LOCUS       sequence-190779-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli MutL 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
FEATURES             Location/Qualifiers
     source          1..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1940..1957
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1991..2008
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2061..2079)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2075..2122
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2159..2614
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2246..2265)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2456..2477
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2706..3521)
                     /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(2706..3521)
                     /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     3429..3448
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3513..3532
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3643..4231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4132..4151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4385..4402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4417..4556
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4482..4501)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4642..4664
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4658..4849)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5658..6740)
                     /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     6702..6721
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6741..6818)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7024..7043
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gccaattcag gtcttaccgc cacaactggc
      121 gaaccagatt gccgcaggtg aggtggtcga gcgacctgcg tcggtagtca aagaactagt
      181 ggaaaacagc ctcgatgcag gtgcgacgcg tatcgatatt gatatcgaac gcggtggggc
      241 gaaacttatc cgcattcgtg ataacggctg cggtatcaaa aaagatgagc tggcgctggc
      301 gctggctcgt catgccacca gtaaaatcgc ctctctggac gatctcgaag ccattatcag
      361 cctgggcttt cgcggtgagg cgctggcgag tatcagttcg gtttcccgcc tgacgctcac
      421 ttcacgcacc gcagaacagc aggaagcctg gcaggcctat gccgaagggc gcgatatgaa
      481 cgtgacggta aaaccggcgg cgcatcctgt ggggacgacg ctggaggtgc tggatctgtt
      541 ctacaacacc ccggcgcggc gcaaattcct gcgcaccgag aaaaccgaat ttaaccacat
      601 tgatgagatc atccgccgca ttgcgctggc gcgtttcgac gtcacgatca acctgtcgca
      661 taacggtaaa attgtgcgtc agtaccgcgc agtgccggaa ggcgggcaaa aagaacggcg
      721 cttaggcgcg atttgcggca ccgcttttct tgaacaagcg ctggcgattg aatggcaaca
      781 cggcgatctc acgctacgcg gctgggtggc cgatccaaat cacaccacgc ccgcactggc
      841 agaaattcag tattgctacg tgaacggtcg catgatgcgc gatcgcctga tcaatcacgc
      901 gatccgccag gcctgcgaag acaaactggg ggccgatcag caaccggcat ttgtgttgta
      961 tctggagatc gacccacatc aggtggacgt caacgtgcac cccgccaaac acgaagtgcg
     1021 tttccatcag tcgcgtctgg tgcatgattt tatctatcag ggcgtgctga gcgtgctaca
     1081 acagcaactg gaaacgccgc taccgctgga cgatgaaccc caacctgcac cgcgttccat
     1141 tccggaaaac cgcgtggcgg cggggcgcaa tcactttgca gaaccggcag ctcgtgagcc
     1201 ggtagctccg cgctacactc ctgcgccagc atcaggcagt cgtccggctg ccccctggcc
     1261 gaatgcgcag ccaggctacc agaaacagca aggtgaagtg tatcgccagc ttttgcaaac
     1321 gcccgcgccg atgcaaaaat taaaagcgcc ggaaccgcag gaacctgcac ttgcggcgaa
     1381 cagtcagagt tttggtcggg tactgactat cgtccattcc gactgtgcgt tgctggagcg
     1441 cgacggcaac atttcacttt tatccttgcc agtggcagaa cgttggctgc gtcaggcaca
     1501 attgacgccg ggtgaagcgc ccgtttgcgc ccagccgctg ctgattccgt tgcggctaaa
     1561 agtttctgcc gaagaaaaat cggcattaga aaaagcgcag tctgccctgg cggaattggg
     1621 tattgatttc cagtcagatg cacagcatgt gaccatcagg gcagtgcctt tacccttacg
     1681 ccaacaaaat ttacaaatct tgattcctga actgataggc tacctggcga agcagtccgt
     1741 attcgaacct ggcaatattg cgcagtggat tgcacgaaat ctgatgagcg aacatgcgca
     1801 gtggtcaatg gcacaggcca taaccctgct ggcggacgtg gaacggttat gtccgcaact
     1861 tgtgaaaacg ccgccgggtg gtctgttaca atctgttgat ttacatccgg cgataaaagc
     1921 cctgaaagat gagtcatcac atcaccatca ccatcatgga gggctctgca caccgtcgcg
     1981 ttaactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     2041 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     2101 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     2161 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2221 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2281 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2341 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2401 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2461 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2521 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2581 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     2641 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     2701 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2761 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2821 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2881 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2941 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     3001 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     3061 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     3121 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     3181 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     3241 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     3301 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     3361 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     3421 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3481 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     3541 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     3601 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3661 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3721 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3781 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3841 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3901 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3961 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4021 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4081 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4141 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4201 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4261 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4321 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4381 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     4441 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     4501 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     4561 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4621 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4681 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     4741 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4801 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4861 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4921 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4981 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     5041 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     5101 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     5161 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     5221 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     5281 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     5341 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     5401 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     5461 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     5521 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     5581 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     5641 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5701 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5761 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5821 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     5881 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     5941 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     6001 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     6061 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     6121 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     6181 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     6241 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     6301 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     6361 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     6421 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     6481 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     6541 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     6601 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     6661 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6721 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6781 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     6841 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     6901 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     6961 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     7021 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     7081 cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat
//
