LOCUS       sequence-208987-        7400 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression in E coli of N-terminally GST-tagged human RNASEH2B 
            and untagged RNASEH2C and A (with D34A and D169A catalytic site 
            mutations) for purification of the RNase H2 trimeric enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7400)
  AUTHORS   Reijns MA, Bubeck D, Gibson LC, Graham SC, Baillie GS, Jones EY, 
            Jackson AP
  TITLE     The structure of the human RNase H2 complex defines key interaction 
            interfaces relevant to enzyme function and human disease.
  JOURNAL   J Biol Chem. 2011 Mar 25;286(12):10530-9. doi: 
            10.1074/jbc.M110.177394. Epub 2010 Dec 22.
  PUBMED    21177854
REFERENCE   2  (bases 1 to 7400)
  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: 
            "10.1074/jbc.M110.177394"; journalName: "J Biol Chem"; date: 
            "2011-03-25- 25"; volume: "286"; issue: "12"; pages: "10530-9"
COMMENT     Sequence Label: pGEX6P1‐hsRNASEH2BCA(D34A/D169A)
FEATURES             Location/Qualifiers
     source          1..7400
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(99..104,105..120,121..127)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    135..151
                     /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             174..827
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     785..807
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             834..857
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             1838..1860
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             2370..2392
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3366..3388)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3533..3551)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3619..3723
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3724..3792,3793..4584)
                     /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(3942..3961)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4755..5343
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5244..5263
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5497..5514
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5587..5664
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5665..6747
                     /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(5684..5703)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6796..6801,6802..6819,6820..6826)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6834..6850
                     /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     6858..6874
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6858..6874
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6870..7397
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6890..6907)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6890..6906)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6899..6921)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga
       61 catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg
      121 tataatgtgt ggaattgtga gcggataaca atttcacaca ggaaacagta ttcatgtccc
      181 ctatactagg ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat
      241 atcttgaaga aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa
      301 acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg
      361 ttaaattaac acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg
      421 gtggttgtcc aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta
      481 gatacggtgt ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc
      541 ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt
      601 taaatggtga tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt
      661 tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta
      721 ttgaagctat cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt
      781 tgcagggctg gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatctggaag
      841 ttctgttcca ggggcccctg ggatccccgg aattcccggg tcgactcgcc gctggcgtgg
      901 actgcgggga cggggttggc gcccggcagc acgtgttcct ggtttcagaa tatttaaaag
      961 atgcttcaaa gaagatgaaa aatgggctaa tgtttgtaaa actggttaac ccctgttcag
     1021 gagaaggagc catttacttg ttcaatatgt gtctacagca gctgtttgaa gtaaaagttt
     1081 tcaaggaaaa acaccattct tggtttataa atcaatcagt tcaatcagga ggtcttctcc
     1141 attttgccac acctgtggat cctctatttc tgcttctcca ctacctcata aaggctgata
     1201 aggaggggaa gtttcagccc cttgatcaag ttgtggtgga taacgtgttt ccaaattgca
     1261 tcttgttgct gaaacttcct ggacttgaga agttacttca tcatgtgaca gaggaaaaag
     1321 gtaatccaga aatagacaac aagaaatatt acaagtacag caaagagaag acattaaagt
     1381 ggctggaaaa aaaggttaat caaactgtgg cagcattaaa aaccaataat gtgaatgtca
     1441 gttcccgggt acagtcaact gcatttttct ctggtgacca agcttccact gacaaggaag
     1501 aggattatat tcgttatgcc catggtctga tatctgacta catccctaaa gaattaagtg
     1561 atgacttatc taaatactta aagcttccag aaccttcagc ctcattgcca aatcctccat
     1621 caaagaaaat aaagttatca gatgagcctg tagaagcaaa agaagattac actaagttta
     1681 atactaaaga tttgaagact gaaaagaaaa atagcaaaat gactgcagct cagaaggctt
     1741 tggctaaagt tgacaagagt ggaatgaaaa gtattgatac cttttttggg gtaaaaaata
     1801 aaaaaaaaat tggaaaggtt tgaaacgacc atcgattttt gtttaacttt aagaaggaga
     1861 tatacatatg gagagcggcg acgaagcggc catcgagagg caccgcgtcc acttgcgctc
     1921 cgccacattg cgcgacgccg tacccgccac actgcatctg ctgccctgcg aggttgcggt
     1981 ggacgggccc gccccggtgg ggcgcttctt cacgcccgcc atccgccagg gccccgaggg
     2041 actcgaagtg tcgtttcggg gccgctgtct acggggagag gaggtggcgg tgccgcctgg
     2101 cctcgtggga tacgtgatgg tgacagaaga gaagaaggtg tcgatgggga agccagaccc
