LOCUS       sequence-208999-        5579 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 C-terminal PIP box 
            deletion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5579)
  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 5579)
  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-Delta-PIP
FEATURES             Location/Qualifiers
     source          1..5579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(96..101,102..117,118..124)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    132..148
                     /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             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1542..1564)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1709..1727)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1795..1899
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1900..1968,1969..2760)
                     /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(2118..2137)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2931..3519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3420..3439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3673..3690
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3763..3840
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3841..4923
                     /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(3860..3879)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4972..4977,4978..4995,4996..5002)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5010..5026
                     /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     5034..5050
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5034..5050
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5046..5573
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5066..5083)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5066..5082)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5075..5097)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat
       61 cataacggtt ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat
      121 aatgtgtgga attgtgagcg gataacaatt tcacacagga aacagtattc atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctggga tccccggaat tcccgggtcg actcatgaag gcaggcatcg
      901 atgaggctgg aaagggctgc gtcatcggcc cactggttgt tgcaggagtg gcttgcagcg
      961 atgaggatag gctgagaaag cttggtgtga aagactccaa aaagctaagt caggggagga
     1021 gagaggaact agccgaggaa ataaggaaaa tctgcagaac ggaggttttg aaagtttctc
     1081 ccgaaaatct cgacgaaagg atggctgcta aaaccataaa cgagattttg aaggagtgct
     1141 acgctgaaat aattctcagg ctgaagccgg aaattgctta tgttgacagt cctgatgtga
     1201 ttcccgagag actttcgagg gagcttgagg agattacggg gttgagagtt gtggccgagc
     1261 acaaggcgga cgagaagtat cccctggtag ctgcggcttc aatcatcgca aaggtggaaa
     1321 gggagcggga gattgagagg ctgaaagaaa aattcgggga tttcggcagc ggctatgcga
     1381 gcgatccgag gacaagagaa gtgctgaagg agtggatagc ttcaggcaga attccgagct
     1441 gcgtgagaat gcgctggaag acggtgtcaa atctgaggta agcggccgca tcgtgactga
     1501 ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     1561 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     1621 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     1681 ggagtgtata attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     1741 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1801 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1861 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     1921 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     1981 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2041 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2101 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     2161 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2221 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2281 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2341 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2401 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     2461 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     2521 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2581 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     2641 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2701 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     2761 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     2821 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     2881 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2941 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3001 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3061 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3121 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3181 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3241 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3301 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3361 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3421 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3481 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3541 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3601 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3661 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3721 ctccttacgc atctgtgcgg tatttcacac cgcataaatt ccgacaccat cgaatggcgc
     3781 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     3841 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     3901 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     3961 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     4021 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     4081 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     4141 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     4201 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     4261 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     4321 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     4381 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     4441 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     4501 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     4561 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     4621 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     4681 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     4741 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     4801 gtctcactgg tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc ctctccccgc
     4861 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4921 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccccagg ctttacactt
     4981 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     5041 cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     5101 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     5161 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     5221 ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc
     5281 ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc
     5341 ccatctacac caacgtgacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga
     5401 atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc
     5461 agacgcgaat tatttttgat ggcgttggaa ttagcttatc gactgcacgg tgcaccaatg
     5521 cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcact
//
