LOCUS       sequence-173016-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli Mutl(R95F) expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7130)
  AUTHORS   Liu J, Hanne J, Britton BM, Bennett J, Kim D, Lee JB, Fishel R
  TITLE     Cascading MutS and MutL sliding clamps control DNA diffusion to 
            activate mismatch repair.
  JOURNAL   Nature. 2016 Nov 16;539(7630):583-587. doi: 10.1038/nature20562.
  PUBMED    27851738
REFERENCE   2  (bases 1 to 7130)
  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"; journalName: 
            "Nature"; date: "2016-11-16- 16"; volume: "539"; issue: "7630"; 
            pages: "583-587"
COMMENT     Sequence Label: pRAF-MutL(R95F)
FEATURES             Location/Qualifiers
     source          1..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1852..1869
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1903..1920
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1973..1991)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1987..2034
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2071..2526
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2158..2177)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2368..2389
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2618..3433)
                     /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             complement(2618..3433)
                     /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     3341..3360
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3425..3444
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3555..4143
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4044..4063
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4297..4314
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4329..4468
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4394..4413)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4554..4576
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4570..4761)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5570..6652)
                     /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
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKFRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6614..6633
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6653..6730)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6936..6955
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7043..7062
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7043..7061
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7062..7086
                     /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             7101..7123
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgccaattc aggtcttacc gccacaactg gcgaaccaga ttgccgcagg tgaggtggtc
       61 gagcgacctg cgtcggtagt caaagaacta gtggaaaaca gcctcgatgc aggtgcgacg
      121 cgtatcgata ttgatatcga acgcggtggg gcgaaactta tccgcattcg tgataacggc
      181 tgcggtatca aaaaagatga gctggcgctg gcgctggctc gtcatgccac cagtaaaatc
      241 gcctctctgg acgatctcga agccattatc agcctgggct ttttcggtga ggcgctggcg
      301 agtatcagtt cggtttcccg cctgacgctc acttcacgca ccgcagaaca gcaggaagcc
      361 tggcaggcct atgccgaagg gcgcgatatg aacgtgacgg taaaaccggc ggcgcatcct
      421 gtggggacga cgctggaggt gctggatctg ttctacaaca ccccggcgcg gcgcaaattc
      481 ctgcgcaccg agaaaaccga atttaaccac attgatgaga tcatccgccg cattgcgctg
      541 gcgcgtttcg acgtcacgat caacctgtcg cataacggta aaattgtgcg tcagtaccgc
      601 gcagtgccgg aaggcgggca aaaagaacgg cgcttaggcg cgatttgcgg caccgctttt
      661 cttgaacaag cgctggcgat tgaatggcaa cacggcgatc tcacgctacg cggctgggtg
      721 gccgatccaa atcacaccac gcccgcactg gcagaaattc agtattgcta cgtgaacggt
      781 cgcatgatgc gcgatcgcct gatcaatcac gcgatccgcc aggcctgcga agacaaactg
      841 ggggccgatc agcaaccggc atttgtgttg tatctggaga tcgacccaca tcaggtggac
      901 gtcaacgtgc accccgccaa acacgaagtg cgtttccatc agtcgcgtct ggtgcatgat
      961 tttatctatc agggcgtgct gagcgtgcta caacagcaac tggaaacgcc gctaccgctg
     1021 gacgatgaac cccaacctgc accgcgttcc attccggaaa accgcgtggc ggcggggcgc
     1081 aatcactttg cagaaccggc agctcgtgag ccggtagctc cgcgctacac tcctgcgcca
     1141 gcatcaggca gtcgtccggc tgccccctgg ccgaatgcgc agccaggcta ccagaaacag
     1201 caaggtgaag tgtatcgcca gcttttgcaa acgcccgcgc cgatgcaaaa attaaaagcg
     1261 ccggaaccgc aggaacctgc acttgcggcg aacagtcaga gttttggtcg ggtactgact
     1321 atcgtccatt ccgactgtgc gttgctggag cgcgacggca acatttcact tttatccttg
     1381 ccagtggcag aacgttggct gcgtcaggca caattgacgc cgggtgaagc gcccgtttgc
     1441 gcccagccgc tgctgattcc gttgcggcta aaagtttctg ccgaagaaaa atcggcatta
     1501 gaaaaagcgc agtctgccct ggcggaattg ggtattgatt tccagtcaga tgcacagcat
     1561 gtgaccatca gggcagtgcc tttaccctta cgccaacaaa atttacaaat cttgattcct
     1621 gaactgatag gctacctggc gaagcagtcc gtattcgaac ctggcaatat tgcgcagtgg
     1681 attgcacgaa atctgatgag cgaacatgcg cagtggtcaa tggcacaggc cataaccctg
     1741 ctggcggacg tggaacggtt atgtccgcaa cttgtgaaaa cgccgccggg tggtctgtta
     1801 caatctgttg atttacatcc ggcgataaaa gccctgaaag atgagtcatc acatcaccat
     1861 caccatcatg gagggctctg cacaccgtcg cgttaactcg agcaccacca ccaccaccac
     1921 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1981 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     2041 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     2101 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2161 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2221 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2281 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2341 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2401 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2461 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     2521 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2581 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     2641 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     2701 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     2761 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     2821 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     2881 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     2941 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     3001 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     3061 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     3121 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     3181 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     3241 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     3301 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     3361 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     3421 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     3481 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3541 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3601 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3661 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3721 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3781 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3841 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3901 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3961 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4021 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4081 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4141 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4201 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4261 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4321 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     4381 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4441 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4501 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4561 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4621 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4681 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4741 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4801 aatgataccg atgaaacgag agaggatgct cacgacacgg gttactgatg atgaacatgc
     4861 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4921 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4981 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     5041 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     5101 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     5161 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     5221 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     5281 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     5341 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     5401 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     5461 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     5521 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     5581 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5641 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     5701 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5761 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5821 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     5881 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5941 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     6001 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     6061 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     6121 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     6181 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     6241 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     6301 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     6361 tcgacaccac cacgctggca cccagttgat cggcgcgaaa tttaatcgcc gcgacaattt
     6421 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     6481 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     6541 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     6601 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     6661 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     6721 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     6781 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     6841 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     6901 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6961 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     7021 atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     7081 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
//
