LOCUS       sequence-172516-        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express D70A 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-D70A-PvuRts1I
FEATURES             Location/Qualifiers
     source          1..6558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     917..936
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(956..1033)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1239..1258
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1342..1361
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1342..1360
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1361..1385
                     /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             1400..1422
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1435..1452
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2396..2414)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2410..2457
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2632..2637,2638..2654,2655..2660))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2691..2709)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2777..2881
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2882..2950,2951..3742)
                     /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(3100..3119)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3913..4501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4402..4421
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4655..4672
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4687..4826
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4752..4771)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4912..4934
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4928..5119)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6431..6558,1..955))
                     /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"
ORIGIN
        1 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
       61 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
      121 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
      181 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
      241 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
      301 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
      361 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
      421 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
      481 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
      541 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
      601 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
      661 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
      721 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
      781 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
      841 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
      901 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
      961 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     1021 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     1081 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     1141 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     1201 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     1261 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     1321 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     1381 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat ggggcatcat
     1441 catcatcatc acgaattcat gtccaaaacc gactatatcc tgcgcgctct gagtaaaatc
     1501 tcccacaaac gctgggaaca ctatattatc aaccgtgtcg tccatacact ggatgaccct
     1561 gatatcgagt tcgtctgtca gcagtgtatc cgtaaagaag gccatctggg caaaatctat
     1621 ctggcggatc tgctgtttcc tcagctgaac ctgtatctgg aaatcgccga ggcacaccat
     1681 gattctaacg atgctcgtaa agccgatgct gttcgccgtc tggatattgt tgaagccacc
     1741 gggtttcagg aagagcgtat tccggcgtct aacattaccc tgtccgaggt gaataaactg
     1801 gtggatgagt ttgtccgcct ggtgaaagac aaaaaagagg agctggagaa ccagggactg
     1861 ttttttcgtt gggactatga cgagcgttat agcgctaaaa aacacatcaa caccggttat
     1921 atggcagttg ggccgaatag cgtattccgt tatcaccgtg acgcgctgca atgctttggt
     1981 tatcgccgtg aaggccatca tcaaagtggt ggttgggctc tgccggctga agttgctcag
     2041 tcaatcggtc tgacgggtcg tgttatggtc tggtttcctc gcctgtatga agctggagag
     2101 tggaaaaatg ccctgagtgc cgacggtaac aaaatcacgg aacagagtct gaatgctacc
     2161 cgtaactatc aagaaacgtg ggattatcgt attgtcatgg cccattctcg tgacgagctg
     2221 aatcgtaccc tgtatcgctt tctgggcgtg tttgccatcg atgtggataa aagcagcgac
     2281 gaagtgaaag tgtttagccg tgtgtattcc cgtgtgaacg tgtatcgtag ccagaactga
     2341 ctcgaggatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     2401 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2461 aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc
     2521 ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt
     2581 tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct
     2641 tatcgatgat aagctgtcaa acatgagaat taattcttga agacgaaagg gcctcgtgat
     2701 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     2761 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2821 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2881 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2941 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3001 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3061 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3121 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3181 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3241 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3301 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3361 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3421 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3481 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3541 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3601 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3661 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3721 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3781 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3841 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3901 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3961 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4021 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     4081 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4141 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4201 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4261 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4321 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4381 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4441 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4501 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4561 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4621 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4681 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     4741 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4801 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4861 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4921 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     4981 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     5041 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     5101 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     5161 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     5221 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     5281 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     5341 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     5401 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5461 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5521 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5581 cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg
     5641 acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc cgcaagaatt
     5701 gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg cttccattca
     5761 ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag
     5821 ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc
     5881 cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg
     5941 aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat
     6001 cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc
     6061 gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt
     6121 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     6181 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     6241 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     6301 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     6361 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     6421 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6481 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     6541 tcaccagtga gacgggca
//
