LOCUS       sequence-426868-        5917 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 5917)
  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 5917)
  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: pET16_Caspase-4_p10
FEATURES             Location/Qualifiers
     source          1..5917
                     /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"
     RBS             424..446
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    462..486
                     /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(486..505)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(487..505)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(589..608)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        814..891
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(911..930)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3285..3476
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3470..3492)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3578..3717
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3633..3652
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3732..3749)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3903..4491)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3983..4002)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4662..5453,5454..5522))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5285..5304
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5523..5627)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5695..5713
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5740..5745,5746..5762,5763..5768)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatcctca attgccagga aagaggtaga aatatcttgt catggacagt
      181 cgttctatgg tgggcatttg agctttggcc cttggagttt caaatgattg ctgtaccttc
      241 cgaaatactt cctctaggtg gcagcaccaa gaatatttct ggaagcatgt gatgagttgt
      301 gtgatgaaga tagagcccat tgtgctgtct ctccaggaca cgttgtgtgg cgttgaagag
      361 cagaaagcaa tgaagtcctt ctccacgtgg gtcttgtaaa cagcgctagc acccatatgt
      421 atatctcctt cttaaagtta aacaaaagag ctctctagag gggaattgtt atccgctcac
      481 aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg
      541 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
      601 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
      661 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac
      721 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc
      781 aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt
      841 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
      901 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
      961 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     1021 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     1081 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     1141 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     1201 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     1261 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     1321 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     1381 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     1441 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     1501 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     1561 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     1621 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     1681 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     1741 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     1801 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     1861 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     1921 gattcattaa tgcagctggc acgacagttt cccgactgga aagcgggcag tgagcgcaac
     1981 gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga
     2041 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     2101 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt
     2161 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc
     2221 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc
     2281 gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg
     2341 ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc
     2401 gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag
     2461 cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcattggacc gctgatcgtc
     2521 acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc
     2581 gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg
     2641 acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca
     2701 atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg
     2761 cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac
     2821 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta
     2881 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa
     2941 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg
     3001 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct
     3061 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga
     3121 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta
     3181 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta
     3241 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa
     3301 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac
     3361 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg
     3421 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     3481 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     3541 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     3601 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     3661 gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     3721 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3781 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3841 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3901 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3961 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4021 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4081 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4141 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4201 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4261 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4321 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     4381 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4441 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4501 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4561 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4621 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4681 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4741 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4801 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4861 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4921 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     4981 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5041 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5101 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5161 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5221 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
     5281 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5341 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5401 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5461 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5521 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5581 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5641 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     5701 cgtatcacga ggccctttcg tcttcaagaa ttctcatgtt tgacagctta tcatcgataa
     5761 gctttaatgc ggtagtttat cacagttaaa ttgctaacgc agtcaggcac cgtgtatgaa
     5821 atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg
     5881 cttggttatg ccggtactgc cgggcctctt gcgggat
//
