LOCUS       sequence-385953-        5557 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for transient mammalian expression of wild type RNase H1 
            tagged with 2xNLS and EGFP that can be used to specifically degrade 
            the nuclear R-loops..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5557)
  AUTHORS   Crossley MP, Song C, Bocek MJ, Choi JH, Kousorous J, Sathirachinda 
            A, Lin C, Brickner JR, Bai G, Lans H, Vermeulen W, Abu-Remaileh M, 
            Cimprich KA
  TITLE     R-loop-derived cytoplasmic RNA-DNA hybrids activate an immune 
            response.
  JOURNAL   Nature. 2023 Jan;613(7942):187-194. doi: 10.1038/s41586-022-05545-9.
            Epub 2022 Dec 21.
  PUBMED    36544021
REFERENCE   2  (bases 1 to 5557)
  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"; doi: 
            "10.1038/s41586-022-05545-9"; journalName: "Nature"; date: 
            "2023-01"; volume: "613"; issue: "7942"; pages: "187-194"
COMMENT     Sequence Label: pEGFP-N2-2XNLS-RNaseH1 delta 1-27 (WT)
FEATURES             Location/Qualifiers
     source          1..5557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             621..641
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             669..689
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1497..1506
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1503..1505,1506..1508,1509..2222)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1503..1505,1506..1508,1509..2219)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1548..1569)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1809..1828)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2156..2177
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2345..2466
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2382..2401)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2436..2455
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2473..2928)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2610..2631)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2822..2841
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2955..3059
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3061..3418
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3061..3252
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3084..3104)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3269..3404
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3453..4247
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3453..4247
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3507..3526)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4117..4136
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4435..4454)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4479..4526
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4855..5443
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5344..5363
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgaatg cccaagaaaa agcggaaagt gggcacgcgt ggcagcttcg
      661 aattcatgcc caagaaaaag cggaaagtgg gcacgcgtgg cttctatgcc gtgaggaggg
      721 gccgcaagac cggggtcttt ctgacctgga atgagtgcag agcacaggtg gaccggtttc
      781 ctgctgccag atttaagaag tttgccacag aggatgaggc ctgggccttt gtcaggaaat
      841 ctgcaagccc ggaagtttca gaagggcatg aaaatcaaca tggacaagaa tcggaggcga
      901 aagccagcaa gcgactccgt gagccactgg atggagatgg acatgaaagc gcagagccgt
      961 atgcaaagca catgaagccg agcgtggagc cggcgcctcc agttagcaga gacacgtttt
     1021 cctacatggg agacttcgtc gtcgtctaca ctgatggctg ctgctccagt aatgggcgta
     1081 gaaggccgcg agcaggaatc ggcgtttact gggggccggg ccatccttta aatgtaggca
     1141 ttagacttcc tgggcggcag acaaaccaaa gagcggaaat tcatgcagcc tgcaaagcca
     1201 ttgaacaagc aaagactcaa aacatcaata aactggttct gtatacagac agtatgttta
     1261 cgataaatgg tataactaac tgggttcaag gttggaagaa aaatgggtgg aagacaagtg
     1321 cagggaaaga ggtgatcaac aaagaggact ttgtggcact ggagaggctt acccagggga
     1381 tggacattca gtggatgcat gttcctggtc attcgggatt tataggcaat gaagaagctg
     1441 acagattagc cagagaagga gctaaacaat cggaagactg gatccaccgg ccggtcgcca
     1501 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1561 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1621 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1681 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1741 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1801 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1861 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1921 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1981 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     2041 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2101 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     2161 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     2221 aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     2281 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     2341 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2401 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2461 atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     2521 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2581 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2641 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     2701 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     2761 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     2821 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     2881 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     2941 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3001 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     3061 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3121 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3181 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3241 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3301 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     3361 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     3421 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3481 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3541 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3601 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     3661 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3721 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3781 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3841 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3901 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3961 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4021 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4081 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4141 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4201 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4261 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4321 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4381 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     4441 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     4501 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     4561 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     4621 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     4681 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     4741 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4801 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4861 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4921 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4981 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5041 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5101 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5161 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5221 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5281 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5341 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5401 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5461 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5521 ccctgattct gtggataacc gtattaccgc catgcat
//
