atattggcca ttagccatat
     5461 tattcattgg ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc
     5521 catatcataa tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt
     5581 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     5641 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     5701 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     5761 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     5821 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     5881 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     5941 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     6001 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     6061 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     6121 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
     6181 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
     6241 tccgcggccg ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta
     6301 ccgcctatag agtctatagg cccaccccct tggcttctta tgcatgctat actgtttttg
     6361 gcttggggtc tatacacccc cgcttcctca tgttataggt gatggtatag cttagcctat
     6421 aggtgtgggt tattgaccat tattgaccac tcccctattg gtgacgatac tttccattac
     6481 taatccataa catggctctt tgccacaact ctctttattg gctatatgcc aatacactgt
     6541 ccttcagaga ctgacacgga ctctgtattt ttacaggatg gggtctcatt tattatttac
     6601 aaattcacat atacaacacc accgtcccca gtgcccgcag tttttattaa acataacgtg
     6661 ggatctccac gcgaatctcg ggtacgtgtt ccggacatgg gctcttctcc ggtagcggcg
     6721 gagcttctac atccgagccc tgctcccatg cctccagcga ctcatggtcg ctcggcagct
     6781 ccttgctcct aacagtggag gccagactta ggcacagcac gatgcccacc accaccagtg
     6841 tgccgcacaa ggccgtggcg gtagggtatg tgtctgaaaa tgagctcggg gagcgggctt
     6901 gcaccgctga cgcatttgga agacttaagg cagcggcaga agaagatgca ggcagctgag
     6961 ttgttgtgtt ctgataagag tcagaggtaa ctcccgttgc ggtgctgtta acggtggagg
     7021 gcagtgtagt ctgagcagta ctcgttgctg ccgcgcgcgc caccagacat aatagctgac
     7081 agactaacag actgttcctt tccatgggtc ttttctgcag tcaccgtcct tgacacg
//
