LOCUS       sequence-269033-        6655 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses mCherry-GFP1-10 in mammalian cell mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6655)
  AUTHORS   Ruan L, Zhou C, Jin E, Kucharavy A, Zhang Y, Wen Z, Florens L, Li R
  TITLE     Cytosolic proteostasis through importing of misfolded proteins into 
            mitochondria.
  JOURNAL   Nature. 2017 Mar 16;543(7645):443-446. doi: 10.1038/nature21695. 
            Epub 2017 Mar 1.
  PUBMED    28241148
REFERENCE   2  (bases 1 to 6655)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature21695"; journalName: "Nature"; date: "2017-03-16- 
            16"; volume: "543"; issue: "7645"; pages: "443-446"
COMMENT     Sequence Label: MTS-mCherry-GFP1-10-Hyg-N1
FEATURES             Location/Qualifiers
     source          1..6655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        69..426
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        69..260
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(92..112)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      277..412
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             434..1459
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             434..1459
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    1576..1657
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1685..1703)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1771..1875
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1876..1944,1945..2736)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2094..2113)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2922..3510
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3411..3430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3685..3988
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3989..4192
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4143..4163
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4497..4506
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    6008..6129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     6099..6118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(6136..6591)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6273..6294)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6485..6504
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(6618..6655,1..67)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
       61 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
      121 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
      181 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
      241 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
      301 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
      361 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
      421 tgcaaagatc gatatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
      481 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
      541 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
      601 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
      661 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
      721 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
      781 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
      841 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
      901 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
      961 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     1021 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     1081 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     1141 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     1201 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     1261 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     1321 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     1381 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     1441 tccgagggca aaggaataga gttctagagg atcataatca gccataccac atttgtagag
     1501 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     1561 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1621 atcacaaatt tcacaaataa agcatttttt tcactgcctc gcgatccaca accatggcga
     1681 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1741 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1801 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1861 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1921 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1981 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2041 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2101 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     2161 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2221 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2281 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2341 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2401 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2461 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2521 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2581 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2641 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2701 gctgagatag gtgcctcact gattaagcat tggtaaggcg cgccaggccc tgccatagcc
     2761 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2821 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2881 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2941 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3001 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3061 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3121 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3181 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3241 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3301 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3361 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3421 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3481 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3541 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3601 gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt
     3661 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     3721 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     3781 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     3841 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     3901 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     3961 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     4021 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     4081 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     4141 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     4201 gaaccgtcag atccgctagc gctaccggac tcagatctcg agctcaagct tcgaattctg
     4261 cagtcgacat ggcctccact cgtgtcctcg cctctcgcct ggcctcccgg atggctgctt
     4321 ccgccaaggt tgcccgccct gctgtccgcg ttgctcaggt cagcaagcgc accatccaga
     4381 ctggctcccc cctccagacc ctcaagcgca cccagatgac ctccatcgtc aacgccacca
     4441 cccgccaggc tttccagaag cgcgcctact cttccggatc cggtgacggt ccggtcgcca
     4501 ccatggtttc taaaggtgaa gaagataata tggctattat taaagaattt atgagattta
     4561 aagttcatat ggaaggttct gttaatggtc atgaatttga aattgaaggt gaaggtgaag
     4621 gtagaccata tgaaggtact caaactgcta aattgaaagt tactaaaggt ggtccattgc
     4681 catttgcttg ggatattttg tctccacaat ttatgtatgg ttctaaagct tatgttaaac
     4741 atccagctga tattccagat tatttgaaat tgtcttttcc agaaggtttt aaatgggaaa
     4801 gagttatgaa ttttgaagat ggtggtgttg ttactgttac tcaagattct tctttgcaag
     4861 atggtgaatt tatttataaa gttaaattga gaggtactaa ttttccatct gatggtccag
     4921 ttatgcaaaa aaaaactatg ggttgggaag cttcttctga aagaatgtat ccagaagatg
     4981 gtgctttgaa aggtgaaatt aaacaaagat tgaaattgaa agatggtggt cattatgatg
     5041 ctgaagttaa aactacttat aaagctaaaa aaccagttca attgccaggt gcttataatg
     5101 ttaatattaa attggatatt acttctcata atgaagatta tactattgtt gaacaatatg
     5161 aaagagctga aggtagacat tctactggtg gtatggatga attgtataaa gaattcggtg
     5221 gtggttctgg cggaggttca atgtctaaag gtgaagaatt gtttactggt gttgttccaa
     5281 ttttggttga attggatggt gatgttaatg gtcataaatt ttctgttaga ggtgaaggtg
     5341 aaggtgatgc tactattggt aaattgactt tgaaatttat ttgtactact ggtaaattgc
     5401 cagttccatg gccaactttg gttactactt tgacttatgg tgttcaatgt ttttctagat
     5461 atccagatca tatgaaacaa catgattttt ttaaatctgc tatgccagaa ggttatgttc
     5521 aagaaagaac tatttctttt aaagatgatg gtaaatataa aactagagct gttgttaaat
     5581 ttgaaggtga tactttggtt aatagaattg aattgaaagg tactgatttt aaagaagatg
     5641 gtaatatttt gggtcataaa ttggaatata attttaattc tcataatgtt tatattactg
     5701 ctgataaaca aaaaaatggt attaaagcta attttactgt tagacataat gttgaagatg
     5761 gttctgttca attggctgat cattatcaac aaaatactcc aattggtgat ggtccagttt
     5821 tgttgccaga taatcattat ttgtctactc aaactgtttt gtctaaagat ccaaatgaaa
     5881 aataaagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     5941 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     6001 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     6061 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     6121 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     6181 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     6241 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     6301 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     6361 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     6421 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     6481 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     6541 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     6601 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattc
//
