LOCUS       sequence-190618-        7768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression construct for S protein-Flag-Streptavidin 
            binding peptide (SFB)-tagged RNF8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7768)
  AUTHORS   Huen MS, Grant R, Manke I, Minn K, Yu X, Yaffe MB, Chen J
  TITLE     RNF8 transduces the DNA-damage signal via histone ubiquitylation and
            checkpoint protein assembly.
  JOURNAL   Cell. 2007 Nov 30;131(5):901-14. Epub 2007 Nov 20.
  PUBMED    18001825
REFERENCE   2  (bases 1 to 7768)
  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: "2007-11-30"; volume: "131(5)"; pages: "901-14. Epub 2007 Nov 
            20"
COMMENT     Sequence Label: pMH-SFB-RNF8
FEATURES             Location/Qualifiers
     source          1..7768
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        13..316
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        317..520
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     471..491
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             580..624
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             658..681
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             685..708
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             718..831
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     protein_bind    904..928
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2389..2413)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2596..2613)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(2675..2695)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2680..3009
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2860..2995
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3019..3618
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3019..3038)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3515..3535
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3839..4308
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4438..4455)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4609..5197)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4689..4708)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5357..6148,6149..6217))
                     /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     5980..5999
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6218..6322)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             6353..6822
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6887..7086
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             7287..7703
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     7633..7655
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     7671..7687
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
       61 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      121 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      181 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      241 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      301 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      361 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      421 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      481 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
      541 ccgctagcgc taccggactc agatctcgag gccaccatga aagaaaccgc tgctgctaaa
      601 ttcgaacgcc agcacatgga cagcggagcc ggcgcaggag ccggcgctga cgcgcctgac
      661 tacaaagacg atgacgacaa gggagattac aaggatgacg atgacaaagg cgcgtcgatg
      721 gacgagaaga ccaccggctg gcggggcggc cacgtggtgg agggcctggc cggcgagctg
      781 gagcagctgc gggccaggct ggagcaccac cctcagggcc agcgggagcc ctccggcggc
      841 tgcaagctgg gcggcgcgcc tgaattctgc agtcgacggt accgcgggcc catcgctcga
      901 gagacaagtt tgtacaaaaa agcaggctcc atgggggagc ccggcttctt cgtcacagga
      961 gaccgcgccg gtggccggag ctggtgcctg cggcgggtgg ggatgagcgc cgggtggctg
     1021 ctgctggaag atgggtgcga ggtgactgta ggacgaggat ttggtgtcac ataccaactg
     1081 gtatcaaaaa tctgccccct gatgatttct cgaaaccact gtgttttgaa gcagaatcct
     1141 gagggccaat ggacaattat ggacaacaag agtctaaatg gtgtttggct gaacagagcg
     1201 cgtctggaac ctttaagggt ctattccatt catcagggag actacatcca acttggagtg
     1261 cctctggaaa ataaggagaa tgcggagtat gaatatgaag ttactgaaga agactgggag
     1321 acaatatatc cttgtctttc cccaaagaat gaccaaatga tagaaaaaaa taaggaattg
     1381 agaactaaaa ggaaattcag tttggatgaa ttagcaggtc ctggagctga aggcccctca
     1441 aatttgaaat ccaaaataaa taaagtgtct tgtgaatctg gtcagccagt gaaatcacag
     1501 gggaaaggtg aagtggccag tacaccctct gacaatttgg atcctaagtt gactgccctt
     1561 gagccaagta agaccacagg ggctcccatt taccctggct tccccaaagt cacagaggtt
     1621 catcatgagc agaaagcctc aaactcttca gcatctcaga gaagcttaca gatgtttaag
     1681 gtgaccatgt ccaggattct gaggctcaaa atacagatgc aggaaaaaca tgaagccgtt
     1741 atgaatgtga aaaagcagac ccaaaagggg aactcaaaga aagttgtgca aatggagcag
     1801 gaacttcagg acttacagtc ccagctgtgt gcagagcagg ctcagcagca ggcaagagtg
     1861 gagcaactag agaagacttt ccaggaagag gaacagcatc ttcagggttt ggagatagcc
     1921 caaggagaaa aggacctgaa gcaacagctg gcccaggctc tgcaggagca ttgggctcta
     1981 atggaagagc taaatcgcag caagaaggac tttgaagcaa tcattcaagc caagaacaaa
     2041 gaattagagc agaccaagga agagaaggag aagatgcaag cacagaagga agaagttctt
     2101 agccacatga atgatgtgct agagaatgag ctccaatgta ttatttgttc agaatacttc
     2161 attgaggctg tcaccttgaa ctgtgcccac agtttctgct cctactgtat caatgaatgg
     2221 atgaagcgga agatagaatg ccccatttgt cggaaggaca ttaagtccaa aacgtactct
     2281 ttggttctgg acaattgcat taataagatg gtaaataatc tgagctcaga agtgaaagaa
     2341 cgacgaattg ttctcattag ggaacgaaaa gcaaagagat tgttctgaac ccagctttct
     2401 tgtacaaagt ggctcgagca gggatccgcc cctctccctc ccccccccct aacgttactg
     2461 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
     2521 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
     2581 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     2641 cgtaggaatt cgccagcaca gtggtcgacc ctgtggaatg tgtgtcagtt agggtgtgga
     2701 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2761 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2821 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2881 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2941 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3001 ttttgcaaaa agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     3061 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     3121 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     3181 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     3241 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     3301 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     3361 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     3421 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     3481 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     3541 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     3601 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca
     3661 cgaccccatg catcgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
     3721 aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg
     3781 aaaaatacat aactgagaat agagaagttc agatcaaggt caggaacaga tggaacagct
     3841 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
     3901 acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     3961 ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag
     4021 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
     4081 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
     4141 aagagcccac aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta
     4201 cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg
     4261 ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcaca tgcagcatgt
     4321 atcaaaatta atttggtttt ttttcttaag tatttacatt aaatggccat agttgcatta
     4381 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4441 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4501 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4561 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4621 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4681 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4741 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4801 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4861 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4921 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4981 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5041 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5101 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5161 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5221 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5281 aaaaaggatc ttcacctaga tccttttgcg gccgcaaatc aatctaaagt atatatgagt
     5341 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5401 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5461 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5521 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5581 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5641 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5701 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5761 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5821 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5881 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5941 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6001 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6061 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6121 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6181 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6241 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6301 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgca gcctgaatat
     6361 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
     6421 ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
     6481 gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat
     6541 cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc
     6601 aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc
     6661 ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg
     6721 tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg
     6781 tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg ctcgtccggg
     6841 atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggcaagc tggccagcaa
     6901 cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc ctgcgtcggt
     6961 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
     7021 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
     7081 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
     7141 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
     7201 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
     7261 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     7321 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     7381 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     7441 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     7501 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     7561 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     7621 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     7681 ccctcactcc ttctctaggc gccggccgga tcccagtgtg gtggtactaa tagtaatcaa
     7741 ttacggggtc attagttcat agcccata
//
