LOCUS       sequence-423165-        6234 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 6234)
  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 6234)
  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: pET28_Caspase-5-enzymatic-domain
FEATURES             Location/Qualifiers
     source          1..6234
                     /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(1085..1105)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1112..1129)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1139..1156)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1175..1197
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1212..1236
                     /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(1236..1255)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1237..1255)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1339..1358)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1564..1641
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1642..2724
                     /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(1661..1680)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3533..3724
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3718..3740)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3826..3965
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3881..3900
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3980..3997)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4151..4739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4231..4250)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4850..4869)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4861..5676
                     /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             4861..5676
                     /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(4934..4953)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5768..6223)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5905..5926)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6117..6136
                     /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 gctcaattgc
      181 caggaaagag gtagaaatct cttgtcaagg ttgctcgttc tatggtgggc atctgggctt
      241 tagcctgtgg aacttcaaat gatttctgta ccttccgaaa tatttccatt aggtggcagc
      301 agcaagaata tttctggaag catgtgatga gttccgtaat gaagatggag ccccttgtgc
      361 ggtctctcca ggacacgtta tgtggtgttg aagaacagaa agcaatgaag tccttctcct
      421 cgtggatctt gcaaacagaa tctgcctcca ggttctcaga tgactgtgaa gagatgagtg
      481 ccaaggatgc tggagagtct ctgacccaga gttccccatg tttttcacct ctgcaggcct
      541 ggacaatgat gaccttgggt ttgtccttta gactgaggca gttgcggttg ttgaatatct
      601 ggaagatggt gtcataaagc agcacatccg gttttttctt tttatgcgca gttccgcaga
      661 ttccctctag gatgccatga gacatgagta ccaagaacgt gctgtcagag gacttgtgct
      721 ctggtctggc agcaaatgcc ctcagcactg actccatatc cctggctgtg agattctttt
      781 cgtcaaccac agtgtagccc aggccttgaa gcagcctttt catccccacg atgtcatagt
      841 gagccccatt ccttgcaggc aggtgatcaa actttgtatt gcatatgatg agagccaggc
      901 gtctgcggtc ctctctcttt tttattggat agatctcatc atgatttttt ttacacagtc
      961 tcaggaattc ttcacgagga caaagtttga gtatatttgt agattctgct gactcaggtg
     1021 gtccagcctc gatttgcaga agaggtttta cactggtgat cttttggtcc atattgaggg
     1081 atccgccctg aaaatacagg ttttccatat ggctgccgcg cggcaccagg ccgctgctgt
     1141 gatgatgatg atgatggctg ctgcccatgg tatatctcct tcttaaagtt aaacaaaatt
     1201 atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
     1261 gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     1321 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     1381 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     1441 gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc
     1501 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc
     1561 ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     1621 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     1681 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     1741 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     1801 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     1861 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     1921 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     1981 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     2041 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     2101 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     2161 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     2221 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     2281 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     2341 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     2401 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     2461 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     2521 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     2581 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     2641 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     2701 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta
     2761 ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg
     2821 ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt
     2881 aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc
     2941 gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt
     3001 cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc
     3061 ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg
     3121 ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg
     3181 ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta
     3241 aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg
     3301 atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc
     3361 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg
     3421 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt
     3481 catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa
     3541 acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct
     3601 ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga
     3661 ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     3721 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     3781 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     3841 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     3901 ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     3961 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4021 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4081 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4141 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4201 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4261 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4321 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4381 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4441 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4501 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4561 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4621 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4681 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4741 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4801 ggattttggt catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     4861 atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat
     4921 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc
     4981 tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc
     5041 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct
     5101 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg
     5161 atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt
     5221 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct
     5281 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg
     5341 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa
     5401 gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca
     5461 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc
     5521 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     5581 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     5641 ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatga gcggatacat
     5701 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5761 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5821 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5881 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5941 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     6001 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     6061 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     6121 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     6181 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gcca
//
