LOCUS       sequence-254780-        6181 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 6181)
  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 6181)
  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: mRIP3 GFP wt
FEATURES             Location/Qualifiers
     source          1..6181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1983..1994
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     regulatory      2137..2146
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2143..2145,2146..2148,2149..2862)
                     /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(2143..2145,2146..2148,2149..2859)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2188..2209)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2449..2468)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2796..2817
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2985..3106
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3022..3041)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3076..3095
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3113..3568)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3250..3271)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3462..3481
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3595..3699
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3701..4058
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3701..3892
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3724..3744)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3909..4044
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4093..4887
                     /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             4093..4887
                     /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(4147..4166)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4757..4776
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5075..5094)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5119..5166
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5495..6083
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5984..6003
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gcgctaccgg actcagatct actatgtctt ctgtcaagtt atggcctact
      661 ggtgcgtcag cggttcctct ggtgagccgt gaagaactga agaagctgga gtttgtgggc
      721 aaaggagggt tcggagtcgt gttccgggca caccacagaa catggaacca tgatgtagca
      781 gtcaagatcg tgaactcgaa gaagatatcc tgggaggtga aggctatggt taatcttcgt
      841 aatgagaacg ttctgctcct gctgggggtc actgaggacc tccagtggga cttcgtgtcc
      901 gggcaggctc tggtgacaag attcatggag aatggctccc tcgcagggct gctgcaaccc
      961 gagtgccctc ggccctggcc actcctctgt cgcctgctgc aggaagtggt gctggggatg
     1021 tgctacctac acagcttgga ccctccgctc ctgcaccggg acctcaagcc ctctaacatt
     1081 ctgctggatc cagagctcca cgccaagcta gcagattttg gcctgtccac gtttcaggga
     1141 gggtcccagt cagggtcagg atcaggatca ggatccaggg actctggggg caccctagcg
     1201 tacttggacc cagagctgtt atttaaagtc aacctgaagg cttctaaagc gagtgatgtc
     1261 tacagctttg ggatcctcgt gtgggcagtg ctggctggca gagaagctga gttggtagac
     1321 aagacttcac taatccggga aacagtgtgt gacaggcaga gtcgtcctcc actgacagag
     1381 ctgcctccag gtagccctga gactcccggc ttggaaaaac tgaaggagtt aatgattcat
     1441 tgctggggtt cccagtccga aaacaggcca tccttccagg actgcgaacc aaaaaccaat
     1501 gaagtttaca atctggtaaa ggacaaggta gatgctgctg tctccgaggt aaagcattat
     1561 ctgtctcagc acagaagcag tggcagaaac ttgtctgcca gagagccaag ccaaagaggc
     1621 acagaaatgg attgcccgag ggaaaccatg gtttctaaaa tgctggaccg cctgcatttg
     1681 gaggaaccct ccggaccagt tcctggaaaa tgtcctgaga ggcaagcaca ggacacatca
     1741 gttgggcctg ccacaccagc aaggacatct tctgaccccg tggctggcac tcctcagatt
     1801 ccacatactt tacccttcag aggcacaaca cctgggccag tctttactga gactcccggt
     1861 cctcaccccc aaaggaatca gggagatgga agacacggca ctccttggta tccctggacc
     1921 ccaccgaatc caatgacagg gccaccggct ctcgtcttca acaactgttc tgaagtgcag
     1981 attgggaact acaactcctt ggtagcacca ccaagaacta ctgcctcaag ttcggccaag
     2041 tatgaccaag cacagttcgg caggggtagg ggctggcagc ccttccacaa gcgaattctg
     2101 cagtcgacgg taccgcgggc ccgggatcca ccggtcgcca ccatggtgag caagggcgag
     2161 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     2221 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     2281 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     2341 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     2401 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     2461 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2521 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     2581 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     2641 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     2701 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     2761 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     2821 gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg cgactctaga
     2881 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     2941 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     3001 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3061 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta
     3121 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     3181 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     3241 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     3301 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     3361 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     3421 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     3481 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     3541 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3601 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3661 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     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 ccgcctcggc ctctgagcta ttccagaagt
     4021 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     4081 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4141 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4201 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4261 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4321 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4381 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4441 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4501 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4561 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4621 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4681 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4741 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4801 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4861 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4921 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4981 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5041 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     5101 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     5161 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     5221 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     5281 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     5341 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5401 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5461 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5521 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5581 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5641 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5701 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5761 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5821 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5881 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5941 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6001 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6061 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6121 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6181 g
//
