LOCUS       sequence-477263-        8586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression and mitochondrial localization of Grx1-roGFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8586)
  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 8586)
  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 mito-GRX1-roGFP2
FEATURES             Location/Qualifiers
     source          1..8586
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(49..68)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        240..619
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        621..819
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     774..794
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             837..1017
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1064..1189
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1682..1915
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2100..2144
                     /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    2442..2559
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2624..3835
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3672..3691
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             join(4518..4520,4521..5234)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FISTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4560..4581)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4821..4840)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5168..5189
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5254..5842
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5307..5327)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5725..5736)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5845..5861)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5846..5862)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6367..6547
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6573..6590)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6579..6690
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(6836..7424)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6916..6935)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7595..8386,8387..8455))
                     /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     8218..8237
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8456..8560)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
       61 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      121 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
      181 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      241 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      301 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      361 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      421 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      481 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      541 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      601 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      661 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      721 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      781 gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt
      841 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
      901 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
      961 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
     1021 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
     1081 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
     1141 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
     1201 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
     1261 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
     1321 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
     1381 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
     1441 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
     1501 caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata
     1561 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag
     1621 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa
     1681 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa
     1741 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt
     1801 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc
     1861 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga
     1921 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga
     1981 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa
     2041 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa
     2101 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca
     2161 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
     2221 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
     2281 tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag
     2341 aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc
     2401 gtgcgccaat tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     2461 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     2521 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     2581 agagatccag tttggttaat taacccgtgt cggctccaga tctggcctcc gcgccgggtt
     2641 ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg ctgccacgtc agacgaaggg
     2701 cgcagcgagc gtcctgatcc ttccgcccgg acgctcagga cagcggcccg ctgctcataa
     2761 gactcggcct tagaacccca gtatcagcag aaggacattt taggacggga cttgggtgac
     2821 tctagggcac tggttttctt tccagagagc ggaacaggcg aggaaaagta gtcccttctc
     2881 ggcgattctg cggagggatc tccgtggggc ggtgaacgcc gatgattata taaggacgcg
     2941 ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg
     3001 gatcgctgtg atcgtcactt ggtgagtagc gggctgctgg gctggccggg gctttcgtgg
     3061 ccgccgggcc gctcggtggg acggaagcgt gtggagagac cgccaagggc tgtagtctgg
     3121 gtccgcgagc aaggttgccc tgaactgggg gttgggggga gcgcagcaaa atggcggctg
     3181 ttcccgagtc ttgaatggaa gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt
     3241 ggggggcatg gtgggcggca agaacccaag gtcttgaggc cttcgctaat gcgggaaagc
     3301 tcttattcgg gtgagatggg ctggggcacc atctggggac cctgacgtga agtttgtcac
     3361 tgactggaga actcggtttg tcgtctgttg cgggggcggc agttatggcg gtgccgttgg
     3421 gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt cacccgttct
     3481 gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg cttttctccg
     3541 tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg cgccggacct
     3601 ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat gtacctatct
     3661 tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga gtgtgttttg
     3721 tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta attttcagtg
     3781 ttagactagt aaattgtccg ctaaattctg gccgtttttg gcttttttgt tagacgaagc
     3841 ttgggcccgg gaattaattc accatgtcta gattaattaa gctagcgatg gcctccactc
     3901 gtgtcctcgc ctctcgcctg gcctcccaga tggctgcttc cgccaaggtt gcccgccctg
     3961 ctgtccgcgt tgctcaggtc agcaagcgca ccatccagac tggctccccc ctccagaccc
     4021 tcaagcgcac ccagatgacc tccatcgtca acgccaccac ccgccaggct ttccagaagc
     4081 gcgcctactc ttccggatcc gctcaagagt ttgtgaactg caaaatccag cctgggaagg
     4141 tggttgtgtt catcaagccc acctgcccgt actgcaggag ggcccaagag atcctcagtc
     4201 aattgcccat caaacaaggg cttctggaat ttgtcgatat cacagccacc aaccacacta
     4261 acgagattca agattatttg caacagctca cgggagcaag aacggtgcct cgagtcttta
     4321 ttggtaaaga ttgtataggc ggatgcagtg atctagtctc tttgcaacag agtggggaac
     4381 tgctgacgcg gctaaagcag attggagctc tgcagactag tggtggttca ggtggtggtg
     4441 gttcaggtgg tggtggttca ggtggaggag gatcaggagg aggaggatca ggaggaggag
     4501 gatcaggagg agaattcgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     4561 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     4621 gcgatgccac ctacggcaag ctgaccctga agttcatctc caccaccggc aagctgcccg
     4681 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     4741 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     4801 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     4861 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     4921 acatcctggg gcacaagctg gagtacaact acaactgcca caacgtctat atcatggccg
     4981 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     5041 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     5101 tgcccgacaa ccactacctg agcacctgct ccgccctgag caaagacccc aacgagaagc
     5161 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     5221 agctgtacaa gtaacgatat caagcttatc gataatcaac ctctggatta caaaatttgt
     5281 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     5341 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     5401 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     5461 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     5521 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     5581 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     5641 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     5701 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     5761 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     5821 tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gagacctaga aaaacatgga
     5881 gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa
     5941 gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac
     6001 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     6061 cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc
     6121 cctgattggc agaactacac accagggcca gggatcagat atccactgac ctttggatgg
     6181 tgctacaagc tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac
     6241 acccgcttgt tacaccctgt gagcctgcat gggatggatg acccggagag agaagtatta
     6301 gagtggaggt ttgacagccg cctagcattt catcacatgg cccgagagct gcatccggac
     6361 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     6421 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     6481 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     6541 tctagcaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     6601 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     6661 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     6721 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgt
     6781 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6841 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6901 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6961 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     7021 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     7081 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     7141 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     7201 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     7261 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     7321 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     7381 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     7441 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7501 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7561 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7621 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7681 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7741 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7801 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7861 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7921 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7981 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     8041 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     8101 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     8161 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     8221 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     8281 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     8341 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     8401 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     8461 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8521 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8581 cacctg
//
