LOCUS       sequence-173051-        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express W215A 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-W215A-PvuRts1I
FEATURES             Location/Qualifiers
     source          1..6558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1390..1409
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1390..1408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1409..1433
                     /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             1448..1470
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1483..1500
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2444..2462)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2458..2505
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2680..2685,2686..2702,2703..2708))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2739..2757)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2825..2929
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2930..2998,2999..3790)
                     /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(3148..3167)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3961..4549
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4450..4469
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4703..4720
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4735..4874
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4800..4819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4960..4982
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4976..5167)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6479..6558,1..1003))
                     /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 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1381 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1441 aaataatttt gtttaacttt aagaaggaga tataccatgg ggcatcatca tcatcatcac
     1501 gaattcatgt ccaaaaccga ctatatcctg cgcgctctga gtaaaatctc ccacaaacgc
     1561 tgggaacact atattatcaa ccgtgtcgtc catacactgg atgaccctga tatcgagttc
     1621 gtctgtcagc agtgtatccg taaagaaggc catctgggca aaatctatct ggcggatctg
     1681 ctgtttcctc agctgaacct gtatctggaa atcgacgagg cacaccatga ttctaacgat
     1741 gctcgtaaag ccgatgctgt tcgccgtctg gatattgttg aagccaccgg gtttcaggaa
     1801 gagcgtattc cggcgtctaa cattaccctg tccgaggtga ataaactggt ggatgagttt
     1861 gtccgcctgg tgaaagacaa aaaagaggag ctggagaacc agggactgtt ttttcgttgg
     1921 gactatgacg agcgttatag cgctaaaaaa cacatcaaca ccggttatat ggcagttggg
     1981 ccgaatagcg tattccgtta tcaccgtgac gcgctgcaat gctttggtta tcgccgtgaa
     2041 ggccatcatc aaagtggtgg ttgggctctg ccggctgaag ttgctcagtc aatcggtctg
     2101 acgggtcgtg ttatggtctg gtttcctcgc ctgtatgaag ctggagaggc gaaaaatgcc
     2161 ctgagtgccg acggtaacaa aatcacggaa cagagtctga atgctacccg taactatcaa
     2221 gaaacgtggg attatcgtat tgtcatggcc cattctcgtg acgagctgaa tcgtaccctg
     2281 tatcgctttc tgggcgtgtt tgccatcgat gtggataaaa gcagcgacga agtgaaagtg
     2341 tttagccgtg tgtattcccg tgtgaacgtg tatcgtagcc agaactgact cgaggatccg
     2401 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     2461 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2521 atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag
     2581 catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg
     2641 cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa
     2701 gctgtcaaac atgagaatta attcttgaag acgaaagggc ctcgtgatac gcctattttt
     2761 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     2821 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2881 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2941 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3001 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3061 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3121 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     3181 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3241 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3301 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3361 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3421 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     3481 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3541 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3601 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     3661 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3721 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3781 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3841 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3901 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3961 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4021 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4081 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4141 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4201 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4261 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4321 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4381 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4441 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4501 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4561 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4621 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4681 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4741 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     4801 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4861 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4921 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4981 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     5041 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     5101 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     5161 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     5221 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5281 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     5341 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     5401 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     5461 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     5521 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     5581 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     5641 acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
     5701 tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa
     5761 ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc
     5821 ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac
     5881 aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag
     5941 cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
     6001 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc
     6061 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa
     6121 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     6181 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     6241 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     6301 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     6361 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     6421 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     6481 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6541 cgcggggaga ggcggttt
//
