LOCUS       sequence-426859-        6163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein overexpression in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6163)
  AUTHORS   Devant P, Dong Y, Mintseris J, Ma W, Gygi SP, Wu H, Kagan JC
  TITLE     Structural insights into cytokine cleavage by inflammatory 
            caspase-4.
  JOURNAL   Nature. 2023 Dec;624(7991):451-459. doi: 10.1038/s41586-023-06751-9.
            Epub 2023 Nov 22.
  PUBMED    37993712
REFERENCE   2  (bases 1 to 6163)
  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: 
            "10.1038/s41586-023-06751-9"; journalName: "Nature"; date: 
            "2023-12"; volume: "624"; issue: "7991"; pages: "451-459"
COMMENT     Sequence Label: pET28a_Pro-IL-1-beta
FEATURES             Location/Qualifiers
     source          1..6163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1014..1034)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1041..1058)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1068..1085)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1104..1126
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1141..1165
                     /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)."
     primer_bind     complement(1165..1184)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1166..1184)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1268..1287)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1493..1570
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1571..2653
                     /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     complement(1590..1609)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3462..3653
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3647..3669)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3755..3894
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3810..3829
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3909..3926)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4080..4668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4160..4179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4779..4798)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4790..5605
                     /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             4790..5605
                     /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     complement(4863..4882)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5697..6152)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5834..5855)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6046..6065
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattctta ggaagacaca aattgcatgg tgaagtcagt tatatcctgg
      241 ccgcctttgg tccctcccag gaagacgggc atgttttctg cttgagaggt gctgatgtac
      301 cagttgggga actgggcaga ctcaaattcc agcttgttat tgatttctat cttgttgaag
      361 acaaatcgct tttccatctt cttctttggg taatttttgg gatctacact ctccagctgt
      421 agagtgggct tatcatcttt caacacgcag gacaggtaca gattcttttc cttgaggccc
      481 aaggccacag gtattttgtc attactttct tctccttgta caaaggacat ggagaacacc
      541 acttgttgct ccatatcctg tccctggagg tggagagctt tcagttcata tggaccagac
      601 atcaccaagc ttttttgctg tgagtcccgg agcgtgcagt tcagtgatcg tacaggtgca
      661 tcgtgcacat aagcctcgtt atcccatgtg tcgaagaaga taggttcttc ttcaaagatg
      721 aagggaaaga aggtgctcag gtcattctcc tggaaggtct gtgggcaggg aaccagcatc
      781 ttcctcagct tgtccatggc cacaacaact gacgcggcct gcctgaagcc cttgctgtag
      841 tggtggtcgg agattcgtag ctggatgccg ccatccagag ggcagaggtc caggtcctgg
      901 aaggagcact tcatctgttt agggccatca gcttcaaaga acaagtcatc ctcattgcca
      961 ctgtaataag ccatcatttc actggcgagc tcaggtactt ctgccatgga tccgccctga
     1021 aaatacaggt tttccatatg gctgccgcgc ggcaccaggc cgctgctgtg atgatgatga
     1081 tgatggctgc tgcccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg
     1141 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     1201 atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
     1261 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     1321 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
     1381 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     1441 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
     1501 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     1561 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     1621 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     1681 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     1741 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     1801 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     1861 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     1921 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     1981 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     2041 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     2101 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     2161 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     2221 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     2281 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     2341 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     2401 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     2461 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     2521 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     2581 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2641 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
     2701 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     2761 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     2821 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     2881 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     2941 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg
     3001 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
     3061 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     3121 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     3181 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     3241 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     3301 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     3361 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     3421 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     3481 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     3541 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     3601 agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     3661 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     3721 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     3781 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     3841 ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     3901 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3961 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4021 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4081 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4141 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4201 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4261 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4321 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4381 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4441 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4501 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4561 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4621 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4681 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4741 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     4801 tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata
     4861 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
     4921 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     4981 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     5041 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa
     5101 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     5161 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     5221 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     5281 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     5341 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     5401 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     5461 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     5521 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     5581 tttgatgctc gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta
     5641 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     5701 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5761 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5821 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5881 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5941 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     6001 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     6061 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     6121 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca
//