     2161 cttgcgggat tccgggactg acgaccaaga ggaggagccg ctggagcggg acttcgaccg
     2221 cttcattgga gccactgcca acttcagccg cttcaccctg tggggtctgg agaccatccc
     2281 tggcccggat gccaaagtgc gtggggcctt aacttggccc agccttgcgg cagcgattca
     2341 cgcacaggtg cccgaggact agggtacctt ttgtttaact ttaagaagga gatatacata
     2401 tggatctcag cgagctggag agagacaata caggccgctg tcgcctgagt tcgcctgtgc
     2461 ccgcggtgtg ccgcaaggag ccttgcgtcc tgggcgtcgc tgaggcgggc aggggccccg
     2521 tgctgggccc catggtctac gccatctgtt attgtcccct gcctcgcctg gcagatctgg
     2581 aggcgctgaa agtggcagac tcaaagaccc tattggagag cgagcgggaa aggctgtttg
     2641 cgaaaatgga ggacacggac tttgtcggct gggcgctgga tgtgctgtct ccaaacctca
     2701 tctctaccag catgcttggg cgggtcaaat acaacctgaa ctccctgtca catgatacag
     2761 ccactgggct tatacagtat gcattggacc agggcgtgaa cgtcacccag gtattcgtgg
     2821 acaccgtagg gatgccagag acataccagg cgcggctgca gcaaagtttt cccgggattg
     2881 aggtgacggt caaggccaaa gcagctgccc tctacccggt ggttagtgct gccagcatct
     2941 gtgccaaggt ggcccgggac caggccgtga agaaatggca gttcgtggag aaactgcagg
     3001 acttggatac tgattatggc tcaggctacc ccaatgatcc caagacaaaa gcgtggttga
     3061 aggagcacgt ggagcctgtg ttcggcttcc cccagtttgt ccggttcagc tggcgcacgg
     3121 cccagaccat cctggagaaa gaggcggaag atgttatatg ggaggactca gcatccgaga
     3181 atcaggaggg actcaggaag atcacatcct acttcctcaa tgaagggtcc caagcccgtc
     3241 cccgttcttc ccaccgatat ttcctggaac gcggcctgga gtcagcaacc agcctctagg
     3301 agctcgcggc cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt
     3361 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     3421 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     3481 atgacccagt cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg
     3541 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3601 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3661 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3721 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3781 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3841 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3901 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3961 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4021 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4081 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4141 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4201 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4261 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4321 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4381 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4441 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4501 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4561 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4621 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4681 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4741 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4801 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4861 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     4921 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4981 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5041 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5101 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5161 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5221 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5281 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5341 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5401 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5461 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5521 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5581 aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     5641 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     5701 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     5761 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     5821 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     5881 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     5941 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     6001 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     6061 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     6121 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     6181 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     6241 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     6301 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     6361 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     6421 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     6481 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     6541 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     6601 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac gctagcgccc
     6661 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     6721 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     6781 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     6841 cggataacaa tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg
     6901 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     6961 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     7021 agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc
     7081 cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact
     7141 ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca
     7201 atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg
     7261 atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaattagct
     7321 tatcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat
     7381 ggctgtgcag gtcgtaaatc
//
