LOCUS       sequence-430010-        7652 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express D210N mutated RNASEH1 which abolishes the catalytic activity
            of RNASEH1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7652)
  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 7652)
  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: 
            "10.1016/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_D210N
FEATURES             Location/Qualifiers
     source          1..7652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(435..711)
                     /label=chicken beta-actin promoter
     enhancer        713..1092
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    1106..1240
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             join(1833..1901,1902..2693)
                     /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     complement(2051..2070)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2864..3452
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3353..3372
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3606..3623
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    3691..3825
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(3831..4026)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3845..3980
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    4085..4106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4121..4126,4127..4144,4145..4151)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4159..4175
                     /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)."
     primer_bind     4164..4186
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4183..4199
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4183..4199
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4246..4470
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4459..4476
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4495..5520)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4495..5520)
                     /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"
     misc_feature    join(5531..5539,5540..5542,5543..6117)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5704..5723)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     5933..5950
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(6161..6202)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(6983..7003)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7043..7062)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          join(7070..7652,1..434)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(7124..7146)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 caccccccca cgcacacacc ccgcacgcag cctttgttcc cctcgcagcc ccccccgcac
       61 cgcggggcac cgcccccggc cgcgctcccc tcgcgcacac tgcggagcgc acaaagcccc
      121 gcgccgcgcc cgcagcgctc acagccgccg ggcagcgcgg agccgcacgc ggcgctcccc
      181 acgcacacac acacgcacgc accccccgag ccgctccccc cgcacaaagg gccctcccgg
      241 agcccctcaa ggctttcacg cagccacaga aaagaaacaa gccgtcatta aaccaagcgc
      301 taattacagc ccggaggaga agggccgtcc cgcccgctca cctgtgggag taacgcggtc
      361 agtcagagcc ggggcgggcg gcgcgaggcg gcggcggagc ggggcacggg gcgaaggcag
      421 cgcgcagcga ctcccgcccg ccgcgcgctt cgctttttat agggccgccg ccgccgccgc
      481 ctcgccataa aaggaaactt tcggagcgcg ccgctctgat tggctgccgc cgcacctctc
      541 cgcctcgccc cgccccgccc ctcgccccgc cccgccccgc ctggcgcgcg cccccccccc
      601 cccccgcccc catcgctgca caaaataatt aaaaaataaa taaatacaaa attgggggtg
      661 gggagggggg ggagatgggg agagtgaagc agaacgtggg gctcacctcg accatggtaa
      721 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact
      781 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
      841 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
      901 gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
      961 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
     1021 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca
     1081 ataatcaatg tcgacgatga tatcagatcc agacatgata agatacattg atgagtttgg
     1141 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     1201 tgctttattt gtaaccatta taagctgcaa taaacaagtt aattcgattc agcttcagaa
     1261 gatggcggag ggcctccaac acagtaattt tcctcccgac tcttaaaata gaaaatgtca
     1321 agtcagttaa gcaggaagtg actaactgac cgcagctggc cgtgcgacat cctcttttaa
     1381 ttagttgcta ggcaactgcc ctccagaggg cagtgtggtt ttgcaagagg aagcaaaaag
     1441 cctctccacc caggcctgga atgtcagtgt ggttttgcaa gaggaagcaa aaagcctctc
     1501 cacccaggcc tggaatgttt ccacccaatc attactatga caacagctgt tttttttagt
     1561 attaagcaga ggccgggggc ccctggcctc cgcttactct ggagaaaaag aagagaggct
     1621 tccagaggca acttgtcaaa acaggactgg cgccttggag gcgctgtggg gccacccaaa
     1681 ttgatataat taagccccaa ccgcctcttc ccgcctcatt tcagcctcac cactatgtga
     1741 atatagctga atacacagtt tattgaataa acattaattt ccaggaaata cagtctttgt
     1801 ttttccaaag cggtcattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1861 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1921 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1981 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2041 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     2101 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2161 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2221 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2281 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2341 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     2401 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2461 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2521 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2581 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2641 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2701 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2761 