LOCUS       sequence-172517-        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express E20A 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-E20A-PvuRts1I
FEATURES             Location/Qualifiers
     source          1..6558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1700..1891
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1885..1907)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1993..2132
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2048..2067
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2147..2164)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2318..2906)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2398..2417)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3077..3868,3869..3937))
                     /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     3700..3719
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3938..4042)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4110..4128
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(4159..4164,4165..4181,4182..4187)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(4362..4409)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4405..4423
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5367..5384)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5397..5419
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5434..5458
                     /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(5458..5477)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5459..5477)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5561..5580)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5786..5863
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5864..6558,1..388)
                     /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(5883..5902)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
       61 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
      121 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
      181 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
      241 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
      301 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
      361 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
      421 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
      481 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
      541 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
      601 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
      661 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
      721 tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct
      781 tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg caggccatgc
      841 tgtccaggca ggtagatgac gaccatcagg gacagcttca aggatcgctc gcggctctta
      901 ccagcctaac ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc gcctcggcga
      961 gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc tgcctccccg
     1021 cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct
     1081 cgctaacgga ttcaccactc caagaattgg agccaatcaa ttcttgcgga gaactgtgaa
     1141 tgcgcaaacc aacccttggc agaacatatc catcgcgtcc gccatctcca gcagccgcac
     1201 gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc
     1261 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     1321 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     1381 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     1441 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     1501 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     1561 taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac
     1621 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     1681 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     1741 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     1801 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     1861 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     1921 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     1981 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     2041 aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc
     2101 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     2161 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2221 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2281 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2341 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2401 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2461 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2521 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2581 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2641 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2701 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     2761 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2821 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2881 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2941 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3001 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3061 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3121 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3181 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3241 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3301 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3361 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     3421 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3481 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3541 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3601 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3661 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     3721 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3781 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3841 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3901 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3961 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4021 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4081 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     4141 aagaattaat tctcatgttt gacagcttat catcgataag ctttaatgcg gtagtttatc
     4201 acagttaaat tgctaacgca gtcaggcacc gtgtatgaaa tctaacaatg cgctcatcgt
     4261 catcctcggc accgtcaccc tggatgctgt aggcataggc ttggttatgc cggtactgcc
     4321 gggcctcttg cgggatatcc ggatatagtt cctcctttca gcaaaaaacc cctcaagacc
     4381 cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc agccaactca
     4441 gcttcctttc gggctttgtt agcagccgga tcctcgagtc agttctggct acgatacacg
     4501 ttcacacggg aatacacacg gctaaacact ttcacttcgt cgctgctttt atccacatcg
     4561 atggcaaaca cgcccagaaa gcgatacagg gtacgattca gctcgtcacg agaatgggcc
     4621 atgacaatac gataatccca cgtttcttga tagttacggg tagcattcag actctgttcc
     4681 gtgattttgt taccgtcggc actcagggca tttttccact ctccagcttc atacaggcga
     4741 ggaaaccaga ccataacacg acccgtcaga ccgattgact gagcaacttc agccggcaga
     4801 gcccaaccac cactttgatg atggccttca cggcgataac caaagcattg cagcgcgtca
     4861 cggtgataac ggaatacgct attcggccca actgccatat aaccggtgtt gatgtgtttt
     4921 ttagcgctat aacgctcgtc atagtcccaa cgaaaaaaca gtccctggtt ctccagctcc
     4981 tcttttttgt ctttcaccag gcggacaaac tcatccacca gtttattcac ctcggacagg
     5041 gtaatgttag acgccggaat acgctcttcc tgaaacccgg tggcttcaac aatatccaga
     5101 cggcgaacag catcggcttt acgagcatcg ttagaatcat ggtgtgcctc gtcgatttcc
     5161 agatacaggt tcagctgagg aaacagcaga tccgccagat agattttgcc cagatggcct
     5221 tctttacgga tacactgctg acagacgaac tcgatatcag ggtcatccag tgtatggacg
     5281 acacggttga taatatagtg tgcccagcgt ttgtgggaga ttttactcag agcgcgcagg
     5341 atatagtcgg ttttggacat gaattcgtga tgatgatgat gatgccccat ggtatatctc
     5401 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
     5461 tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc
     5521 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     5581 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     5641 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     5701 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     5761 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
     5821 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     5881 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     5941 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     6001 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     6061 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     6121 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     6181 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     6241 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     6301 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     6361 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     6421 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     6481 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     6541 tcaacaaacc atgcaaat
//
