LOCUS       sequence-172522-        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express E71A 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-E71A-PvuRts1I
FEATURES             Location/Qualifiers
     source          1..6558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     978..997
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1017..1094)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1300..1319
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1403..1422
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1403..1421
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1422..1446
                     /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             1461..1483
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1496..1513
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2457..2475)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2471..2518
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2693..2698,2699..2715,2716..2721))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2752..2770)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2838..2942
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2943..3011,3012..3803)
                     /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(3161..3180)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3974..4562
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4463..4482
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4716..4733
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4748..4887
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4813..4832)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4973..4995
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4989..5180)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6492..6558,1..1016))
                     /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 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
       61 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
      121 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
      181 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
      241 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
      301 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
      361 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
      421 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
      481 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
      541 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
      601 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
      661 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
      721 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
      781 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
      841 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
      901 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
      961 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     1021 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     1081 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     1141 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     1201 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     1261 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     1321 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     1381 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     1441 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tggggcatca
     1501 tcatcatcat cacgaattca tgtccaaaac cgactatatc ctgcgcgctc tgagtaaaat
     1561 ctcccacaaa cgctgggaac actatattat caaccgtgtc gtccatacac tggatgaccc
     1621 tgatatcgag ttcgtctgtc agcagtgtat ccgtaaagaa ggccatctgg gcaaaatcta
     1681 tctggcggat ctgctgtttc ctcagctgaa cctgtatctg gaaatcgacg cggcacacca
     1741 tgattctaac gatgctcgta aagccgatgc tgttcgccgt ctggatattg ttgaagccac
     1801 cgggtttcag gaagagcgta ttccggcgtc taacattacc ctgtccgagg tgaataaact
     1861 ggtggatgag tttgtccgcc tggtgaaaga caaaaaagag gagctggaga accagggact
     1921 gttttttcgt tgggactatg acgagcgtta tagcgctaaa aaacacatca acaccggtta
     1981 tatggcagtt gggccgaata gcgtattccg ttatcaccgt gacgcgctgc aatgctttgg
     2041 ttatcgccgt gaaggccatc atcaaagtgg tggttgggct ctgccggctg aagttgctca
     2101 gtcaatcggt ctgacgggtc gtgttatggt ctggtttcct cgcctgtatg aagctggaga
     2161 gtggaaaaat gccctgagtg ccgacggtaa caaaatcacg gaacagagtc tgaatgctac
     2221 ccgtaactat caagaaacgt gggattatcg tattgtcatg gcccattctc gtgacgagct
     2281 gaatcgtacc ctgtatcgct ttctgggcgt gtttgccatc gatgtggata aaagcagcga
     2341 cgaagtgaaa gtgtttagcc gtgtgtattc ccgtgtgaac gtgtatcgta gccagaactg
     2401 actcgaggat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     2461 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2521 gaaaggagga actatatccg gatatcccgc aagaggcccg gcagtaccgg cataaccaag
     2581 cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc attgttagat
     2641 ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc gcattaaagc
     2701 ttatcgatga taagctgtca aacatgagaa ttaattcttg aagacgaaag ggcctcgtga
     2761 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     2821 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2881 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2941 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3001 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3061 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3121 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3181 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3241 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3301 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3361 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3421 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3481 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3541 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3601 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3661 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3721 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3781 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3841 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3901 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3961 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4021 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4081 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4141 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4201 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4261 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4321 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4381 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4441 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4501 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4561 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4621 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4681 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4741 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     4801 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     4861 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4921 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4981 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     5041 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     5101 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     5161 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     5221 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     5281 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     5341 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     5401 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     5461 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     5521 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     5581 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     5641 acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct ggagatggcg
     5701 gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat
     5761 tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg gcttccattc
     5821 aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata
     5881 gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg
     5941 ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt
     6001 gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca
     6061 tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg
     6121 cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct
     6181 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     6241 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     6301 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     6361 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     6421 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     6481 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     6541 gaatcggcca acgcgcgg
//
