LOCUS       sequence-208997-        5606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression in E. coli, and affinity purification of N-terminally
            GST-tagged Archaeoglobus fulgidus RNase HII with D101N catalytic 
            site mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5606)
  AUTHORS   Bubeck D, Reijns MA, Graham SC, Astell KR, Jones EY, Jackson AP
  TITLE     PCNA directs type 2 RNase H activity on DNA replication and repair 
            substrates.
  JOURNAL   Nucleic Acids Res. 2011 May;39(9):3652-66. doi: 10.1093/nar/gkq980. 
            Epub 2011 Jan 17.
  PUBMED    21245041
REFERENCE   2  (bases 1 to 5606)
  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: 
            "10.1093/nar/gkq980"; journalName: "Nucleic Acids Res"; date: 
            "2011-05"; volume: "39"; issue: "9"; pages: "3652-66"
COMMENT     Sequence Label: pGEX6P1‐Afu‐rnhB D101N
FEATURES             Location/Qualifiers
     source          1..5606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(79..84,85..100,101..107)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    115..131
                     /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             154..807
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     765..787
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             814..837
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1552..1574)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1719..1737)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1805..1909
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1910..1978,1979..2770)
                     /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(2128..2147)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2941..3529
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3430..3449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3683..3700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3773..3850
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3851..4933
                     /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(3870..3889)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4982..4987,4988..5005,5006..5012)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5020..5036
                     /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     5044..5060
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5044..5060
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5056..5583
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5076..5093)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5076..5092)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5085..5107)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa
       61 atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt ggaattgtga
      121 gcggataaca atttcacaca ggaaacagta ttcatgtccc ctatactagg ttattggaaa
      181 attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga aaaatatgaa
      241 gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt
      301 ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac acagtctatg
      361 gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt
      421 gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt ttcgagaatt
      481 gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct acctgaaatg
      541 ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga tcatgtaacc
      601 catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga cccaatgtgc
      661 ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat cccacaaatt
      721 gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg gcaagccacg
      781 tttggtggtg gcgaccatcc tccaaaatcg gatctggaag ttctgttcca ggggcccctg
      841 ggatccccgg aattcccggg tcgactcatg aaggcaggca tcgatgaggc tggaaagggc
      901 tgcgtcatcg gcccactggt tgttgcagga gtggcttgca gcgatgagga taggctgaga
      961 aagcttggtg tgaaagactc caaaaagcta agtcagggga ggagagagga actagccgag
     1021 gaaataagga aaatctgcag aacggaggtt ttgaaagttt ctcccgaaaa tctcgacgaa
     1081 aggatggctg ctaaaaccat aaacgagatt ttgaaggagt gctacgctga aataattctc
     1141 aggctgaagc cggaaattgc ttatgttaac agtcctgatg tgattcccga gagactttcg
     1201 agggagcttg aggagattac ggggttgaga gttgtggccg agcacaaggc ggacgagaag
     1261 tatcccctgg tagctgcggc ttcaatcatc gcaaaggtgg aaagggagcg ggagattgag
     1321 aggctgaaag aaaaattcgg ggatttcggc agcggctatg cgagcgatcc gaggacaaga
     1381 gaagtgctga aggagtggat agcttcaggc agaattccga gctgcgtgag aatgcgctgg
     1441 aagacggtgt caaatctgag gcagaagacg cttgacgatt tctaagagct cgcggccgca
     1501 tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     1561 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     1621 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     1681 tagcgatagc ggagtgtata attcttgaag acgaaagggc ctcgtgatac gcctattttt
     1741 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     1801 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1861 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1921 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1981 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2041 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2101 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     2161 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2221 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2281 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2341 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2401 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2461 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2521 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2581 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2641 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2701 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2761 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2821 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2881 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2941 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3001 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3061 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3121 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3181 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3241 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3301 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3361 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3421 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3481 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3541 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3601 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3661 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3721 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataaatt ccgacaccat
     3781 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     3841 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     3901 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     3961 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     4021 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     4081 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     4141 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     4201 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     4261 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     4321 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     4381 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     4441 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     4501 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     4561 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     4621 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     4681 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     4741 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     4801 gctgttgccc gtctcactgg tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc
     4861 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     4921 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccccagg
     4981 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     5041 acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt
     5101 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     5161 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     5221 aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg
     5281 gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt
     5341 tacgatgcgc ccatctacac caacgtgacc tatcccatta cggtcaatcc gccgtttgtt
     5401 cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta
     5461 caggaaggcc agacgcgaat tatttttgat ggcgttggaa ttagcttatc gactgcacgg
     5521 tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg
     5581 taaatcactg cataattcgt gtcgct
//
