LOCUS       sequence-456552-        6725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6725)
  AUTHORS   Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, Chan FK
  TITLE     Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates 
            programmed necrosis and virus-induced inflammation.
  JOURNAL   Cell. 2009 Jun 12;137(6):1112-23.
  PUBMED    19524513
REFERENCE   2  (bases 1 to 6725)
  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: "Cell."; 
            date: "2009-06-12"; volume: "137(6)"; pages: "1112-23"
COMMENT     Sequence Label: hRIP1 HA GFP wt
FEATURES             Location/Qualifiers
     source          1..6725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             604..630
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2665..2674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2671..2673,2674..2676,2677..3390)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2671..2673,2674..2676,2677..3387)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2716..2737)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2977..2996)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3324..3345
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3513..3634
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3550..3569)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3604..3623
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3641..4096)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3778..3799)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3990..4009
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4123..4227
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4229..4586
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4229..4420
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4252..4272)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4437..4572
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4621..5415
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4621..5415
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4675..4694)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5285..5304
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5603..5622)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5647..5694
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6023..6611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6512..6531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagccacc
      601 atgtacccat acgacgtccc agactacgct gaattccaac cagacatgtc cttgaatgtc
      661 attaagatga aatccagtga cttcctggag agtgcagaac tggacagcgg aggcttcggg
      721 aaggtgtctc tgtgtttcca cagaacccag ggactcatga tcatgaaaac agtgtacaag
      781 gggcccaact gcattgagca caacgaggcc ctcttggagg aggcgaagat gatgaacaga
      841 ctgagacaca gccgggtggt gaagctcctg ggcgtcatca tagaggaagg gaagtactcc
      901 ctggtgatgg agtacatggg gaagggcaac ctgatgcacg tgctgaaagc cgagatgagt
      961 actccgcttt ctgtaaaagg aaggataatt ttggaaatca ttgaaggaat gtgctactta
     1021 catggaaaag gcgtgataca caaggacctg aagcctgaaa atatccttgt tgataatgac
     1081 ttccacatta agatcgcaga cctcggcctt gcctccttta agatgtggag caaactgaat
     1141 aatgaagagc acaatgagct gagggaagtg gacggcaccg ctaagaagaa tggcggcacc
     1201 ctctactaca tggcgcccga gcacctgaat gacgtcaacg caaagcccac agagaagtcg
     1261 gatgtgtaca gctttgctgt agtactctgg gcgatatttg caaataagga gccatatgaa
     1321 aatgctatct gtgagcagca gttgataatg tgcataaaat ctgggaacag gccagatgtg
     1381 gatgacatca ctgagtactg cccaagagaa attatcagtc tcatgaagct ctgctgggaa
     1441 gcgaatccgg aagctcggcc gacatttcct ggcattgaag aaaaatttag gcctttttat
     1501 ttaagtcaat tagaagaaag tgtagaagag gacgtgaaga gtttaaagaa agagtattca
     1561 aacgaaaatg cagttgtgaa gagaatgcag tctcttcaac ttgattgtgt ggcagtacct
     1621 tcaagccggt caaattcagc cacagaacag cctggttcac tgcacagttc ccagggactt
     1681 gggatgggtc ctgtggagga gtcctggttt gctccttccc tggagcaccc acaagaagag
     1741 aatgagccca gcctgcagag taaactccaa gacgaagcca actaccatct ttatggcagc
     1801 cgcatggaca ggcagacgaa acagcagccc agacagaatg tggcttacaa cagagaggag
     1861 gaaaggagac gcagggtctc ccatgaccct tttgcacagc aaagacctta cgagaatttt
     1921 cagaatacag agggaaaagg cactgcttat tccagtgcag ccagtcatgg taatgcagtg
     1981 caccagccct cagggctcac cagccaacct caagtactgt atcagaacaa tggattatat
     2041 agctcacatg gctttggaac aagaccactg gatccaggaa cagcaggtcc cagagtttgg
     2101 tacaggccaa ttccaagtca tatgcctagt ctgcataata tcccagtgcc tgagaccaac
     2161 tatctaggaa atacacccac catgccattc agctccttgc caccaacaga tgaatctata
     2221 aaatatacca tatacaatag tactggcatt cagattggag cctacaatta tatggagatt
     2281 ggtgggacga gttcatcact actagacagc acaaatacga acttcaaaga agagccagct
     2341 gctaagtacc aagctatctt tgataatacc actagtctga cggataaaca cctggaccca
     2401 atcagggaaa atctgggaaa gcactggaaa aactgtgccc gtaaactggg cttcacacag
     2461 tctcagattg atgaaattga ccatgactat gagcgagatg gactgaaaga aaaggtttac
     2521 cagatgctcc aaaagtgggt gatgagggaa ggcataaagg gagccacggt ggggaagctg
     2581 gcccaggcgc tccaccagtg ttccaggatc gaccttctga gcagcttgat ttacgtcagc
     2641 cagaacgccc gggatccacc ggtcgccacc atggtgagca agggcgagga gctgttcacc
     2701 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2761 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     2821 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     2881 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2941 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3001 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     3061 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     3121 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     3181 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     3241 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     3301 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     3361 actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc ataatcagcc
     3421 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     3481 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     3541 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3601 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     3661 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     3721 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     3781 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     3841 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     3901 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3961 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4021 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4081 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4141 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4201 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     4261 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4321 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4381 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4441 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4501 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4561 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     4621 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4681 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4741 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4801 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4861 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4921 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4981 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     5041 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     5101 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     5161 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     5221 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     5281 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     5341 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     5401 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     5461 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5521 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5581 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     5641 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     5701 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     5761 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     5821 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     5881 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5941 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6001 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6061 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6661 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     6721 tgcat
//
