LOCUS       sequence-212932-        6686 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli ClpX with a C-terminal His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6686)
  AUTHORS   Dotiwala F, Sen Santara S, Binker-Cosen AA, Li B, Chandrasekaran S, 
            Lieberman J
  TITLE     Granzyme B Disrupts Central Metabolism and Protein Synthesis in 
            Bacteria to Promote an Immune Cell Death Program.
  JOURNAL   Cell. 2017 Nov 16;171(5):1125-1137.e11. doi: 
            10.1016/j.cell.2017.10.004. Epub 2017 Oct 26.
  PUBMED    29107333
REFERENCE   2  (bases 1 to 6686)
  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: 
            "10.1016/j.cell.2017.10.004"; journalName: "Cell"; date: 
            "2017-11-16- 16"; volume: "171"; issue: "5"; pages: "1125-1137.e11"
COMMENT     Sequence Label: pET21a - Ec-ClpX
FEATURES             Location/Qualifiers
     source          1..6686
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     101..120
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        101..119
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    120..144
                     /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             159..181
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             189..221
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1518..1535
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1588..1606)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1602..1649
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1686..2141
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1773..1792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1983..2004
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2167..2271
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2272..2340,2341..3132)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2490..2509)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3303..3891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3792..3811
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4045..4062
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4077..4216
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4142..4161)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4302..4324
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4318..4509)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5318..6400)
                     /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     6362..6381
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6401..6478)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6684..6686,1..17)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
       61 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
      121 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
      181 atatacatat ggctagcatg actggtggac agcaaatggg tcgcggatcc atgacagata
      241 aacgcaaaga tggctcaggc aaattgctgt attgctcttt ttgcggcaaa agccagcatg
      301 aagtgcgcaa gctgattgcc ggtccatccg tgtatatctg cgacgaatgt gttgatttat
      361 gtaacgacat cattcgcgaa gagattaaag aagttgcacc gcatcgtgaa cgcagtgcgc
      421 taccgacgcc gcatgaaatt cgcaaccacc tggacgatta cgttatcggc caggaacagg
      481 cgaaaaaagt gctggcggtc gcggtataca accattacaa acgtctgcgc aacggcgata
      541 ccagcaatgg cgtcgagttg ggcaaaagta acattctgct gatcggtccg accggttccg
      601 gtaaaacgct gctggctgaa acgctggcgc gcctgctgga tgttccgttc accatggccg
      661 acgcgactac actgaccgaa gccggttatg tgggtgaaga cgttgaaaac atcattcaga
      721 agctgttgca gaaatgcgac tacgatgtcc agaaagcaca gcgtggtatt gtctacatcg
      781 atgaaatcga caagatttct cgtaagtcag acaacccgtc cattacccga gacgtttccg
      841 gtgaaggcgt acagcaggca ctgttgaaac tgatcgaagg tacggtagct gctgttccac
      901 cgcaaggtgg gcgtaaacat ccgcagcagg aattcttgca ggttgatacc tctaagatcc
      961 tgtttatttg tggcggtgcg tttgccggtc tggataaagt gatttcccac cgtgtagaaa
     1021 ccggctccgg cattggtttt ggcgcgacgg taaaagcgaa gtccgacaaa gcaagcgaag
     1081 gcgagctgct ggcgcaggtt gaaccggaag atctgatcaa gtttggtctt atccctgagt
     1141 ttattggtcg tctgccggtt gtcgcaacgt tgaatgaact gagcgaagaa gctctgattc
     1201 agatcctcaa agagccgaaa aacgccctga ccaagcagta tcaggcgctg tttaatctgg
     1261 aaggcgtgga tctggaattc cgtgacgagg cgctggatgc tatcgctaag aaagcgatgg
     1321 cgcgtaaaac cggtgcccgt ggcctgcgtt ccatcgtaga agccgcactg ctcgatacca
     1381 tgtacgatct gccgtccatg gaagacgtcg aaaaagtggt tatcgacgag tcggtaattg
     1441 atggtcaaag cgaaccgttg ctgatttatg gcaagccgga agcgcaacag gcatctggtg
     1501 aagcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     1561 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1621 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1681 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1741 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1801 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1861 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1921 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1981 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2041 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2101 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     2161 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2221 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     2281 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2341 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     2401 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     2461 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     2521 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2581 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2641 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2701 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2761 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     2821 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2881 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2941 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3001 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3061 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3121 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3181 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3241 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3301 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3361 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3421 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3481 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3541 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3601 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3661 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3721 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3781 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3841 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3901 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3961 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4021 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4081 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     4141 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4201 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4261 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4321 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4381 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4441 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4501 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4561 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4621 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4681 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4741 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4801 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     4861 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     4921 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     4981 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     5041 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     5101 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5161 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     5221 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5281 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5341 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5401 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5461 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5521 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     5581 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     5641 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     5701 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     5761 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     5821 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     5881 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     5941 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     6001 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     6061 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     6121 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     6181 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     6241 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     6301 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     6361 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     6421 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     6481 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     6541 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     6601 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     6661 tggcgagccc gatcttcccc atcggt
//
