LOCUS       Exported                7684 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 7684)
  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 7684)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7684
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     5..22
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6..22
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        588..967
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        968..1171
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1122..1142
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1168..1192
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2339..2413
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2435..2509
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2531..2605
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2627..2701
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2756..3472
                     /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(2801..2822)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3062..3081)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3409..3430
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3494..4201
                     /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     3904..3923
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4327..4461
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4364..4383)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4418..4437
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4484..4500)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4484..4500)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4497..4519)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4508..4524
                     /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(4532..4562)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4714..4731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4885..5473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4965..4984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5644..6504)
                     /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     6267..6286
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6561..7074)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6756..6777)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6968..6987
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7246..7264
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7302..7324)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7424..7443
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(7511..7530)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     7675..13
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttgtaaa acgacggcca gtgcccgtgt caaggacggt gactgcagtg aataataaaa
       61 tgtgtgtttg tccgaaatac gcgttttgag atttctgtcg ccgactaaat tcatgtcgcg
      121 cgatagtggt gtttatcgcc gatagagatg gcgatattgg aaaaatcgat atttgaaaat
      181 atggcatatt gaaaatgtcg ccgatgtgag tttctgtgta actgatatcg ccatttttcc
      241 aaaagtgatt tttgggcata cgcgatatct ggcgatagcg cttatatcgt ttacggggga
      301 tggcgataga cgactttggt gacttgggcg attctgtgtg tcgcaaatat cgcagtttcg
      361 atataggtga cagacgatat gaggctatat cgccgataga ggcgacatca agctggcaca
      421 tggccaatgc atatcgatct atacattgaa tcaatattgg ccattagcca tattattcat
      481 tggttatata gcataaatca atattggcta ttggccattg catacgttgt atccatatca
      541 taatatgtac atttatattg gctcatgtcc aacattaccg ccatgttgac attgattatt
      601 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      661 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      721 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      781 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      841 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      901 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      961 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     1021 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     1081 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     1141 tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag
     1201 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccgcgg
     1261 ccgggaacgg tgcattggaa cgcggattcc ccgtgccaag agtgacgtaa gtaccgccta
     1321 tagagtctat aggcccaccc ccttggcttc ttatgcatgc tatactgttt ttggcttggg
     1381 gtctatacac ccccgcttcc tcatgttata ggtgatggta tagcttagcc tataggtgtg
     1441 ggttattgac cattattgac cactccccta ttggtgacga tactttccat tactaatcca
     1501 taacatggct ctttgccaca actctcttta ttggctatat gccaatacac tgtccttcag
     1561 agactgacac ggactctgta tttttacagg atggggtctc atttattatt tacaaattca
     1621 catatacaac accaccgtcc ccagtgcccg cagtttttat taaacataac gtgggatctc
     1681 cacgcgaatc tcgggtacgt gttccggaca tgggctcttc tccggtagcg gcggagcttc
     1741 tacatccgag ccctgctccc atgcctccag cgactcatgg tcgctcggca gctccttgct
     1801 cctaacagtg gaggccagac ttaggcacag cacgatgccc accaccacca gtgtgccgca
     1861 caaggccgtg gcggtagggt atgtgtctga aaatgagctc ggggagcggg cttgcaccgc
     1921 tgacgcattt ggaagactta aggcagcggc agaagaagat gcaggcagct gagttgttgt
     1981 gttctgataa gagtcagagg taactcccgt tgcggtgctg ttaacggtgg agggcagtgt
     2041 agtctgagca gtactcgttg ctgccgcgcg cgccaccaga cataatagct gacagactaa
     2101 cagactgttc ctttccatgg gtcttttctg cagtcaccgt ccttgacacg aagcttcccg
     2161 gccacagcaa tcgatccatg acacaaccag tgacaagcag tcccccagtg aaaccgcatc
     2221 cgcatccgag tccgataccg ataaagattc tgaatcggag tgagtgccgc gtccgagagc
     2281 gttccctgtc cacgtccacc atcggcggag caggtgtgcc tgaggcctct agaccaccat
     2341 gagcgtgctg acaccattgt tgctcagagg cctcactggt tcagccaggc ggctgccagt
     2401 cccaagagct aagatccata gtctgggaga tccaatgtca gttctcactc cccttttgct
     2461 taggggactg accggaagcg ctcggcggtt gcctgtccca cgcgccaaga ttcactccct
     2521 gggcgaccct atgagtgtcc ttactcctct tttgctgaga ggcttgacag ggagcgctcg
     2581 gaggctccca gtgcctcgcg caaagatcca ttctctcgga gatcccatgt ccgtcctcac
     2641 tcctctgctg ctgcgcgggc ttacaggttc cgctcgccgc ctgcctgttc ctcgggcaaa
     2701 gatacacagt ctgggcgatc ctcaccggcg tggaggagca tctggtggag ctagtatggt
     2761 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2821 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2881 gctgaccctg aagttcatct ccaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2941 gaccactctg tcttacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     3001 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     3061 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     3121 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     3181 ggagtacaac tacaactgcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     3241 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     3301 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3361 gagcacctgc tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3421 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtata agggaggttc
     3481 aggaggtcga tccgtgagca agggcgagga gaataacatg gccatcatca aggagttcat
     3541 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     3601 gggcgagggc cgcccctacg aggcctttca gaccgctaag ctgaaggtga ccaagggtgg
     3661 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggtcta
     3721 cattaagcac ccagccgaca tccccgacta cttcaagctg tccttccccg agggcttcag
     3781 gtgggagcgc gtgatgaact tcgaggacgg cggcattatt cacgttaacc aggactcctc
     3841 cctgcaggac ggcgtgttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     3901 cggccccgta atgcagaaga agaccatggg ctgggaggcc tccgaggagc ggatgtaccc
     3961 cgaggacggc gccctgaaga gcgagatcaa gaagaggctg aagctgaagg acggcggcca
     4021 ctacgccgcc gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     4081 ctacatcgtc gacatcaagt tggacatcgt gtcccacaac gaggactaca ccatcgtgga
     4141 acagtacgaa cgcgccgagg gtcgtcactc taccggcggc atggacgagc tgtacaagta
     4201 ataaactcga cgaattcatt gatcataatc agccatacca catttgtaga ggttttactt
     4261 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     4321 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4381 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4441 gtatcttatc atgtctggat ccgtaccgag ctcgcgtaat catgtcatag ctgtttcctg
     4501 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4561 aagcctgggg tgcctaatga gtgagctaac tacattaatt gcgttgcgct cactgcccgc
     4621 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4681 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4741 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4801 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4861 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4921 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4981 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5041 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5101 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5161 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5221 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5281 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5341 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5401 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5461 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5521 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5581 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5641 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5701 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5761 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5821 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5881 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5941 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6001 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6061 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6121 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6181 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6241 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6301 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6361 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6421 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6481 gacacggaaa tgttgaatac tcatactctt cctttttcaa taagcggccg cggccatgcc
     6541 ggccactagt ctcgagggcc accccggttg ataatcagaa aagccccaaa aacaggaaga
     6601 ttgtataagc aaatatttaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     6661 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     6721 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     6781 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     6841 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     6901 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     6961 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     7021 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtggggat
     7081 ccctcgagtt attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7141 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7201 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     7261 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     7321 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     7381 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     7441 gtactgagag tgcaccatat gaagacgtcg cctcctcact acttctggaa tagctcagag
     7501 gccgaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagggcgc cattcgccat
     7561 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     7621 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     7681 cacg
//
