LOCUS       sequence-182824-        6768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6768)
  AUTHORS   Ding J, Wang K, Liu W, She Y, Sun Q, Shi J, Sun H, Wang DC, Shao F
  TITLE     Pore-forming activity and structural autoinhibition of the gasdermin
            family.
  JOURNAL   Nature. 2016 Jul 7;535(7610):111-6. Epub 2016 Jun 8.
  PUBMED    27281216
REFERENCE   2  (bases 1 to 6768)
  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"; journalName: "Nature."; 
            date: "2016-07-7"; volume: "535(7610)"; pages: "111-6. Epub 2016 Jun
            8"
COMMENT     Sequence Label: pET28a-PFO
FEATURES             Location/Qualifiers
     source          1..6768
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..147
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1634..1651
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1704..1722)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1718..1765
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1802..2256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     2098..2119
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2348..3163)
                     /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="MSHIQWETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3071..3090
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3155..3174
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3285..3873
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3774..3793
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4027..4044
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4059..4198
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4124..4143)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(4303..4491)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5300..6382)
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSPVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVPRLESGQ"
     primer_bind     6344..6363
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6383..6460)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6666..6685
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcgaga atctttattt tcagggccgg ccaatgtcaa aggatataac agataaaaat
      181 caaagtattg attctggaat atcaagctta agttacaata gaaatgaagt tttagctagt
      241 aatggagata aaattgaaag ttttgttcca aaggaaggta aaaagactgg taataaattt
      301 atagttgtag aacgtcaaaa aagatccctt acaacatcac cagtagatat atcaataatt
      361 gattctgtaa atgaccgtac atatccagga gcattacaac ttgcagataa agcctttgtg
      421 gaaaatagac ctacaatctt aatggtaaaa agaaagccta ttaacattaa tatagattta
      481 ccaggattaa agggtgaaaa tagtataaag gttgatgatc caacctatgg aaaagtttct
      541 ggagcaattg atgagttagt gtctaagtgg aatgaaaagt attcatctac acatacttta
      601 ccagcaagaa ctcaatattc agaatctatg gtttatagta aatcacaaat atcaagtgcc
      661 cttaatgtta atgctaaagt ccttgaaaac tcacttggag tagactttaa tgcagtagca
      721 aacaatgaga aaaaagttat gattttagca tataaacaaa tattctatac agtaagtgca
      781 gacttaccta agaatccatc agatcttttt gatgacagtg ttacatttaa tgatttaaaa
      841 caaaagggag taagtaatga agcacctcca cttatggttt caaatgtagc ttatggaaga
      901 actatatatg ttaagttaga aactacttct agtagtaaag atgtacaagc tgctttcaaa
      961 gctcttataa agaacactga tataaaaaat agtcaacaat ataaagatat ttatgaaaat
     1021 agttccttca cagcagtagt tttaggagga gatgcacaag aacataacaa agttgtaact
     1081 aaggactttg atgaaataag aaaagtaatt aaagacaatg caacttttag tacaaaaaac
     1141 ccagcatatc caatatctta tactagtgtt ttcttaaaag ataactcagt tgctgctgtt
     1201 cacaataaaa cagattatat agaaacaact tctacagagt attctaaggg aaaaataaac
     1261 ttagatcata gtggagccta tgttgcacag tttgaagtag catgggatga agtttcatat
     1321 gacaaagaag gaaatgaagt tttaactcat aaaacatggg atggaaatta tcaagataaa
     1381 acagctcact attcaacagt aatacctctt gaagccaatg caagaaatat aagaataaag
     1441 gcaagagagt gtacaggtct tgcttgggaa tggtggagag atgttataag tgaatatgat
     1501 gttccattaa caaataatat aaatgtttca atatggggaa ctactttata ccctggatct
     1561 agtattactt acaattaagg cgcgccagga tccgaattcg agctccgtcg acaagcttgc
     1621 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     1681 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1741 taaacgggtc ttgaggggtt ctttgctgaa aggaggaact atatccggat tggcgaatgg
     1801 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1861 gctacacttg ccagcgccct agcgcccgct cctttgcttt cttcccttcc tttctcgcca
     1921 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1981 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2041 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2101 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2161 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2221 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     2281 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     2341 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     2401 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     2461 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     2521 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     2581 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     2641 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     2701 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     2761 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     2821 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     2881 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     2941 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     3001 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     3061 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     3121 atcgcggcct agagcaagac gtttcccatt gaatatggct cataacaccc cttgtattac
     3181 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     3241 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3301 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3361 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3421 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3481 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3541 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3601 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3661 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3721 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3781 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3841 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3901 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3961 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4021 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4081 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     4141 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4201 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4261 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cggaggtttt caccgtcatc
     4321 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4381 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4441 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4501 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4561 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4621 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4681 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4741 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4801 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4861 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4921 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     4981 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     5041 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     5101 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     5161 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     5221 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5281 tacattaatt gcgttgcgct cactgcccgc tttccagtcg gggaacctgt cgtgccagct
     5341 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5401 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5461 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5521 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5581 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5641 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5701 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5761 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca ggacttaatg
     5821 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5881 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5941 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     6001 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     6061 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     6121 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     6181 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     6241 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     6301 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6361 cgtataacgt tactggtttc acactcacca ccctgaattg actctcttcc gggcgctatc
     6421 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6481 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6541 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6601 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6661 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6721 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
