LOCUS       sequence-173048-        6111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express C-term His tagged PvuRts1I in ER2566 strain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6111)
  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 6111)
  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: pET28a-PvuRts1I
FEATURES             Location/Qualifiers
     source          1..6111
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..32
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(112..129)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(283..871)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(363..382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(982..1001)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             993..1808
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             993..1808
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1066..1085)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1900..2355)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2037..2058)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2249..2268
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2392..2439)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2435..2453
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2506..2523)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3418..3440
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3455..3479
                     /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     complement(3479..3498)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3480..3498)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3582..3601)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3807..3884
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3885..4967
                     /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(3904..3923)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5776..5967
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5961..5983)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    join(6069..6111,1..97)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat
       61 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
      121 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
      181 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
      241 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
      301 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      361 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      421 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      481 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      541 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      601 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
      661 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
      721 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
      781 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
      841 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
      901 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt
      961 ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt
     1021 gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
     1081 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
     1141 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
     1201 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
     1261 ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat
     1321 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     1381 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
     1441 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     1501 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac
     1561 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     1621 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     1681 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     1741 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     1801 ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     1861 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     1921 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1981 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     2041 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2101 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     2161 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2221 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2281 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2341 cgctacaggg cgcgtcccat tcgccaatcc ggatatagtt cctcctttca gcaaaaaacc
     2401 cctcaagacc cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc
     2461 agccaactca gcttcctttc gggctttgtt agcagccgga tctcagtggt ggtggtggtg
     2521 gtgctcgagg ttctggctac gatacacgtt cacacgggaa tacacacggc taaacacttt
     2581 cacttcgtcg ctgcttttat ccacatcgat ggcaaacacg cccagaaagc gatacagggt
     2641 acgattcagc tcgtcacgag aatgggccat gacaatacga taatcccacg tttcttgata
     2701 gttacgggta gcattcagac tctgttccgt gattttgtta ccgtcggcac tcagggcatt
     2761 tttccactct ccagcttcat acaggcgagg aaaccagacc ataacacgac ccgtcagacc
     2821 gattgactga gcaacttcag ccggcagagc ccaaccacca ctttgatgat ggccttcacg
     2881 gcgataacca aagcattgca gcgcgtcacg gtgataacgg aatacgctat tcggcccaac
     2941 tgccatataa ccggtgttga tgtgtttttt agcgctataa cgctcgtcat agtcccaacg
     3001 aaaaaacagt ccctggttct ccagctcctc ttttttgtct ttcaccaggc ggacaaactc
     3061 atccaccagt ttattcacct cggacagggt aatgttagac gccggaatac gctcttcctg
     3121 aaacccggtg gcttcaacaa tatccagacg gcgaacagca tcggctttac gagcatcgtt
     3181 agaatcatgg tgtgcctcgt cgatttccag atacaggttc agctgaggaa acagcagatc
     3241 cgccagatag attttgccca gatggccttc tttacggata cactgctgac agacgaactc
     3301 gatatcaggg tcatccagtg tatggacgac acggttgata atatagtgtt cccagcgttt
     3361 gtgggagatt ttactcagag cgcgcaggat atagtcggtt ttggagccca tggtatatct
     3421 ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc
     3481 ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat
     3541 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
     3601 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
     3661 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
     3721 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     3781 gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta
     3841 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     3901 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     3961 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4021 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4081 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4141 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     4201 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     4261 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     4321 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     4381 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     4441 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     4501 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     4561 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     4621 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     4681 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     4741 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     4801 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     4861 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     4921 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     4981 aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc
     5041 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact
     5101 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc
     5161 gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc
     5221 ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag
     5281 caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg
     5341 aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc
     5401 gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg
     5461 gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt
     5521 atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta
     5581 ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc
     5641 gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt
     5701 atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct
     5761 tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc
     5821 agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca
     5881 gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt
     5941 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     6001 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     6061 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac t
//
