LOCUS       sequence-254924-        6361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Escherichia coli RssB E134G:E135S:E136G:E137S variant as 
            an N-terminal 10His fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6361)
  AUTHORS   Dorich V, Brugger C, Tripathi A, Hoskins JR, Tong S, Suhanovsky MM, 
            Sastry A, Wickner S, Gottesman S, Deaconescu AM
  TITLE     Structural basis for inhibition of a response regulator of sigma(S) 
            stability by a ClpXP antiadaptor.
  JOURNAL   Genes Dev. 2019 Jun 1;33(11-12):718-732. doi: 
            10.1101/gad.320168.118. Epub 2019 Apr 11.
  PUBMED    30975721
REFERENCE   2  (bases 1 to 6361)
  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"; journalName: "Genes 
            Dev."; date: "2019-06-1"; volume: "33(11-12):718-732. doi"; pages: "
            10.1101/gad.320168.118. Epub 2019 Apr 11"
COMMENT     Sequence Label: pMS22
FEATURES             Location/Qualifiers
     source          1..6361
                     /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             100..129
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             139..162
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1226..1243
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1296..1314)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1310..1357
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1394..1849
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1691..1712
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1941..2756)
                     /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(1941..2756)
                     /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     2664..2683
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2748..2767
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2878..3466
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3367..3386
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3620..3637
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3652..3791
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3717..3736)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3877..3899
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3893..4084)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4893..5975)
                     /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     5937..5956
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5976..6053)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6259..6278
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcaccat
      121 caccatcaca gcagcggcct ggaagttctg ttccaggggc cccatatgac gcagccattg
      181 gtcggaaaac agattctcat tgttgaagat gagcaggtat ttcgctcgct tctggattca
      241 tggttttcct cattgggagc gacaacggta ctggcggctg atggggtgga tgcccttgag
      301 ttgctgggag gtttcactcc agacctgatg atatgtgata tcgcgatgcc acgaatgaac
      361 gggcttaaac tgctggagca tatacgtaac agaggcgacc agaccccagt tctggtgata
      421 tctgccactg aaaatatggc agatattgcc aaagcgttac gtctgggcgt tgaagatgtt
      481 ttgctgaaac cagttaaaga tctgaatcgc ttgcgcgaga tggtttttgc ctgtctctat
      541 cccagcatgt ttaattcgcg cgttgggtca gggtccaggc tttttcgcga ctgggatgca
      601 atggttgata accctgccgc agcggcgaaa ttattacagg aactacaacc gccggttcag
      661 caggtgattt cccattgccg ggttaattat cgtcaattgg ttgccgcgga caaacccggc
      721 ctggtgcttg atattgccgc actttcggaa aacgatctgg cattttattg ccttgatgtc
      781 acccgagctg gacataatgg cgtacttgct gccttgttat tacgcgcatt gtttaacgga
      841 ttattacagg aacagcttgc acaccaaaat caacggttgc cagagttggg cgcgttattg
      901 aagcaggtaa accatttact tcgtcaggcc aatctgccgg ggcagtttcc gctattagtt
      961 ggctattatc atcgcgaact gaaaaatctc attctggttt ctgcgggtct gaatgcgacg
     1021 ttaaataccg gcgaacacca ggtgcaaatc agtaatggtg ttccgttagg cactttaggt
     1081 aacgcttatt tgaatcaatt gagccagcga tgcgatgcct ggcaatgcca aatatgggga
     1141 accggtggtc gactgcgctt gatgttgtct gcagaatgag gatccgaatt cgagctccgt
     1201 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     1261 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     1321 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1381 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1441 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1501 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1561 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1621 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1681 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1741 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1801 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     1861 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1921 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     1981 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     2041 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     2101 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     2161 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     2221 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     2281 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     2341 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     2401 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     2461 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     2521 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     2581 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     2641 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     2701 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     2761 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     2821 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2881 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2941 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3001 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3061 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3121 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3181 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3241 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3301 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3361 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3421 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3481 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3541 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3601 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3661 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     3721 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3781 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3841 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3901 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     3961 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4021 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     4081 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     4141 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     4201 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4261 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4321 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4381 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4441 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4501 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     4561 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4621 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4681 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4741 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4801 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4861 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4921 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4981 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     5041 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     5101 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     5161 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5221 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5281 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5341 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5401 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5461 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5521 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5581 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5641 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5701 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5761 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5821 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     5881 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     5941 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     6001 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     6061 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     6121 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     6181 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     6241 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     6301 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     6361 t
//
