LOCUS       sequence-173052-        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express H74A variant 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-H74A-PvuRts1I
FEATURES             Location/Qualifiers
     source          1..6558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(172..1254)
                     /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     1216..1235
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1255..1332)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1538..1557
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1641..1660
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1641..1659
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1660..1684
                     /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             1699..1721
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1734..1751
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2695..2713)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2709..2756
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2931..2936,2937..2953,2954..2959))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2990..3008)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3076..3180
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3181..3249,3250..4041)
                     /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(3399..3418)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4212..4800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4701..4720
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4954..4971
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4986..5125
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5051..5070)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5211..5233
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5227..5418)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
       61 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
      121 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
      181 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
      241 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
      301 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
      361 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
      421 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
      481 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
      541 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
      601 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
      661 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
      721 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
      781 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
      841 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
      901 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
      961 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     1021 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     1081 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     1141 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     1201 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     1261 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     1321 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     1381 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     1441 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     1501 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     1561 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     1621 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     1681 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg gggcatcatc
     1741 atcatcatca cgaattcatg tccaaaaccg actatatcct gcgcgctctg agtaaaatct
     1801 cccacaaacg ctgggaacac tatattatca accgtgtcgt ccatacactg gatgaccctg
     1861 atatcgagtt cgtctgtcag cagtgtatcc gtaaagaagg ccatctgggc aaaatctatc
     1921 tggcggatct gctgtttcct cagctgaacc tgtatctgga aatcgacgag gcacacgctg
     1981 attctaacga tgctcgtaaa gccgatgctg ttcgccgtct ggatattgtt gaagccaccg
     2041 ggtttcagga agagcgtatt ccggcgtcta acattaccct gtccgaggtg aataaactgg
     2101 tggatgagtt tgtccgcctg gtgaaagaca aaaaagagga gctggagaac cagggactgt
     2161 tttttcgttg ggactatgac gagcgttata gcgctaaaaa acacatcaac accggttata
     2221 tggcagttgg gccgaatagc gtattccgtt atcaccgtga cgcgctgcaa tgctttggtt
     2281 atcgccgtga aggccatcat caaagtggtg gttgggctct gccggctgaa gttgctcagt
     2341 caatcggtct gacgggtcgt gttatggtct ggtttcctcg cctgtatgaa gctggagagt
     2401 ggaaaaatgc cctgagtgcc gacggtaaca aaatcacgga acagagtctg aatgctaccc
     2461 gtaactatca agaaacgtgg gattatcgta ttgtcatggc ccattctcgt gacgagctga
     2521 atcgtaccct gtatcgcttt ctgggcgtgt ttgccatcga tgtggataaa agcagcgacg
     2581 aagtgaaagt gtttagccgt gtgtattccc gtgtgaacgt gtatcgtagc cagaactgac
     2641 tcgaggatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     2701 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2761 aaggaggaac tatatccgga tatcccgcaa gaggcccggc agtaccggca taaccaagcc
     2821 tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt
     2881 catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt
     2941 atcgatgata agctgtcaaa catgagaatt aattcttgaa gacgaaaggg cctcgtgata
     3001 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     3061 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3121 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3181 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3241 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3301 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3361 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3421 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3481 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3541 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3601 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3661 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3721 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3781 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3841 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3901 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3961 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4021 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4081 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4141 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4201 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4261 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4321 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4381 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4441 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4501 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4561 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4621 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4681 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4741 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4801 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4861 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4921 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4981 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     5041 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     5101 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     5161 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5221 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     5281 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     5341 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5401 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     5461 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5521 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5581 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     5641 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     5701 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     5761 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     5821 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     5881 ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg agatggcgga
     5941 cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg
     6001 attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag
     6061 gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg
     6121 gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc
     6181 gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag cgatccttga
     6241 agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc
     6301 ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgcg
     6361 aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc
     6421 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     6481 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     6541 aaaatgaccc agagcgct
//
