LOCUS       sequence-214533-        6244 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 6244)
  TITLE     Yuan Lab Plasmids
REFERENCE   2  (bases 1 to 6244)
  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-delN
FEATURES             Location/Qualifiers
     source          1..6244
                     /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(1944..1962)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1945..1962)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1982..1999)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1988..2212
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2344..2932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2424..2443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3261..3308
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3333..3352
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3540..4334)
                     /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(3540..4334)
                     /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(3651..3670)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4261..4280
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4369..4726)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4383..4518
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4535..4726
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     4683..4703
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(4786..5577,5578..5646))
                     /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     5409..5428
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5647..5751)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5778..6233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5865..5884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6075..6096
                     /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 cggccgacat ttcctggcat tgaagaaaaa tttaggcctt tttatttaag tcaattagaa
      781 gaaagtgtag aagaggacgt gaagagttta aagaaagagt attcaaacga aaatgcagtt
      841 gtgaagagaa tgcagtctct tcaacttgat tgtgtggcag taccttcaag ccggtcaaat
      901 tcagccacag aacagcctgg ttcactgcac agttcccagg gacttgggat gggtcctgtg
      961 gaggagtcct ggtttgctcc ttccctggag cacccacaag aagagaatga gcccagcctg
     1021 cagagtaaac tccaagacga agccaactac catctttatg gcagccgcat ggacaggcag
     1081 acgaaacagc agcccagaca gaatgtggct tacaacagag aggaggaaag gagacgcagg
     1141 gtctcccatg acccttttgc acagcaaaga ccttacgaga attttcagaa tacagaggga
     1201 aaaggcactg cttattccag tgcagccagt catggtaatg cagtgcacca gccctcaggg
     1261 ctcaccagcc aacctcaagt actgtatcag aacaatggat tatatagctc acatggcttt
     1321 ggaacaagac cactggatcc aggaacagca ggtcccagag tttggtacag gccaattcca
     1381 agtcatatgc ctagtctgca taatatccca gtgcctgaga ccaactatct aggaaataca
     1441 cccaccatgc cattcagctc cttgccacca acagatgaat ctataaaata taccatatac
     1501 aatagtactg gcattcagat tggagcctac aattatatgg agattggtgg gacgagttca
     1561 tcactactag acagcacaaa tacgaacttc aaagaagagc cagctgctaa gtaccaagct
     1621 atctttgata ataccactag tctgacggat aaacacctgg acccaatcag ggaaaatctg
     1681 ggaaagcact ggaaaaactg tgcccgtaaa ctgggcttca cacagtctca gattgatgaa
     1741 attgaccatg actatgagcg agatggactg aaagaaaagg tttaccagat gctccaaaag
     1801 tgggtgatga gggaaggcat aaagggagcc acggtgggga agctggccca ggcgctccac
     1861 cagtgttcca ggatcgacct tctgagcagc ttgatttacg tcagccagaa cctcgagtag
     1921 atgactagtc tagagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca
     1981 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2041 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2101 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2161 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2221 gcggaaagaa ccagtggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2281 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2341 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2401 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2461 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2521 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2581 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2641 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2701 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2761 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     2821 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2881 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2941 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3001 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3061 taaatcaatc taaagtatat atgagtaacc tgaggctatg gcagggcctg ccgccccgac
     3121 gttggctgcg agccctgggc cttcacccga acttgggggg tggggtgggg aaaaggaaga
     3181 aacgcgggcg tattggcccc aatggggtct cggtggggta tcgacagagt gccagccctg
     3241 ggaccgaacc ccgcgtttat gaacaaacga cccaacaccg tgcgttttat tctgtctttt
     3301 tattgccgtc atagcgcggg ttccttccgg tattgtctcc ttccgtgttt cagttagcct
     3361 ccccctaggg tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc
     3421 ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa
     3481 atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct
     3541 cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata
     3601 ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg
     3661 gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat
     3721 ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg
     3781 acgagatcct cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg
     3841 agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta
     3901 cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc
     3961 gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga
     4021 gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca
     4081 gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc
     4141 gctgcctcgt cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc
     4201 gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt
     4261 gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca
     4321 tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcgatctt tgcaaaagcc
     4381 taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggccg aggcggcctc
     4441 ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg
     4501 cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag
     4561 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc
     4621 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     4681 acaccctaac tgacacacat tccacagctg gttctttccg cctcaggact cttccttttt
     4741 caataaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4801 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4861 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4921 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4981 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5041 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5101 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5161 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5221 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5281 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5341 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5401 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5461 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5521 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5581 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5641 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5701 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5761 ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5821 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5881 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5941 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     6001 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6061 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     6121 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6181 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttacgcgcg
     6241 cgtt
//
