LOCUS       sequence-173050-        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express E228A 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-E228A-PvuRts1I
FEATURES             Location/Qualifiers
     source          1..6558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1779..1970
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1964..1986)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2072..2211
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2127..2146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2226..2243)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2397..2985)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2477..2496)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3156..3947,3948..4016))
                     /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     3779..3798
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4017..4121)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4189..4207
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(4238..4243,4244..4260,4261..4266)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(4441..4488)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4484..4502
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5446..5463)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5476..5498
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5513..5537
                     /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(5537..5556)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5538..5556)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5640..5659)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5865..5942
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5943..6558,1..467)
                     /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(5962..5981)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
       61 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
      121 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
      181 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
      241 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
      301 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
      361 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      421 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      481 aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc
      541 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact
      601 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc
      661 gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc
      721 ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag
      781 caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct ggcgttcgcg
      841 acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg catcgggatg
      901 cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg acagcttcaa
      961 ggatcgctcg cggctcttac cagcctaact tcgatcattg gaccgctgat cgtcacggcg
     1021 atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg cgccgcccta
     1081 taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga
     1141 atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat
     1201 tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg
     1261 ccatctccag cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc
     1321 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
     1381 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
     1441 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     1501 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     1561 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     1621 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     1681 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     1741 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     1801 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     1861 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     1921 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     1981 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     2041 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     2101 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     2161 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     2221 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2281 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2341 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2401 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2461 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2521 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2581 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2641 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2701 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2761 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2821 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2881 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2941 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3001 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3061 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3121 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3181 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3241 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3301 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3361 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3421 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     3481 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3541 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3601 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3661 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3721 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat
     3781 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3841 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3901 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3961 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4021 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4081 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4141 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     4201 acgaggccct ttcgtcttca agaattaatt ctcatgtttg acagcttatc atcgataagc
     4261 tttaatgcgg tagtttatca cagttaaatt gctaacgcag tcaggcaccg tgtatgaaat
     4321 ctaacaatgc gctcatcgtc atcctcggca ccgtcaccct ggatgctgta ggcataggct
     4381 tggttatgcc ggtactgccg ggcctcttgc gggatatccg gatatagttc ctcctttcag
     4441 caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc
     4501 ggtggcagca gccaactcag cttcctttcg ggctttgtta gcagccggat cctcgagtca
     4561 gttctggcta cgatacacgt tcacacggga atacacacgg ctaaacactt tcacttcgtc
     4621 gctgctttta tccacatcga tggcaaacac gcccagaaag cgatacaggg tacgattcag
     4681 ctcgtcacga gaatgggcca tgacaatacg ataatcccac gtttcttgat agttacgggt
     4741 agcattcaga ctctgtgccg tgattttgtt accgtcggca ctcagggcat ttttccactc
     4801 tccagcttca tacaggcgag gaaaccagac cataacacga cccgtcagac cgattgactg
     4861 agcaacttca gccggcagag cccaaccacc actttgatga tggccttcac ggcgataacc
     4921 aaagcattgc agcgcgtcac ggtgataacg gaatacgcta ttcggcccaa ctgccatata
     4981 accggtgttg atgtgttttt tagcgctata acgctcgtca tagtcccaac gaaaaaacag
     5041 tccctggttc tccagctcct cttttttgtc tttcaccagg cggacaaact catccaccag
     5101 tttattcacc tcggacaggg taatgttaga cgccggaata cgctcttcct gaaacccggt
     5161 ggcttcaaca atatccagac ggcgaacagc atcggcttta cgagcatcgt tagaatcatg
     5221 gtgtgcctcg tcgatttcca gatacaggtt cagctgagga aacagcagat ccgccagata
     5281 gattttgccc agatggcctt ctttacggat acactgctga cagacgaact cgatatcagg
     5341 gtcatccagt gtatggacga cacggttgat aatatagtgt tcccagcgtt tgtgggagat
     5401 tttactcaga gcgcgcagga tatagtcggt tttggacatg aattcgtgat gatgatgatg
     5461 atgccccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
     5521 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat
     5581 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
     5641 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
     5701 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
     5761 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
     5821 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
     5881 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     5941 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     6001 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     6061 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     6121 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     6181 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     6241 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     6301 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     6361 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     6421 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     6481 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     6541 gctggcataa atatctca
//
