LOCUS       sequence-208976-        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, allowing affinity 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
FEATURES             Location/Qualifiers
     source          1..7400
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(89..94,95..110,111..117)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    125..141
                     /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             164..817
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     775..797
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             824..847
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             1828..1850
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             2360..2382
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3356..3378)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3523..3541)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3609..3713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3714..3782,3783..4574)
                     /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(3932..3951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4745..5333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5234..5253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5487..5504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5577..5654
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5655..6737
                     /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(5674..5693)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6786..6791,6792..6809,6810..6816)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6824..6840
                     /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     6848..6864
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6848..6864
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6860..7387
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6880..6897)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6880..6896)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6889..6911)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg
       61 gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt
      121 ggaattgtga gcggataaca atttcacaca ggaaacagta ttcatgtccc ctatactagg
      181 ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga
      241 aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt
      301 tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac
      361 acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc
      421 aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt
      481 ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct
      541 acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga
      601 tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga
      661 cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat
      721 cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg
      781 gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatctggaag ttctgttcca
      841 ggggcccctg ggatccccgg aattcccggg tcgactcgcc gctggcgtgg actgcgggga
      901 cggggttggc gcccggcagc acgtgttcct ggtttcagaa tatttaaaag atgcttcaaa
      961 gaagatgaaa aatgggctaa tgtttgtaaa actggttaac ccctgttcag gagaaggagc
     1021 catttacttg ttcaatatgt gtctacagca gctgtttgaa gtaaaagttt tcaaggaaaa
     1081 acaccattct tggtttataa atcaatcagt tcaatcagga ggtcttctcc attttgccac
     1141 acctgtggat cctctatttc tgcttctcca ctacctcata aaggctgata aggaggggaa
     1201 gtttcagccc cttgatcaag ttgtggtgga taacgtgttt ccaaattgca tcttgttgct
     1261 gaaacttcct ggacttgaga agttacttca tcatgtgaca gaggaaaaag gtaatccaga
     1321 aatagacaac aagaaatatt acaagtacag caaagagaag acattaaagt ggctggaaaa
     1381 aaaggttaat caaactgtgg cagcattaaa aaccaataat gtgaatgtca gttcccgggt
     1441 acagtcaact gcatttttct ctggtgacca agcttccact gacaaggaag aggattatat
     1501 tcgttatgcc catggtctga tatctgacta catccctaaa gaattaagtg atgacttatc
     1561 taaatactta aagcttccag aaccttcagc ctcattgcca aatcctccat caaagaaaat
     1621 aaagttatca gatgagcctg tagaagcaaa agaagattac actaagttta atactaaaga
     1681 tttgaagact gaaaagaaaa atagcaaaat gactgcagct cagaaggctt tggctaaagt
     1741 tgacaagagt ggaatgaaaa gtattgatac cttttttggg gtaaaaaata aaaaaaaaat
     1801 tggaaaggtt tgaaacgacc atcgattttt gtttaacttt aagaaggaga tatacatatg
     1861 gagagcggcg acgaagcggc catcgagagg caccgcgtcc acttgcgctc cgccacattg
     1921 cgcgacgccg tacccgccac actgcatctg ctgccctgcg aggttgcggt ggacgggccc
     1981 gccccggtgg ggcgcttctt cacgcccgcc atccgccagg gccccgaggg actcgaagtg
     2041 tcgtttcggg gccgctgtct acggggagag gaggtggcgg tgccgcctgg cctcgtggga
     2101 tacgtgatgg tgacagaaga gaagaaggtg tcgatgggga agccagaccc cttgcgggat
     2161 tccgggactg acgaccaaga ggaggagccg ctggagcggg acttcgaccg cttcattgga
     2221 gccactgcca acttcagccg cttcaccctg tggggtctgg agaccatccc tggcccggat
     2281 gccaaagtgc gtggggcctt aacttggccc agccttgcgg cagcgattca cgcacaggtg
     2341 cccgaggact agggtacctt ttgtttaact ttaagaagga gatatacata tggatctcag
     2401 cgagctggag agagacaata caggccgctg tcgcctgagt tcgcctgtgc ccgcggtgtg
     2461 ccgcaaggag ccttgcgtcc tgggcgtcga tgaggcgggc aggggccccg tgctgggccc
     2521 catggtctac gccatctgtt attgtcccct gcctcgcctg gcagatctgg aggcgctgaa
     2581 agtggcagac tcaaagaccc tattggagag cgagcgggaa aggctgtttg cgaaaatgga
     2641 ggacacggac tttgtcggct gggcgctgga tgtgctgtct ccaaacctca tctctaccag
     2701 catgcttggg cgggtcaaat acaacctgaa ctccctgtca catgatacag ccactgggct
     2761 tatacagtat gcattggacc agggcgtgaa cgtcacccag gtattcgtgg acaccgtagg
     2821 gatgccagag acataccagg cgcggctgca gcaaagtttt cccgggattg aggtgacggt
     2881 caaggccaaa gcagatgccc tctacccggt ggttagtgct gccagcatct gtgccaaggt
     2941 ggcccgggac caggccgtga agaaatggca gttcgtggag aaactgcagg acttggatac
     3001 tgattatggc tcaggctacc ccaatgatcc caagacaaaa gcgtggttga aggagcacgt
     3061 ggagcctgtg ttcggcttcc cccagtttgt ccggttcagc tggcgcacgg cccagaccat
     3121 cctggagaaa gaggcggaag atgttatatg ggaggactca gcatccgaga atcaggaggg
     3181 actcaggaag atcacatcct acttcctcaa tgaagggtcc caagcccgtc cccgttcttc
     3241 ccaccgatat ttcctggaac gcggcctgga gtcagcaacc agcctctagg agctcgcggc
     3301 cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3361 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3421 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3481 cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat
     3541 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     3601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     3961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     4261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4861 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     4921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5521 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata aattccgaca
     5581 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     5641 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     5701 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     5761 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     5821 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     5881 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     5941 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     6001 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     6061 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     6121 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     6181 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     6241 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     6301 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     6361 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     6421 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     6481 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     6541 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     6601 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac gctagcgccc aatacgcaaa
     6661 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     6721 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccc
     6781 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     6841 tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg
     6901 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     6961 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     7021 cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc cggaaagctg
     7081 gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca
     7141 cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca atccgccgtt
     7201 tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg atgaaagctg
     7261 gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaattagct tatcgactgc
     7321 acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag
     7381 gtcgtaaatc actgcataat
//
