LOCUS       sequence-423131-        5860 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 5860)
  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 5860)
  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-alpha
FEATURES             Location/Qualifiers
     source          1..5860
                     /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(711..731)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(738..755)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(765..782)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             801..823
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    838..862
                     /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(862..881)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(863..881)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(965..984)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1190..1267
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1268..2350
                     /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(1287..1306)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3159..3350
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3344..3366)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3452..3591
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3507..3526
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3606..3623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3777..4365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3857..3876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4476..4495)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4487..5302
                     /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             4487..5302
                     /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(4560..4579)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5394..5849)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5531..5552)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5743..5762
                     /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 aaacagcata
      241 gttaacccaa agtcagtagt gttggctttc cccagttctt gggtaaggat gagaggacag
      301 cctccttcag agctgacagc gatgaaccag ccagggaaag ccacagactc gaaggtggag
      361 ttcctgccac tctggctgtg gtagaagaga aaggacttca caggctcggg ttggttgtac
      421 aaatccatta tatccttttc cttcagctgc agtgtgggct ggtccccgac tttagcacac
      481 atcaggcaga gattgagtcc attcaggccc aggtagatgg ggttccctct gtctttctca
      541 agggtctcca catgtcggca tgagattaag gcaatagtga ctggagacat acggtccttc
      601 ctcgggactg ctatgagcgt ctggtcctga agaacccaca cccgatgatt gatatcctga
      661 atgctcccct gctgaggtgt gtcaattttc aatgcttttt ccatggatcc gccctgaaaa
      721 tacaggtttt ccatatggct gccgcgcggc accaggccgc tgctgtgatg atgatgatga
      781 tggctgctgc ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga
      841 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc
      901 tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
      961 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
     1021 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc
     1081 atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg
     1141 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga
     1201 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     1261 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     1321 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     1381 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     1441 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     1501 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     1561 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     1621 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     1681 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     1741 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     1801 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     1861 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     1921 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     1981 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     2041 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     2101 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     2161 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     2221 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     2281 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2341 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc
     2401 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
     2461 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
     2521 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc
     2581 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg
     2641 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc
     2701 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
     2761 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
     2821 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     2881 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     2941 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     3001 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     3061 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     3121 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     3181 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     3241 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     3301 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     3361 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     3421 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     3481 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     3541 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     3601 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3661 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3721 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3781 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3841 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3901 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3961 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4021 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4081 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4141 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4201 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4261 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4321 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4381 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4441 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     4501 acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg
     4561 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     4621 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     4681 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     4741 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     4801 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     4861 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     4921 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     4981 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     5041 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     5101 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     5161 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     5221 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     5281 gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt
     5341 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     5401 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5461 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     5521 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     5581 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     5641 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5701 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5761 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     5821 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
