LOCUS       sequence-477265-        8700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression and mitohcondrial localization of Timer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8700)
  AUTHORS   Goldman, C., Kareva, T., Sarrafha, L., Schuldt, B.R., Sahasrabudhe, 
            A., Ahfeldt, T.,, Blanchard, J.W
  TITLE     Genetically encoded and modular subcellular organelle probes reveal 
            dysfunction in lysosomes and mitochondria driven by PRKN knockout
  JOURNAL   iScience 28 (2025)
REFERENCE   2  (bases 1 to 8700)
  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"; journalName: "iScience 28
            (2025)"
COMMENT     Sequence Label: pFUW PuroR-P2A-COX8ASig-Timer
FEATURES             Location/Qualifiers
     source          1..8700
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2622..3833
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3670..3689
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3886..4482
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3886..3905)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4382..4402
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4492..4548
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4561..4635
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             join(4669..4671,4672..5346)
                     /codon_start=1
                     /product="green-to-red fluorescent timer derivative of 
                     DsRed (Terskikh et al., 2000)"
                     /label=Timer
                     /note="mammalian codon-optimized"
                     /translation="VRSSKNVIKESMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(4849..4869)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     5261..5284
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    5366..5954
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5419..5439)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5837..5848)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5957..5973)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5958..5974)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6479..6659
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6685..6702)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6691..6802
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(6948..7536)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7028..7047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7707..8498,8499..8567))
                     /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     8330..8349
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8568..8672)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta acccgtgtcg gctccagatc tggcctccgc gccgggtttt
     2641 ggcgcctccc gcgggcgccc ccctcctcac ggcgagcgct gccacgtcag acgaagggcg
     2701 cagcgagcgt cctgatcctt ccgcccggac gctcaggaca gcggcccgct gctcataaga
     2761 ctcggcctta gaaccccagt atcagcagaa ggacatttta ggacgggact tgggtgactc
     2821 tagggcactg gttttctttc cagagagcgg aacaggcgag gaaaagtagt cccttctcgg
     2881 cgattctgcg gagggatctc cgtggggcgg tgaacgccga tgattatata aggacgcgcc
     2941 gggtgtggca cagctagttc cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga
     3001 tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc tggccggggc tttcgtggcc
     3061 gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt
     3121 ccgcgagcaa ggttgccctg aactgggggt tggggggagc gcagcaaaat ggcggctgtt
     3181 cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg
     3241 ggggcatggt gggcggcaag aacccaaggt cttgaggcct tcgctaatgc gggaaagctc
     3301 ttattcgggt gagatgggct ggggcaccat ctggggaccc tgacgtgaag tttgtcactg
     3361 actggagaac tcggtttgtc gtctgttgcg ggggcggcag ttatggcggt gccgttgggc
     3421 agtgcacccg tacctttggg agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt
     3481 tggcttataa tgcagggtgg ggccacctgc cggtaggtgt gcggtaggct tttctccgtc
     3541 gcaggacgca gggttcgggc ctagggtagg ctctcctgaa tcgacaggcg ccggacctct
     3601 ggtgagggga gggataagtg aggcgtcagt ttctttggtc ggttttatgt acctatcttc
     3661 ttaagtagct gaagctccgg ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg
     3721 aagtttttta ggcacctttt gaaatgtaat catttgggtc aatatgtaat tttcagtgtt
     3781 agactagtaa attgtccgct aaattctggc cgtttttggc ttttttgtta gacgaagctt
     3841 gggcccggga attaattcac catgtctaga ttaattaagc tagcgatgac cgagtacaag
     3901 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     3961 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     4021 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     4081 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     4141 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     4201 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     4261 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     4321 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     4381 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     4441 gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccggaagcgg agctactaac
     4501 ttcagcctgt tgaaacaagc aggggatgtc gaagagaatc ccgggccatt gaagacaaaa
     4561 atgtccgtcc tgacgccgct gctgctgcgg ggcttgacag gctcggcccg gcggctccca
     4621 gtgccgcgcg ccaagatcca ttcgttgggg gatccccggg taccggtcgt gcgctcctcc
     4681 aagaacgtca tcaaggagtc catgcgcttc aaggtgcgca tggagggcac cgtgaacggc
     4741 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcca caacaccgtg
     4801 aagctgaagg tgaccaaggg cggccccctg cccttcgcct gggacatcct gtccccccag
     4861 ttccagtacg gctccaaggt gtacgtgaag caccccgccg acatccccga ctacaagaag
     4921 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     4981 gcgaccgtga cccaggactc ctccctgcag gacggctgct tcatctacaa ggtgaagttc
     5041 atcggcgtga acttcccctc cgacggcccc gtgatgcaga agaagaccat gggctgggag
     5101 gcctccaccg agcgcctgta cccccgcgac ggcgtgctga agggcgagat ccacaaggcc
     5161 ctgaagctga aggacggcgg ccactacctg gtggagttca agtccatcta catggccaag
     5221 aagcccgtgc agctgcccgg ctactactac gtggacacca agctggacat cacctcccac
     5281 aacgaggact acaccatcgt ggagcagtac gagcgcaccg agggccgcca ccacctgttc
     5341 ctgtagcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     5401 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     5461 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     5521 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     5581 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     5641 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     5701 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     5761 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     5821 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     5881 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     5941 ggccgcctcc ccgcatcgat accgtcgacc tcgagaccta gaaaaacatg gagcaatcac
     6001 aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga
     6061 ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc
     6121 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     6181 acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg
     6241 gcagaactac acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa
     6301 gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt
     6361 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag
     6421 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg
     6481 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     6541 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     6601 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     6661 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     6721 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     6781 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     6841 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gtgagcaaaa
     6901 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6961 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     7021 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     7081 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     7141 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     7201 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     7261 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     7321 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     7381 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     7441 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     7501 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     7561 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     7621 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     7681 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7741 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7801 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7861 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7921 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7981 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     8041 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     8101 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     8161 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     8221 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     8281 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     8341 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     8401 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     8461 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     8521 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     8581 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     8641 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
