LOCUS       sequence-212872-        7716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express WKKD mutated RNASEH1 in mammalian cell. The combination of 
            three specific mutations (W43A, K59A, K60A) in the binding domain 
            prevents the enzyme from binding to RNA/DNA hybrids.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7716)
  AUTHORS   Chen L, Chen JY, Zhang X, Gu Y, Xiao R, Shao C, Tang P, Qian H, Luo 
            D, Li H, Zhou Y, Zhang DE, Fu XD
  TITLE     R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops 
            with Transcriptional Pausing at Gene Promoters.
  JOURNAL   Mol Cell. 2017 Nov 16;68(4):745-757.e5. doi: 
            10.1016/j.molcel.2017.10.008. Epub 2017 Nov 2.
  PUBMED    29104020
REFERENCE   2  (bases 1 to 7716)
  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: 
            "10.1016/j.molcel.2017.10.008"; journalName: "Mol Cell"; date: 
            "2017-11-16- 16"; volume: "68"; issue: "4"; pages: "745-757.e5"
COMMENT     Sequence Label: ppyCAG_RNaseH1_WKKD
FEATURES             Location/Qualifiers
     source          1..7716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(35..609,610..612,613..621)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(202..219)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     429..448
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             632..1657
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             632..1657
                     /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    1745..1969
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2016..2032)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2016..2032)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2029..2051)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2040..2056
                     /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(join(2064..2070,2071..2088,2089..2094))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2109..2130
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2189..2384
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2235..2370
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2390..2524
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2592..2609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2763..3351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2843..2862)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3522..4313,4314..4382))
                     /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     4145..4164
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     polyA_signal    4975..5109
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        5123..5502
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          5782..6798
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     6722..6744
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     6806..6825
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             6865..6885
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7666..7707
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
ORIGIN
        1 gctcgataag cttgatatcg aattccgccc cccccccctc tccctccccc ccccctaacg
       61 ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca
      121 ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga
      181 gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga
      241 aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca
      301 ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag
      361 atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa
      421 gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac
      481 cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga
      541 ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac
      601 gatgataagc ttgccacaac ccaacccaaa gatgaaaaag cctgaactca ccgcgacgtc
      661 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
      721 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
      781 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
      841 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
      901 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
      961 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     1021 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     1081 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     1141 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     1201 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     1261 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     1321 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     1381 gcggaggcat ccggagcttg caggatcgcc taggctccgg gcgtatatgc tccgcattgg
     1441 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     1501 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     1561 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     1621 ccgacgcccc agcactcgtc cgagggcaaa ggaatagtcg gagctcgctg atcagcctcg
     1681 actgtgcctt ctagttgccc gtccgagggc aaaggaatag tcggagctcg ctgatcagcc
     1741 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     1801 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     1861 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     1921 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     1981 gaaagaacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     2041 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     2101 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     2161 tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc
     2221 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     2281 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     2341 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat
     2401 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2461 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2521 gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2581 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2641 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2701 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2761 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2821 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2881 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2941 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3001 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3061 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3121 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3181 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3241 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3301 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3361 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3421 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3481 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3541 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3601 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3661 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3721 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3781 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3841 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3901 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3961 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4021 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4081 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4141 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4201 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4261 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4321 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4381 atactcttcc tttttcaatg accgctttgg aaaaacaaag actgtatttc ctggaaatta
     4441 atgtttattc aataaactgt gtattcagct atattcacat agtggtgagg ctgaaatgag
     4501 gcgggaagag gcggttgggg cttaattata tcaatttggg tggccccaca gcgcctccaa
     4561 ggcgccagtc ctgttttgac aagttgcctc tggaagcctc tcttcttttt ctccagagta
     4621 agcggaggcc aggggccccc ggcctctgct taatactaaa aaaaacagct gttgtcatag
     4681 taatgattgg gtggaaacat tccaggcctg ggtggagagg ctttttgctt cctcttgcaa
     4741 aaccacactg acattccagg cctgggtgga gaggcttttt gcttcctctt gcaaaaccac
     4801 actgccctct ggagggcagt tgcctagcaa ctaattaaaa gaggatgtcg cacggccagc
     4861 tgcggtcagt tagtcacttc ctgcttaact gacttgacat tttctatttt aagagtcggg
     4921 aggaaaatta ctgtgttgga ggccctccgc catcttctga agctgaatcg aattaacttg
     4981 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5041 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     5101 gtctggatct gatatcatcg tcgacattga ttattgacta gttattaata gtaatcaatt
     5161 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     5221 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     5281 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     5341 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     5401 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     5461 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
     5521 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
     5581 ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccggggggg
     5641 gggggggggg gggggggggg gggggggggg gggggggggg gggcggcggc agccaatcag
     5701 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
     5761 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
     5821 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
     5881 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt
     5941 tcttttctgt ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg
     6001 agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg
     6061 ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg
     6121 cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac
     6181 aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc
     6241 gggctgcaac cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt
     6301 gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag
     6361 gtgggggtgc cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc
     6421 ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta
     6481 tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa
     6541 tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc
     6601 aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct
     6661 ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg
     6721 gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc
     6781 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
     6841 ggcaaagaat tcctcgagac catgcccaag aagaagagga aggtgtcctt ctatgccgtg
     6901 aggaggggcc gcaagaccgg ggtctttctg accgcgaatg agtgcagagc acaggtggac
     6961 cggtttcctg ctgccagatt tgcggcgttt gccacagagg atgaggcctg ggcctttgtc
     7021 aggaaatctg caagcccgga agtttcagaa gggcatgaaa atcaacatgg acaagaatcg
     7081 gaggcgaaag ccagcaagcg actccgtgag ccactggatg gagatggaca tgaaagcgca
     7141 gagccgtatg caaagcacat gaagccgagc gtggagccgg cgcctccagt tagcagagac
     7201 acgttttcct acatgggaga cttcgtcgtc gtctacactg atggctgctg ctccagtaat
     7261 gggcgtagaa ggccgcgagc aggaatcggc gtttactggg ggccgggcca tcctttaaat
     7321 gtaggcatta gacttcctgg gcggcagaca aaccaaagag cggaaattca tgcagcctgc
     7381 aaagccattg aacaagcaaa gactcaaaac atcaataaac tggttctgta tacaaacagt
     7441 atgtttacga taaatggtat aactaactgg gttcaaggtt ggaagaaaaa tgggtggaag
     7501 acaagtgcag ggaaagaggt gatcaacaaa gaggactttg tggcactgga gaggcttacc
     7561 caggggatgg acattcagtg gatgcatgtt cctggtcatt cgggatttat aggcaatgaa
     7621 gaagctaaca gattagccag agaaggagct aaacaatcgg aagacggtaa gcctatccct
     7681 aaccctctcc tcggtctcga ttctacgtga gcggcc
//
