LOCUS       sequence-214530-        5923 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 5923)
  TITLE     Yuan Lab Plasmids
REFERENCE   2  (bases 1 to 5923)
  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-delC
FEATURES             Location/Qualifiers
     source          1..5923
                     /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(1623..1641)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1624..1641)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1661..1678)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1667..1891
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2023..2611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2103..2122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2940..2987
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3012..3031
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3219..4013)
                     /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(3219..4013)
                     /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(3330..3349)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3940..3959
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4048..4405)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4062..4197
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4214..4405
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     4362..4382
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(4465..5256,5257..5325))
                     /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     5088..5107
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5326..5430)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5457..5912
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5544..5563)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5754..5775
                     /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 ctcgagtaga tgactagtct agagggccct
     1621 attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt
     1681 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1741 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1801 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     1861 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagtggcggt
     1921 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1981 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2041 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2101 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2161 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2221 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2281 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2341 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2401 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2461 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2521 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2581 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2641 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2701 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2761 tgagtaacct gaggctatgg cagggcctgc cgccccgacg ttggctgcga gccctgggcc
     2821 ttcacccgaa cttggggggt ggggtgggga aaaggaagaa acgcgggcgt attggcccca
     2881 atggggtctc ggtggggtat cgacagagtg ccagccctgg gaccgaaccc cgcgtttatg
     2941 aacaaacgac ccaacaccgt gcgttttatt ctgtcttttt attgccgtca tagcgcgggt
     3001 tccttccggt attgtctcct tccgtgtttc agttagcctc cccctagggt gggcgaagaa
     3061 ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc
     3121 gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg
     3181 cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg
     3241 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
     3301 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
     3361 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
     3421 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc
     3481 atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
     3541 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
     3601 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
     3661 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
     3721 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
     3781 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ttgcagttca
     3841 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
     3901 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
     3961 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac
     4021 gatcctcatc ctgtctcttg atcgatcttt gcaaaagcct aggcctccaa aaaagcctcc
     4081 tcactacttc tggaatagct cagaggccga ggcggcctcg gcctctgcat aaataaaaaa
     4141 aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg
     4201 cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct
     4261 gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct
     4321 ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt
     4381 ccacagctgg ttctttccgc ctcaggactc ttcctttttc aataaatcaa tctaaagtat
     4441 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4501 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4561 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4621 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4681 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4741 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4801 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4861 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4921 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4981 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5041 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5101 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5161 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5221 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5281 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5341 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5401 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc
     5461 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5521 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5581 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5641 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     5701 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5761 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5821 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5881 gaattttaac aaaatattaa cgcttacaat ttacgcgcgc gtt
//
