LOCUS Exported 7066 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses Gsdmd-NT 4A mutant in mammalian cells. ACCESSION . VERSION . KEYWORDS Flag-Gsdmd-NT 4A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7066) AUTHORS Liu X, Zhang Z, Ruan J, Pan Y, Magupalli VG, Wu H, Lieberman J TITLE Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. JOURNAL Nature. 2016 Jul 6;535(7610):153-8. doi: 10.1038/nature18629. PUBMED 27383986 REFERENCE 2 (bases 1 to 7066) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7066 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 126..148 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 140..157 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 141..157 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" enhancer 318..697 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 698..901 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 852..872 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 945..968 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" polyA_signal 1825..2447 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2024..2043) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" primer_bind complement(2471..2491) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2489..2786 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind 2718..2737 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 2840..3634 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(2894..2913) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3504..3523 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4286..4420 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4323..4342) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4377..4396 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4425..4444) /label=T7 /note="T7 promoter, forward primer" promoter complement(4426..4444) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4458..4474) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4458..4474) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4471..4493) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4482..4498 /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)." promoter complement(4506..4536) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4551..4572 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4689..4706) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4860..5448) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4940..4959) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5619..6479) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6242..6261 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6480..6584) /gene="bla" /label=AmpR promoter rep_origin 6611..7066 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6698..6717) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6908..6929 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct 61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg 121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa 181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta 241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg 361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 901 tcgtttagtg aaccgtcaga attaattcag atcgatctac catggactac aaagacgatg 961 acgacaagct tgcggccgca atgccatcgg cctttgagaa agtggtcaag aatgtgatca 1021 aggaggtaag cggcagcaga ggcgatctca ttccggtgga cagcctgcgg aactccacca 1081 gcttcaggcc ctactgcctt ctgaacagga aattttcaag ctcaaggttc tggaaacccc 1141 gttattcatg tgtcaacctg tcaatcaagg acatcctgga gcccagtgct ccagaaccag 1201 aaccggagtg ttttggctcc ttcaaagtct ctgatgtcgt cgatgggaac attcagggca 1261 gagtgatgtt gtcaggcatg ggagaaggga aaatttctgg tggggctgca gtgtctgaca 1321 gttccagtgc ctccatgaat gtgtgtatac tgcgtgtgac tcagaagacc tgggagacca 1381 tgcagcatga agcgcacctt cagcagcctg agaacgcaat cctgcaacag cttgcgagtg 1441 ctggggatga cctgtttgtg gtgaccgagg tgctgcagac aaaggaggaa gtgcagatca 1501 ctgaggtcca cagccaagag ggctcaggcc agtttacgct gcctggagct ttatgcttga 1561 agggtgaagg caagggccac caaagccgga agaagatggt gaccattcct gcaggcagca 1621 tcctggcatt ccgagtggcc caactgctta ttggctctaa atgggatatc cttctcgtct 1681 cagatgagaa acagaggacc tttgagccct cctcaggtga cagaaaagca gtgggccaga 1741 ggcaccatgg cctcaatgtg cttgctgcgc tttgttccat cggaaagcag ctcagtctcc 1801 tgtcagatta gtctagagga tcccgggtgg catccctgtg acccctcccc agtgcctctc 1861 ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc 1921 atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg 1981 agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc 2041 tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt 2101 ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa 2161 tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc 2221 taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc 2281 actgctccct tccctgtcct tctgatttta aaataactat accagcagga ggacgtccag 2341 acacagcata ggctacctgg ccatgcccaa ccggtgggac atttgagttg cttgcttggc 2401 actgtcctct catgcgttgg gtccactcag tagatgcctg ttgaattggg tacgcggcca 2461 gcttggctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 2521 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 2581 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 2641 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 2701 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc 2761 cagaagtagt gaggaggctt ttttggagga attgatcagc ttgggatctg atcaagagac 2821 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc 2881 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc 2941 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc 3001 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg 3061 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt 3121 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc 3181 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga 3241 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga 3301 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct 3361 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc 3421 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt 3481 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg 3541 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat 3601 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc 3661 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa 3721 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat 3781 ctcatgctgg agttcttcgc ccaccccggg ctcgatcccc tcgcgagttg gttcagctgc 3841 tgcctgaggc tggacgacct cgcggagttc taccggcagt gcaaatccgt cggcatccag 3901 gaaaccagca gcggctatcc gcgcatccat gcccccgaac tgcaggagtg gggaggcacg 3961 atggccgctt tggtcgaccc ggacgggacg ctcctgcgcc tgatacagaa cgaattgctt 4021 gcaggcatct catgagtgtg tcttcccgtt ttccgcctga ggtcactgcg tggatggagc 4081 gctggcgcct gctgcgcgac ggcgagctgc tcaccaccca ctcgccaagc tggaaccgta 4141 aaaaggccgc gttgctggcg tttttccata ggctccgccg atcataatca gccataccac 4201 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca 4261 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata 4321 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 4381 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc aattccctat agtgagtcgt 4441 attaaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 4501 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 4561 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 4621 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 4681 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 4741 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 4801 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 4861 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 4921 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 4981 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 5041 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 5101 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 5161 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 5221 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 5281 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 5341 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 5401 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 5461 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 5521 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 5581 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 5641 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 5701 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 5761 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 5821 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 5881 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 5941 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 6001 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 6061 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 6121 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 6181 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 6241 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 6301 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 6361 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 6421 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 6481 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 6541 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 6601 gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 6661 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 6721 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 6781 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 6841 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 6901 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 6961 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 7021 caaaaattta acgcgaattt taacaaaata ttaacgctta caattt //