LOCUS       sequence-423136-        5893 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 5893)
  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 5893)
  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-36-gamma
FEATURES             Location/Qualifiers
     source          1..5893
                     /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(201..230)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(744..764)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(771..788)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(798..815)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             834..856
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    871..895
                     /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(895..914)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(896..914)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(998..1017)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1223..1300
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1301..2383
                     /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(1320..1339)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3192..3383
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3377..3399)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3485..3624
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3540..3559
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3639..3656)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3810..4398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3890..3909)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4509..4528)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4520..5335
                     /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             4520..5335
                     /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(4593..4612)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5427..5882)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5564..5585)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5776..5795
                     /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 cgaattccta cagatcctct tctgagatga gtttttgttc gtcatttata
      241 tttaattcaa aggcagtgtt gtatgacttc ccaagttctg aagtcagaat gatgggctgg
      301 tctctcttgg aggaggcaat gaaccagtcc gggaaggcca cagactcaag ggtggaggtc
      361 ctaccagtct tggcacggta gaaaaggaag ggtttcacgg gctcgggttg gccatacaga
      421 tccatgatct tctgctcttt tagctgcaat gtgggctgtt ctccaacctt ctcacaatac
      481 aaacacattt ctggattctg gattcccaaa taaatggggt cccctctgcc ttgctcaaga
      541 gcctctggat acttgcatgt gataacagca acagtgactg gggtcacact gtcacttcgt
      601 ggaactgcca caaggttctg accctgaagg gtccacactt gctgattcaa atcattaata
      661 gtcccagtaa taggtttaca cattgattga tagacggccc ttcctccacc atcagcgtct
      721 cctggagtgc ctctcatgga tccgccctga aaatacaggt tttccatatg gctgccgcgc
      781 ggcaccaggc cgctgctgtg atgatgatga tgatggctgc tgcccatggt atatctcctt
      841 cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat
      901 agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg
      961 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
     1021 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
     1081 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
     1141 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     1201 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
     1261 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     1321 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     1381 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     1441 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     1501 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     1561 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     1621 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     1681 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     1741 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     1801 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     1861 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     1921 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     1981 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     2041 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     2101 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     2161 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     2221 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     2281 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     2341 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2401 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     2461 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     2521 tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
     2581 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc
     2641 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg
     2701 ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga
     2761 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     2821 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     2881 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     2941 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     3001 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     3061 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     3121 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     3181 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     3241 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     3301 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     3361 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3421 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3481 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     3541 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
     3601 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3661 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3721 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3781 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3841 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3901 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3961 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4021 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4081 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4141 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4201 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4261 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4321 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4381 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4441 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa
     4501 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     4561 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     4621 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     4681 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     4741 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     4801 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     4861 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     4921 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     4981 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     5041 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     5101 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     5161 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     5221 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     5281 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt
     5341 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5401 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     5461 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     5521 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     5581 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     5641 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     5701 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     5761 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     5821 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     5881 gtcccattcg cca
//