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2821 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2881 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2941 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3001 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3061 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3121 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3181 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3241 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3301 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3361 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3421 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3481 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3541 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3601 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     3661 ccgcgcgttg gccgattcat taatgcagcg gatccagaca tgataagata cattgatgag
     3721 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     3781 gctattgctt tatttgtaac cattataagc tgcaataaac aagttagctt tttgcaaaag
     3841 cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc cgaggcggcc
     3901 tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg
     3961 ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg
     4021 agatgcggat ccgctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     4081 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     4141 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     4201 atgattacgc caagcttggg ctgcaggttc tttccgcctc agaagccata gagcccaccg
     4261 catccccagc atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc
     4321 caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt
     4381 aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa
     4441 caacagatgg ctggcaacta gaaggcacag tcgaggctga tcagcgagct ccgactattc
     4501 ctttgccctc ggacgagtgc tggggcgtcg gtttccacta tcggcgagta cttctacaca
     4561 gccatcggtc cagacggccg cgcttctgcg ggcgatttgt gtacgcccga cagtcccggc
     4621 tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat cgaaattgcc
     4681 gtcaaccaag ctctgataga gttggtcaag accaatgcgg agcatatacg cccggagcct
     4741 aggcgatcct gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct gctccataca
     4801 agccaaccac ggcctccaga agaagatgtt ggcgacctcg tattgggaat ccccgaacat
     4861 cgcctcgctc cagtcaatga ccgctgttat gcggccattg tccgtcagga cattgttgga
     4921 gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag tcctcggccc aaagcatcag
     4981 ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg tccatcacag tttgccagtg
     5041 atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt attgaccgat
     5101 tccttgcggt ccgaatgggc cgaacccgct cgtctggcta agatcggccg cagcgatcgc
     5161 atccatggcc tccgcgaccg gctgcagaac agcgggcagt tcggtttcag gcaggtcttg
     5221 caacgtgaca ccctgtgcac ggcgggagat gcaataggtc aggctctcgc tgaattcccc
     5281 aatgtcaagc acttccggaa tcgggagcgc ggccgatgca aagtgccgat aaacataacg
     5341 atctttgtag aaaccatcgg cgcagctatt tacccgcagg acatatccac gccctcctac
     5401 atcgaagctg aaagcacgag attcttcgcc ctccgagagc tgcatcaggt cggagacgct
     5461 gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg gtgagttcag gctttttcat
     5521 ctttgggttg ggttgtggca agcttatcat cgtgtttttc aaaggaaaac cacgtccccg
     5581 tggttcgggg ggcctagacg tttttttaac ctcgactaaa cacatgtaaa gcatgtgcac
     5641 cgaggcccca gatcagatcc catacaatgg ggtaccttct gggcatcctt cagccccttg
     5701 ttgaatacgc ttgaggagag ccatttgact ctttccacaa ctatccaact cacaacgtgg
     5761 cactggggtt gtgccgcctt tgcaggtgta tcttatacac gtggcttttg gccgcagagg
     5821 cacctgtcgc caggtggggg gttccgctgc ctgcaaaggg tcgctacaga cgttgtttgt
     5881 cttcaagaag cttccagagg aactgcttcc ttcacgacat tcaacagacc ttgcattcct
     5941 ttggcgagag gggaaagacc cctaggaatg ctcgtcaaga agacagggcc aggtttccgg
     6001 gccctcacat tgccaaaaga cggcaatatg gtggaaaata acatatagac aaacgcacac
     6061 cggccttatt ccaagcggct tcggccagta acgttagggg ggggggaggg agaggggggg
     6121 ggggcggaat tcgatatcaa gcttatcgag cggccgctca cgtagaatcg agaccgagga
     6181 gagggttagg gataggctta ccgtcttccg attgtttagc tccttctctg gctaatctgt
     6241 cagcttcttc attgcctata aatcccgaat gaccaggaac atgcatccac tgaatgtcca
     6301 tcccctgggt aagcctctcc agtgccacaa agtcctcttt gttgatcacc tctttccctg
     6361 cacttgtctt ccacccattt ttcttccaac cttgaaccca gttagttata ccatttatcg
     6421 taaacatact gtttgtatac agaaccagtt tattgatgtt ttgagtcttt gcttgttcaa
     6481 tggctttgca ggctgcatga atttccgctc tttggtttgt ctgccgccca ggaagtctaa
     6541 tgcctacatt taaaggatgg cccggccccc agtaaacgcc gattcctgct cgcggccttc
     6601 tacgcccatt actggagcag cagccatcag tgtagacgac gacgaagtct cccatgtagg
     6661 aaaacgtgtc tctgctaact ggaggcgccg gctccacgct cggcttcatg tgctttgcat
     6721 acggctctgc gctttcatgt ccatctccat ccagtggctc acggagtcgc ttgctggctt
     6781 tcgcctccga ttcttgtcca tgttgatttt catgcccttc tgaaacttcc gggcttgcag
     6841 atttcctgac aaaggcccag gcctcatcct ctgtggcaaa cttcttaaat ctggcagcag
     6901 gaaaccggtc cacctgtgct ctgcactcat tccaggtcag aaagaccccg gtcttgcggc
     6961 ccctcctcac ggcatagaag gacaccttcc tcttcttctt gggcatggtc tcgaggaatt
     7021 ctttgccaaa atgatgagac agcacaataa ccagcacgtt gcccaggagc tgtaggaaaa
     7081 agaagaaggc atgaacatgg ttagcagagg ctctagagcc gccggtcaca cgccagaagc
     7141 cgaaccccgc cctgccccgt cccccccgaa ggcagccgtc cccccgcgga cagccccgag
     7201 gctggagagg gagaagggga cggcggcgcg gcgacgcacg aaggccctcc ccgcccattt
     7261 ccttcctgcc ggcgccgcac cgcttcgccc cgcgcccgct agagggggtg cggcggcgcc
     7321 tcccagattt cggctccgca cagatttggg acaaaggaag tccctgcgcc ctctcgcacg
     7381 attaccataa aaggcaatgg ctgcggctcg ccgcgcctcg acagccgccg gcgctccggg
     7441 ggccgccgcg cccctccccc gagccctccc cggcccgagg cggccccgcc ccgcccggca
     7501 cccccacctg ccgccacccc ccgcccggca cggcgagccc cgcgccacgc cccgtacgga
     7561 gccccgcacc cgaagccggg ccgtgctcag caactcgggg aggggggtgc agggggggtt
     7621 gcagcccgac cgacgcgccc acaccccctg ct
//
