LOCUS       sequence-214535-        7074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of 3xFlag-hRIPK3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7074)
  TITLE     Yuan Lab Plasmids
REFERENCE   2  (bases 1 to 7074)
  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"
COMMENT     Sequence Label: pCINEO-3xFlag-hRIPK3
FEATURES             Location/Qualifiers
     source          1..7074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..592
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     543..563
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     589..613
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          753..885
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     868..887
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        930..948
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             957..1022
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2641..2762
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2652..2671)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2706..2725
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2948..3403
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3035..3054)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3245..3266
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3520..3877
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3520..3711
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3543..3563)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3728..3863
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3928..4722
                     /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(3982..4001)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4592..4611
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4786..4834
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4875..4894)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4994..5016
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5054..5072)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5140..5244
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5245..5313,5314..6105)
                     /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     complement(5463..5482)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6276..6864
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6765..6784
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggcattgatt attgactagt 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 ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctgcgatcgc ccgccccgtt
      541 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt
      601 gaaccgtcag atcactagaa gctttattgc ggtagtttat cacagttaaa ttgctaacgc
      661 agtcagtgct tctgacacaa cagtctcgaa cttaagctgc agtgactctc ttaaggtagc
      721 cttgcagaag ttggtcgtga ggcactgggc aggtaagtat caaggttaca agacaggttt
      781 aaggagacca atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg
      841 cacctattgg tcttactgac atccactttg cctttctctc cacaggtgtc cactcccagt
      901 tcaattacag ctcttaaggc tagagtactt aatacgactc actataggct agcatggact
      961 acaaagacca tgacggtgat tataaagatc atgatatcga ttacaaggat gacgatgaca
     1021 agcttggatc cctcgagatg tcgtgcgtca agttatggcc cagcggtgcc cccgccccct
     1081 tggtgtccat cgaggaactg gagaaccagg agctcgtcgg caaaggcggg ttcggcacag
     1141 tgttccgggc gcaacatagg aagtggggct acgatgtggc ggtcaagatc gtaaactcga
     1201 aggcgatatc cagggaggtc aaggccatgg caagtctgga taacgaattc gtgctgcgcc
     1261 tagaaggggt tatcgagaag gtgaactggg accaagatcc caagccggct ctggtgacta
     1321 aattcatgga gaacggctcc ttgtcggggc tgctgcagtc ccagtgccct cggccctggc
     1381 cgctcctttg ccgcctgctg aaagaagtgg tgcttgggat gttttacctg cacgaccaga
     1441 acccggtgct cctgcaccgg gacctcaagc catccaacgt cctgctggac ccagagctgc
     1501 acgtcaagct ggcagatttt ggcctgtcca catttcaggg aggctcacag tcagggacag
     1561 ggtccgggga gccagggggc accctgggct acttggcccc agaactgttt gttaacgtaa
     1621 accggaaggc ctccacagcc agtgacgtct acagcttcgg gatcctaatg tgggcagtgc
     1681 ttgctggaag agaagttgag ttgccaaccg aaccatcact cgtgtacgaa gcagtgtgca
     1741 acaggcagaa ccggccttca ttggctgagc tgccccaagc cgggcctgag actcccggct
     1801 tagaaggact gaaggagcta atgcagctct gctggagcag tgagcccaag gacagaccct
     1861 ccttccagga atgcctacca aaaactgatg aagtcttcca gatggtggag aacaatatga
     1921 atgctgctgt ctccacggta aaggatttcc tgtctcagct caggagcagc aataggagat
     1981 tttctatccc agagtcaggc caaggaggga cagaaatgga tggctttagg agaaccatag
     2041 aaaaccagca ctctcgtaat gatgtcatgg tttctgagtg gctaaacaaa ctgaatctag
     2101 aggagcctcc cagctctgtt cctaaaaaat gcccgagcct taccaagagg agcagggcac
     2161 aagaggagca ggttccacaa gcctggacag caggcacatc ttcagattcg atggcccaac
     2221 ctccccagac tccagagacc tcaactttca gaaaccagat gcccagccct acctcaactg
     2281 gaacaccaag tcctggaccc cgagggaatc agggggctga gagacaaggc atgaactggt
     2341 cctgcaggac cccggagcca aatccagtaa cagggcgacc gctcgttaac atatacaact
     2401 gctctggggt gcaagttgga gacaacaact acttgactat gcaacagaca actgccttgc
     2461 ccacatgggg cttggcacct tcgggcaagg ggaggggctt gcagcacccc ccaccagtag
     2521 gttcgcaaga aggccctaaa gatcctgaag cctggagcag gccacagggt tggtataatc
     2581 atagcgggaa ataagcggcc gcttcccttt agtgagggtt aatgcttcga gcagacatga
     2641 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     2701 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     2761 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt
     2821 tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatccg ataaggatcg atccgggctg
     2881 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     2941 cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     3001 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     3061 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3121 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3181 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3241 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3301 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3361 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc
     3421 tccttacgca tctgtgcggt atttcacacc gcatacgcgg atctgcgcag caccatggcc
     3481 tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct
     3541 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3601 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3661 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3721 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3781 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3841 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag cttgattctt ctgacacaac
     3901 agtctcgaac ttaaggctag agccaccatg attgaacaag atggattgca cgcaggttct
     3961 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4021 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4081 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4141 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4201 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4261 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4321 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4381 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4441 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4501 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4561 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4621 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4681 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4741 aaatgaccga ccaagcgacg cccaacctgc catcacgatg gccgcaataa aatatcttta
     4801 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagcgataag gattcgcgta
     4861 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     4921 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4981 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5041 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5101 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5161 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5221 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5281 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5341 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5401 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5461 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5521 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5581 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5641 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5701 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5761 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5821 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5881 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5941 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6001 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6061 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6121 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6181 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6241 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6301 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6361 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6421 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6481 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6541 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6601 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6661 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6721 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6781 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6841 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6901 ttttgctggc cttttgctca catggctcga cagatcttca atattggcca ttagccatat
     6961 tattcattgg ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc
     7021 tatatcataa tatgtacatt tatattggct catgtccaat atgaccgcca tgtt
//
