LOCUS       sequence-423152-        6244 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 6244)
  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 6244)
  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_Caspase-4_lemur_catalytic domain
FEATURES             Location/Qualifiers
     source          1..6244
                     /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(1095..1115)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1122..1139)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1149..1166)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1185..1207
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1222..1246
                     /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(1246..1265)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1247..1265)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1349..1368)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1574..1651
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1652..2734
                     /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(1671..1690)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3543..3734
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3728..3750)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3836..3975
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3891..3910
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3990..4007)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4161..4749)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4241..4260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4860..4879)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4871..5686
                     /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             4871..5686
                     /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(4944..4963)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5778..6233)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5915..5936)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6127..6146
                     /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 cgaattctca atttccagga aagaggtaga agcatcttgt catggacagt
      241 cgttcaatgg tgggcatctg ggctcgaaca cttggtgttt caaatgattg ttgtaccttc
      301 cgaaacactt cctgtaggtg acagcaccaa gagtatttct ggaagcatgt gatgagctgt
      361 gtgatgaaga gggagccctt tgtgatgtct ctccaggaca tgttatgtgg ggtggaagag
      421 cagaaagcaa tgaagtcctt ctccacgtgg gtcttgcaaa cagcatcttc ctccaggttc
      481 tcaggggact gtgagaagct gtctgccaca gggttctcag gtaactgtga ggagctggct
      541 gccgaggctt ctggagagtc tctgacccac acttccccac ggtttgcacc tctgcaggcc
      601 tggacaatga tgaccttggg tttgtccttt agaatagggc agttgcggtt gttgagtatc
      661 cggaagacgg tgtcataagg cagcacatcc catgttccct cactgtgctt cgttccacag
      721 attccatcca ggatgccatg agacatgagc accacgaagg tgctgtctga ggacttgtgc
      781 tctggtcggg cagcaaattc ccgcagcaca gactccatat ccctggctgt gagtttctct
      841 tttacatcca cactgtagcc caggccctca agcagcccct tcatccccac aatgtcatgg
      901 tcagccccat ttctccgctc aagatggtca aactctgtgt tgcatatgat gagagccagg
      961 cgagtgcgat cctttttctc ctttatgggg tagatctcat cagcccgttc tttactcagt
     1021 ctcaagaagt ctttacgagg gcaaagcttg agggtatctg taggttctgc tgactcagct
     1081 ggctcagcgg atccgccctg aaaatacagg ttttccatat ggctgccgcg cggcaccagg
     1141 ccgctgctgt gatgatgatg atgatggctg ctgcccatgg tatatctcct tcttaaagtt
     1201 aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg
     1261 tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc
     1321 accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat
     1381 cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc
     1441 gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg
     1501 ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag
     1561 cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg
     1621 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     1681 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     1741 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     1801 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     1861 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     1921 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     1981 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     2041 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     2101 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     2161 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     2221 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     2281 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     2341 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     2401 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     2461 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     2521 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     2581 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     2641 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2701 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     2761 tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct
     2821 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     2881 tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc
     2941 gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg
     3001 ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg
     3061 ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag
     3121 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     3181 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     3241 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     3301 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     3361 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     3421 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     3481 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     3541 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     3601 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     3661 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     3721 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     3781 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     3841 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     3901 gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga
     3961 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4021 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4081 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4141 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4201 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4261 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4321 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4381 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4441 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4501 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4561 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     4621 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4681 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4741 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4801 tcacgttaag ggattttggt catgaacaat aaaactgtct gcttacataa acagtaatac
     4861 aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc
     4921 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     4981 tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     5041 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     5101 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     5161 caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     5221 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     5281 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     5341 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     5401 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     5461 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     5521 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     5581 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     5641 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatga
     5701 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5761 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5821 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5881 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5941 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6001 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     6061 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     6121 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     6181 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     6241 gcca
//
