LOCUS Exported 7417 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION To overexpress RIPK1 in Mammalian Cells. ACCESSION . VERSION . KEYWORDS pcDNA3-FLAG-RIPK1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7417) AUTHORS Seo J, Lee EW, Sung H, Seong D, Dondelinger Y, Shin J, Jeong M, Lee HK, Kim JH, Han SY, Lee C, Seong JK, Vandenabeele P, Song J TITLE CHIP controls necroptosis through ubiquitylation- and lysosome-dependent degradation of RIPK3. JOURNAL Nat Cell Biol. 2016 Mar;18(3):291-302. doi: 10.1038/ncb3314. Epub 2016 Feb 22. PUBMED 26900751 REFERENCE 2 (bases 1 to 7417) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7417 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 898..907 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 907..930 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" primer_bind complement(3011..3028) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3017..3241 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3287..3715 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3374..3393) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3584..3605 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3724..3744) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3729..4058 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3909..4044 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3971..3990 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4125..4919 /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(4179..4198) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4789..4808 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5093..5214 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5130..5149) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5184..5203 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5263..5279) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5263..5279) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5276..5298) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5287..5303 /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(5311..5341) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5356..5377 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5494..5511) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5665..6250) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5745..5764) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6421..7281) /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 7044..7063 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7282..7386) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgccgcc 901 accatggatt acaaggatga cgacgataag agcgcgggcg gatccgaatt catgcaacca 961 gacatgtcct tgaatgtcat taagatgaaa tccagtgact tcctggagag tgcagaactg 1021 gacagcggag gctttgggaa ggtgtctctg tgtttccaca gaacccaggg actcatgatc 1081 atgaaaacag tgtacaaggg gcccaactgc attgagcaca acgaggccct cttggaggag 1141 gcgaagatga tgaacagact gagacacagc cgggtggtga agctcctggg cgtcatcata 1201 gaggaaggga agtactccct ggtgatggag tacatggaga agggcaacct gatgcacgtg 1261 ctgaaagccg agatgagtac tccgctttct gtaaaaggaa ggataatttt ggaaatcatt 1321 gaaggaatgt gctacttaca tggaaaaggc gtgatacaca aggacctgaa gcctgaaaat 1381 atccttgttg ataatgactt ccacattaag atcgcagacc tcggccttgc ctcctttaag 1441 atgtggagca aactgaataa tgaagagcac aatgagctga gggaagtgga cggcaccgct 1501 aagaagaatg gcggcaccct ctactacatg gcgcccgagc acctgaatga cgtcaacgca 1561 aagcccacag agaagtcgga tgtgtacagc tttgctgtag tactctgggc gatatttgca 1621 aataaggagc catatgaaaa tgctatctgt gagcagcagt tgataatgtg cataaaatct 1681 gggaacaggc cagatgtgga tgacatcact gagtactgcc caagagaaat tatcagtctc 1741 atgaagctct gctgggaagc gaatccggaa gctcggccga catttcctgg cattgaagaa 1801 aaatttaggc ctttttattt aagtcaatta gaagaaagtg tagaagagga cgtgaagagt 1861 ttaaagaaag agtattcaaa cgaaaatgca gttgtgaaga gaatgcagtc tcttcaactt 1921 gattgtgtgg cagtaccttc aagccggtca aattcagcca cagaacagcc tggttcactg 1981 cacagttccc agggacttgg gatgggtcct gtggaggagt cctggtttgc tccttccctg 2041 gagcacccac aagaagagaa tgagcccagc ctgcagagta aactccaaga cgaagccaac 2101 taccatcttt atggcagccg catggacagg cagacgaaac agcagcccag acagaatgtg 2161 gcttacaaca gagaggagga aaggagacgc agggtctccc atgacccttt tgcacagcaa 2221 agaccttacg agaattttca gaatacagag ggaaaaggca ctgcttattc cagtgcagcc 2281 agtcatggta atgcagtgca ccagccctca gggctcacca gccaacctca agtactgtat 2341 cagaacaatg gattatatag ctcacatggc tttggaacaa gaccactgga tccaggaaca 2401 gcaggtccca gagtttggta caggccaatt ccaagtcata tgcctagtct gcataatatc 2461 ccagtgcctg agaccaacta tctaggaaat acacccacca tgccattcag ctccttgcca 2521 ccaacagatg aatctataaa atataccata tacaatagta ctggcattca gattggagcc 2581 tacaattata tggagattgg tgggacgagt tcatcactac tagacagcac aaatacgaac 2641 ttcaaagaag agccagctgc taagtaccaa gctatctttg ataataccac tagtctgacg 2701 gataaacacc tggacccaat cagggaaaat ctgggaaagc actggaaaaa ctgtgcccgt 2761 aaactgggct tcacacagtc tcagattgat gaaattgacc atgactatga gcgagatgga 2821 ctgaaagaaa aggtttacca gatgctccaa aagtgggtga tgagggaagg cataaaggga 2881 gccacggtgg ggaagctggc ccaggcgctc caccagtgtt ccaggatcga ccttctgagc 2941 agcttgattt acgtcagcca gaactaactc gagcatgcat ctagagggcc cgtttaaacc 3001 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 3061 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 3121 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 3181 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 3241 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc 3301 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 3361 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 3421 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 3481 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 3541 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 3601 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg 3661 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc 3721 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta 3781 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag 3841 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa 3901 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac 3961 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt 4021 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat 4081 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 4141 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 4201 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 4261 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc 4321 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 4381 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 4441 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 4501 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 4561 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 4621 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg 4681 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 4741 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 4801 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 4861 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag 4921 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt 4981 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg 5041 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt 5101 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 5161 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 5221 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt 5281 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 5341 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 5401 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 5461 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 5521 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 5581 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 5641 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 5701 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 5761 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 5821 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 5881 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 5941 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 6001 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 6061 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 6121 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 6181 acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 6241 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 6301 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 6361 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 6421 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 6481 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 6541 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 6601 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 6661 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 6721 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 6781 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 6841 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 6901 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 6961 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 7021 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 7081 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 7141 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 7201 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 7261 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 7321 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 7381 tccgcgcaca tttccccgaa aagtgccacc tgacgtc //