LOCUS       sequence-212919-        6686 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli ClpX with a C-terminal His tag and a mutation from
            D144 to L144.
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 D144L
FEATURES             Location/Qualifiers
     source          1..6686
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             30..52
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             60..92
                     /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             1389..1406
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1459..1477)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1473..1520
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1557..2012
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1644..1663)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1854..1875
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2038..2142
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2143..2211,2212..3003)
                     /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(2361..2380)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3174..3762
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3663..3682
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3916..3933
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3948..4087
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4013..4032)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4173..4195
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4189..4380)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5189..6271)
                     /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     6233..6252
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6272..6349)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6555..6574
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6658..6677
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6658..6676
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    join(6677..6686,1..15)
                     /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)."
ORIGIN
        1 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
       61 tggctagcat gactggtgga cagcaaatgg gtcgcggatc catgacagat aaacgcaaag
      121 atggctcagg caaattgctg tattgctctt tttgcggcaa aagccagcat gaagtgcgca
      181 agctgattgc cggtccatcc gtgtatatct gcgacgaatg tgttgattta tgtaacgaca
      241 tcattcgcga agagattaaa gaagttgcac cgcatcgtga acgcagtgcg ctaccgacgc
      301 cgcatgaaat tcgcaaccac ctggacgatt acgttatcgg ccaggaacag gcgaaaaaag
      361 tgctggcggt cgcggtatac aaccattaca aacgtctgcg caacggcgat accagcaatg
      421 gcgtcgagtt gggcaaaagt aacattctgc tgatcggtcc gaccggttcc ggtaaaacgc
      481 tgctggctga aacgctggcg cgcctgctgg atgttccgtt caccatggcc ctagcgacta
      541 cactgaccga agccggttat gtgggtgaag acgttgaaaa catcattcag aagctgttgc
      601 agaaatgcga ctacgatgtc cagaaagcac agcgtggtat tgtctacatc gatgaaatcg
      661 acaagatttc tcgtaagtca gacaacccgt ccattacccg agacgtttcc ggtgaaggcg
      721 tacagcaggc actgttgaaa ctgatcgaag gtacggtagc tgctgttcca ccgcaaggtg
      781 ggcgtaaaca tccgcagcag gaattcttgc aggttgatac ctctaagatc ctgtttattt
      841 gtggcggtgc gtttgccggt ctggataaag tgatttccca ccgtgtagaa accggctccg
      901 gcattggttt tggcgcgacg gtaaaagcga agtccgacaa agcaagcgaa ggcgagctgc
      961 tggcgcaggt tgaaccggaa gatctgatca agtttggtct tatccctgag tttattggtc
     1021 gtctgccggt tgtcgcaacg ttgaatgaac tgagcgaaga agctctgatt cagatcctca
     1081 aagagccgaa aaacgccctg accaagcagt atcaggcgct gtttaatctg gaaggcgtgg
     1141 atctggaatt ccgtgacgag gcgctggatg ctatcgctaa gaaagcgatg gcgcgtaaaa
     1201 ccggtgcccg tggcctgcgt tccatcgtag aagccgcact gctcgatacc atgtacgatc
     1261 tgccgtccat ggaagacgtc gaaaaagtgg ttatcgacga gtcggtaatt gatggtcaaa
     1321 gcgaaccgtt gctgatttat ggcaagccgg aagcgcaaca ggcatctggt gaagcggccg
     1381 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     1441 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1501 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1561 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1621 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1681 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1741 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1801 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1861 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1921 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1981 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2041 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2101 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     2161 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     2221 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     2281 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     2341 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     2401 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     2461 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     2521 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     2581 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2641 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     2701 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2761 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2821 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2881 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2941 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3001 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3061 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3121 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3181 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3241 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3301 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3361 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3421 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3481 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3541 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3601 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3661 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3721 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3781 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3841 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3901 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3961 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4021 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4081 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4141 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4201 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4261 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4321 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4381 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4441 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4501 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4561 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4621 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4681 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     4741 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     4801 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     4861 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     4921 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     4981 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5041 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5101 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5161 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5221 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5281 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5341 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     5401 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     5461 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     5521 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     5581 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     5641 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     5701 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     5761 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     5821 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     5881 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     5941 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6001 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6061 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6121 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6181 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6241 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6301 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     6361 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     6421 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     6481 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     6541 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     6601 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     6661 tacgactcac tataggggaa ttgtga
//
