LOCUS       sequence-172515-        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express N-term His tagged PvuRts1I in ER2566 strain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6558)
  AUTHORS   Kazrani AA, Kowalska M, Czapinska H, Bochtler M
  TITLE     Crystal structure of the 5hmC specific endonuclease PvuRts1I.
  JOURNAL   Nucleic Acids Res. 2014 May;42(9):5929-36. doi: 10.1093/nar/gku186. 
            Epub 2014 Mar 14.
  PUBMED    24634440
REFERENCE   2  (bases 1 to 6558)
  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/gku186"; journalName: "Nucleic Acids Res"; date: 
            "2014-05"; volume: "42"; issue: "9"; pages: "5929-36"
COMMENT     Sequence Label: pET15b-PvuRts1I
FEATURES             Location/Qualifiers
     source          1..6558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      696..1284
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1185..1204
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1438..1455
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1470..1609
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1535..1554)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1695..1717
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1711..1902)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3214..4296)
                     /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     4258..4277
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4297..4374)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4580..4599
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4683..4702
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4683..4701
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4702..4726
                     /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)."
     RBS             4741..4763
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4776..4793
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5737..5755)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5751..5798
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5973..5978,5979..5995,5996..6001))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6032..6050)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6118..6222
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6223..6291,6292..6558,1..525)
                     /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(6441..6460)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
       61 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
      121 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
      181 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
      241 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
      301 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
      361 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
      421 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
      481 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
      541 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
      601 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
      661 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
      721 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
      781 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
      841 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
      901 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
      961 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1021 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1081 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1141 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1201 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     1261 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     1321 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     1381 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     1441 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     1501 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     1561 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     1621 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     1681 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     1741 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     1801 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     1861 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     1921 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     1981 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     2041 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     2101 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     2161 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     2221 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     2281 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     2341 cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg agatgcgccg
     2401 cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt tggtttgcgc
     2461 attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc
     2521 gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac
     2581 gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg
     2641 ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga agttaggctg
     2701 gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac
     2761 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg
     2821 aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc
     2881 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     2941 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     3001 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     3061 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     3121 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3181 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3241 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3301 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3361 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3421 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3481 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3541 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3601 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3661 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     3721 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3781 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     3841 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     3901 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     3961 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4021 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4081 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4141 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4201 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4261 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4321 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     4381 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     4441 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     4501 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     4561 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     4621 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     4681 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     4741 tttgtttaac tttaagaagg agatatacca tggggcatca tcatcatcat cacgaattca
     4801 tgtccaaaac cgactatatc ctgcgcgctc tgagtaaaat ctcccacaaa cgctgggaac
     4861 actatattat caaccgtgtc gtccatacac tggatgaccc tgatatcgag ttcgtctgtc
     4921 agcagtgtat ccgtaaagaa ggccatctgg gcaaaatcta tctggcggat ctgctgtttc
     4981 ctcagctgaa cctgtatctg gaaatcgacg aggcacacca tgattctaac gatgctcgta
     5041 aagccgatgc tgttcgccgt ctggatattg ttgaagccac cgggtttcag gaagagcgta
     5101 ttccggcgtc taacattacc ctgtccgagg tgaataaact ggtggatgag tttgtccgcc
     5161 tggtgaaaga caaaaaagag gagctggaga accagggact gttttttcgt tgggactatg
     5221 acgagcgtta tagcgctaaa aaacacatca acaccggtta tatggcagtt gggccgaata
     5281 gcgtattccg ttatcaccgt gacgcgctgc aatgctttgg ttatcgccgt gaaggccatc
     5341 atcaaagtgg tggttgggct ctgccggctg aagttgctca gtcaatcggt ctgacgggtc
     5401 gtgttatggt ctggtttcct cgcctgtatg aagctggaga gtggaaaaat gccctgagtg
     5461 ccgacggtaa caaaatcacg gaacagagtc tgaatgctac ccgtaactat caagaaacgt
     5521 gggattatcg tattgtcatg gcccattctc gtgacgagct gaatcgtacc ctgtatcgct
     5581 ttctgggcgt gtttgccatc gatgtggata aaagcagcga cgaagtgaaa gtgtttagcc
     5641 gtgtgtattc ccgtgtgaac gtgtatcgta gccagaactg actcgaggat ccggctgcta
     5701 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     5761 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     5821 gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag
     5881 ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact
     5941 gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca
     6001 aacatgagaa ttaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     6061 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     6121 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     6181 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6241 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6301 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6361 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6421 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     6481 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6541 acagaaaagc atcttacg
//
