LOCUS       sequence-423141-        6199 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 6199)
  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 6199)
  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-alpha
FEATURES             Location/Qualifiers
     source          1..6199
                     /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(1050..1070)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1077..1094)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1104..1121)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1140..1162
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1177..1201
                     /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(1201..1220)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1202..1220)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1304..1323)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1529..1606
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1607..2689
                     /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(1626..1645)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3498..3689
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3683..3705)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3791..3930
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3846..3865
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3945..3962)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4116..4704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4196..4215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4815..4834)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4826..5641
                     /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             4826..5641
                     /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(4899..4918)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5733..6188)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5870..5891)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6082..6101
                     /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 cagatcctct tctgagatga gtttttgttc cgcctggttt
      241 tccagtatct gaaagtcagt gatagagggt ggcccccctg ccaagcacac ccagtagtct
      301 tgctttgtgg caataaacaa gtttggatgg gcaactgatg tgaaatagtt cttagtgccg
      361 tgagtttccc agaagaagag gaggttggtc tcactacctg tgatggtttt gggtatctca
      421 ggcatctcct tcagcagcac tggttggtct tcatcttggg cagtcacata caattgagtt
      481 tttgagattc ttagaatcac ggtaatttta gcatcatcct ttgatgactt ataagcaccc
      541 atgtcaaatt tcactgcttc atccagatta tgtaatgcag cagccgtgag gtactgatca
      601 ttggctcgaa ttatactttg attgagggcg tcattcagga tgaactcgta tttgatgatc
      661 ctcataaagt tgtatttcac attgctcagg aagctaaaag gtgctgacct aggcttgatg
      721 atttcttcct ctgagtcatt ggcgatggcc tccaggtcat catcagtgat ggattggctt
      781 aaactcaacc gtctcttctt cagaaccttc ccgttggttg ctactaccac catgctctcc
      841 ttgaaggtaa gcttggatgt tttagaggtt tcagagatac tcagagacac agattgatcc
      901 atgcagcctt catggagtgg gccatagctt acatgataga aggatttctg attcagagac
      961 agatgatcaa tggaggaact gtcttcttca ttttcactgt aacagttctt caggtcttca
     1021 aacatgtctg gaactttggc catggatccg ccctgaaaat acaggttttc catatggctg
     1081 ccgcgcggca ccaggccgct gctgtgatga tgatgatgat ggctgctgcc catggtatat
     1141 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
     1201 ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc
     1261 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
     1321 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
     1381 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc
     1441 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
     1501 tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg
     1561 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     1621 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     1681 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     1741 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     1801 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     1861 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     1921 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     1981 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     2041 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     2101 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     2161 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     2221 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     2281 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     2341 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     2401 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     2461 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     2521 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     2581 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2641 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     2701 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
     2761 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     2821 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
     2881 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
     2941 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
     3001 agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt
     3061 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
     3121 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
     3181 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
     3241 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
     3301 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
     3361 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
     3421 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
     3481 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
     3541 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
     3601 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
     3661 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     3721 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     3781 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     3841 ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac
     3901 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     3961 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4021 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4081 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4141 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4201 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4261 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4321 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4381 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4441 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4501 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     4561 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4621 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4681 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4741 cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta
     4801 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag
     4861 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     4921 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt
     4981 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     5041 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     5101 tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga
     5161 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     5221 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     5281 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     5341 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     5401 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     5461 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     5521 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     5581 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     5641 agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5701 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     5761 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     5821 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     5881 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     5941 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     6001 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     6061 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     6121 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     6181 gggcgcgtcc cattcgcca
//
