LOCUS       sequence-423142-        6196 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 6196)
  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 6196)
  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-33
FEATURES             Location/Qualifiers
     source          1..6196
                     /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(1047..1067)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1074..1091)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1101..1118)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1137..1159
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1174..1198
                     /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(1198..1217)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1199..1217)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1301..1320)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1526..1603
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1604..2686
                     /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(1623..1642)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3495..3686
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3680..3702)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3788..3927
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3843..3862
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3942..3959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4113..4701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4193..4212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4812..4831)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4823..5638
                     /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             4823..5638
                     /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(4896..4915)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5730..6185)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5867..5888)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6079..6098
                     /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 agtttcagag
      241 agcttaaaca agatattttc agtacacaaa ttctcagaag agtctacttt aatcagagca
      301 agatgattat cctttacacc tataaacact ccaggatcag tcttgcattc aaatgaaaca
      361 cagttggagt gcatattatg aaggacaaag aaggcctggt ctggcagtgg tttttcacac
      421 ttatggagct ccacagagtg ttccttgttg ttggcatgca accagaagtc ttttgtagga
      481 ctcagggtta ccattaacat cttaccatca acaccgtcac ctgattcatt tgaggggtgt
      541 tgagactcat agtaactcag taacacctta tctttctttt catctttttt caagtcttca
      601 acatatatct cataactttc atcctccaaa gcaaaagtaa tggattgatc attgtatgtg
      661 cttagagaag caagatactc tgtaataggt gaaattcctg tgatacttga atcatgaagt
      721 gctctagtat atttctggac ccctgatata ccaaaggcaa agcactccac agtagactgc
      781 tgttgacagg cagcgagtac cagatgtctt ttgtgctttc tacctgtttt cagtgaaggc
      841 cttttggtgg tttctctcct aaagtaacag gcctcctttt ttatcataag gccagagcgg
      901 agcttcataa agtacatggg gcaaacttct ttggccttct gttgggattt tcccagcttg
      961 aaacacaagg ctttgcttgc tgtgttcttc cactttgctg tggaaatttt gttggttgaa
     1021 tacttcattt taggcttcat ggatccgccc tgaaaataca ggttttccat atggctgccg
     1081 cgcggcacca ggccgctgct gtgatgatga tgatgatggc tgctgcccat ggtatatctc
     1141 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
     1201 tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc
     1261 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     1321 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     1381 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     1441 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     1501 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
     1561 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     1621 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     1681 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     1741 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     1801 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     1861 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     1921 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     1981 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     2041 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     2101 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     2161 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     2221 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     2281 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     2341 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     2401 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     2461 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     2521 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     2581 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     2641 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     2701 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca
     2761 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     2821 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg
     2881 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct
     2941 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc
     3001 aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga
     3061 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg
     3121 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg
     3181 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta
     3241 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     3301 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg
     3361 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta
     3421 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt
     3481 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca
     3541 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag
     3601 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt
     3661 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     3721 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     3781 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     3841 tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga
     3901 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     3961 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4021 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4081 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4141 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4201 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4261 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4321 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4381 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4441 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4501 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     4561 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4621 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4681 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4741 tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat
     4801 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc
     4861 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     4921 cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt
     4981 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     5041 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     5101 tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata
     5161 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     5221 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     5281 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     5341 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     5401 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     5461 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     5521 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     5581 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga
     5641 attaattcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5701 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     5761 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     5821 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     5881 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     5941 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     6001 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     6061 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     6121 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     6181 cgcgtcccat tcgcca
//
