LOCUS       sequence-214537-        7104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the indicated Flag-hRIPK1 mutant. Please 
            note Flag tag is N-terminal..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7104)
  TITLE     Yuan Lab Plasmids
REFERENCE   2  (bases 1 to 7104)
  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: pcDNA3.1-hRIPK1-Flag-K599R
FEATURES             Location/Qualifiers
     source          1..7104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..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"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             691..714
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2804..2822)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2805..2822)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2842..2859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2848..3072
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3204..3792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3284..3303)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4121..4168
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4193..4212
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4400..5194)
                     /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             complement(4400..5194)
                     /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(4511..4530)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5121..5140
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5229..5586)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5243..5378
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5395..5586
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5543..5563
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5646..6437,6438..6506))
                     /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     6269..6288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6507..6611)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6638..7093
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6725..6744)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6935..6956
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gacattgatt 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 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 agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccggat cccccgggaa tcaaaacatg gattacaaag acgatgacga taaagaattc
      721 aggatgcaac cagacatgtc cttgaatgtc attaagatga aatccagtga cttcctggag
      781 agtgcagaac tggacagcgg aggctttggg aaggtgtctc tgtgtttcca cagaacccag
      841 ggactcatga tcatgaaaac agtgtacaag gggcccaact gcattgagca caacgaggcc
      901 ctcttggagg aggcgaagat gatgaacaga ctgagacaca gccgggtggt gaagctcctg
      961 ggcgtcatca tagaggaagg gaagtactcc ctggtgatgg agtacatgga gaagggcaac
     1021 ctgatgcacg tgctgaaagc cgagatgagt actccgcttt ctgtaaaagg aaggataatt
     1081 ttggaaatca ttgaaggaat gtgctactta catggaaaag gcgtgataca caaggacctg
     1141 aagcctgaaa atatccttgt tgataatgac ttccacatta agatcgcaga cctcggcctt
     1201 gcctccttta agatgtggag caaactgaat aatgaagagc acaatgagct gagggaagtg
     1261 gacggcaccg ctaagaagaa tggcggcacc ctctactaca tggcgcccga gcacctgaat
     1321 gacgtcaacg caaagcccac agagaagtcg gatgtgtaca gctttgctgt agtactctgg
     1381 gcgatatttg caaataagga gccatatgaa aatgctatct gtgagcagca gttgataatg
     1441 tgcataaaat ctgggaacag gccagatgtg gatgacatca ctgagtactg cccaagagaa
     1501 attatcagtc tcatgaagct ctgctgggaa gcgaatccgg aagctcggcc gacatttcct
     1561 ggcattgaag aaaaatttag gcctttttat ttaagtcaat tagaagaaag tgtagaagag
     1621 gacgtgaaga gtttaaagaa agagtattca aacgaaaatg cagttgtgaa gagaatgcag
     1681 tctcttcaac ttgattgtgt ggcagtacct tcaagccggt caaattcagc cacagaacag
     1741 cctggttcac tgcacagttc ccagggactt gggatgggtc ctgtggagga gtcctggttt
     1801 gctccttccc tggagcaccc acaagaagag aatgagccca gcctgcagag taaactccaa
     1861 gacgaagcca actaccatct ttatggcagc cgcatggaca ggcagacgaa acagcagccc
     1921 agacagaatg tggcttacaa cagagaggag gaaaggagac gcagggtctc ccatgaccct
     1981 tttgcacagc aaagacctta cgagaatttt cagaatacag agggaaaagg cactgcttat
     2041 tccagtgcag ccagtcatgg taatgcagtg caccagccct cagggctcac cagccaacct
     2101 caagtactgt atcagaacaa tggattatat agctcacatg gctttggaac aagaccactg
     2161 gatccaggaa cagcaggtcc cagagtttgg tacaggccaa ttccaagtca tatgcctagt
     2221 ctgcataata tcccagtgcc tgagaccaac tatctaggaa atacacccac catgccattc
     2281 agctccttgc caccaacaga tgaatctata aaatatacca tatacaatag tactggcatt
     2341 cagattggag cctacaatta tatggagatt ggtgggacga gttcatcact actagacagc
     2401 acaaatacga acttcaaaga agagccagct gctaagtacc aagctatctt tgataatacc
     2461 actagtctga cggataaaca cctggaccca atcagggaaa atctgggaaa gcactggaga
     2521 aactgtgccc gtaaactggg cttcacacag tctcagattg atgaaattga ccatgactat
     2581 gagcgagatg gactgaaaga aaaggtttac cagatgctcc aaaagtgggt gatgagggaa
     2641 ggcataaagg gagccacggt ggggaagctg gcccaggcgc tccaccagtg ttccaggatc
     2701 gaccttctga gcagcttgat ttacgtcagc cagaacctga attctgcaga tatccatcac
     2761 actggcggcc gctcgagtag atgactagtc tagagggccc tattctatag tgtcacctaa
     2821 atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2881 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2941 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     3001 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     3061 tgggctctat ggcttctgag gcggaaagaa ccagtggcgg taatacggtt atccacagaa
     3121 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3181 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3241 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3301 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3361 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3421 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3481 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3541 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3601 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3661 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3721 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3781 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3841 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3901 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaacc tgaggctatg
     3961 gcagggcctg ccgccccgac gttggctgcg agccctgggc cttcacccga acttgggggg
     4021 tggggtgggg aaaaggaaga aacgcgggcg tattggcccc aatggggtct cggtggggta
     4081 tcgacagagt gccagccctg ggaccgaacc ccgcgtttat gaacaaacga cccaacaccg
     4141 tgcgttttat tctgtctttt tattgccgtc atagcgcggg ttccttccgg tattgtctcc
     4201 ttccgtgttt cagttagcct ccccctaggg tgggcgaaga actccagcat gagatccccg
     4261 cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag
     4321 aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt
     4381 tcgaacccca gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     4441 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
     4501 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     4561 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     4621 aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgctcgcc ttgagcctgg
     4681 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     4741 gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     4801 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     4861 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     4921 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg
     4981 tggccagcca cgatagccgc gctgcctcgt cttgcagttc attcagggca ccggacaggt
     5041 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     5101 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     5161 gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt
     5221 gatcgatctt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     5281 tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc
     5341 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact
     5401 atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     5461 gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
     5521 ggggagcctg gggactttcc acaccctaac tgacacacat tccacagctg gttctttccg
     5581 cctcaggact cttccttttt caataaatca atctaaagta tatatgagta aacttggtct
     5641 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5701 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5761 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5821 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5881 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5941 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6001 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6061 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6121 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6181 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6241 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6301 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6361 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6421 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6481 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6541 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6601 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta
     6661 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     6721 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     6781 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     6841 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     6901 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     6961 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     7021 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     7081 acgcttacaa tttacgcgcg cgtt
//
