LOCUS       sequence-270281-        5887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of catalytic inactive RNase H fused to micrococcal 
            nuclease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5887)
  AUTHORS   Yan Q, Shields EJ, Bonasio R, Sarma K
  TITLE     Mapping Native R-Loops Genome-wide Using a Targeted Nuclease 
            Approach.
  JOURNAL   Cell Rep. 2019 Oct 29;29(5):1369-1380.e5. doi: 
            10.1016/j.celrep.2019.09.052.
  PUBMED    31665646
REFERENCE   2  (bases 1 to 5887)
  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.celrep.2019.09"; journalName: "Cell Rep"; date: 
            "2019-10-29- 29"; volume: "29"; issue: "5"; pages: "1369-1380.e5"
COMMENT     Sequence Label: GST-RH▲-MNase
FEATURES             Location/Qualifiers
     source          1..5887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             75..602
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(95..111)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(104..126)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(704..709,710..725,726..732)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    740..756
                     /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)."
     CDS             779..1432
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1390..1412
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1439..1462
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1943..2389
                     /codon_start=1
                     /product="micrococcal nuclease from Staphylococcus aureus"
                     /label=MNase
                     /translation="ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK
                     HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALV
                     RQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ"
     primer_bind     complement(2458..2480)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2625..2643)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2711..2815
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2816..2884,2885..3676)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3034..3053)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3847..4435
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4336..4355
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4589..4606
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4679..4756
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4757..5839
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4776..4795)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta caacgtcgtg
      121 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
      181 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      241 atggcgaatg gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg
      301 agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt
      361 acgatgcgcc catctacacc aacgtgacct atcccattac ggtcaatccg ccgtttgttc
      421 ccacggagaa tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac
      481 aggaaggcca gacgcgaatt atttttgatg gcgttggaat tagcttatcg actgcacggt
      541 gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt
      601 aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc
      661 gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg
      721 gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagtattcat
      781 gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt
      841 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg
      901 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg
      961 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat
     1021 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga
     1081 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga
     1141 ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac
     1201 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt
     1261 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa
     1321 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg
     1381 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct
     1441 ggaagttctg ttccaggggc ccctgggatc catgctgaaa cagggggaaa tcttcacccg
     1501 gggcagctgt ctgggcaatc ctggccctgg cggatacggc gccatcctga gataccgggg
     1561 cagagagaaa accttcagcg ccggctacac ccggaccacc aacaaccgga tgagactgat
     1621 ggccgccatc gtggccctgg aagccctgaa agaacactgc gaagtgatcc tgagcaccga
     1681 cagccagtac gtgcggcagg gcatcaccca gtggattcac aactggaaga agcggggctg
     1741 gaaaaccgcc gacaagaaac ccgtgaagaa cgtggacctg tggcagagac tggatgccgc
     1801 cctgggacag caccagatca agtgggagtg ggtcaagggc cacgccggcc accctgagaa
     1861 tgagagatgt gacgagctgg ccagagccgc cgctatgaac cccaccctgg aagataccgg
     1921 ctaccaggtg gaagtggaat tcgcaacttc aactaaaaaa ttacataaag aacctgcgac
     1981 tttaattaaa gcgattgatg gtgatacggt taaattaatg tacaaaggtc aaccaatgac
     2041 attcagacta ttattggttg atacacctga aacaaagcat cctaaaaaag gtgtagagaa
     2101 atatggtcct gaagcaagtg catttacgaa aaaaatggta gaaaatgcaa agaaaattga
     2161 agtcgagttt gacaaaggtc aaagaactga taaatatgga cgtggcttag cgtatattta
     2221 tgctgatgga aaaatggtaa acgaagcttt agttcgtcaa ggcttggcta aagttgctta
     2281 tgtttacaaa cctaacaata cacatgaaca acatttaaga aaaagtgaag cacaagcgaa
     2341 aaaagagaaa ttaaatattt ggagcgaaga caacgctgat tcaggtcaat aactcgagcg
     2401 gccgcatcgt gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     2461 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     2521 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     2581 gtcacgtagc gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct
     2641 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     2701 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2761 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2821 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2881 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2941 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3001 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     3061 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3121 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3181 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3241 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3301 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     3361 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3421 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3481 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3541 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3601 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3661 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3721 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3781 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3841 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3901 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3961 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4021 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4081 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4141 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4201 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4261 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4321 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4381 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4441 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4501 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4561 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4621 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca taaattccga
     4681 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     4741 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     4801 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     4861 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     4921 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     4981 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     5041 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     5101 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     5161 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     5221 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     5281 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     5341 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     5401 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     5461 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     5521 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     5581 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     5641 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     5701 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc acgctagcgc ccaatacgca
     5761 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5821 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     5881 cccaggc
//
