LOCUS       sequence-192444-        8438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression construct for S protein-Flag-Streptavidin 
            binding peptide (SFB)-tagged RNF169.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8438)
  AUTHORS   Chen J, Feng W, Jiang J, Deng Y, Huen MS
  TITLE     Ring finger protein RNF169 antagonizes the ubiquitin-dependent 
            signaling cascade at sites of DNA damage.
  JOURNAL   J Biol Chem. 2012 Aug 10;287(33):27715-22. doi: 
            10.1074/jbc.M112.373530. Epub 2012 Jun 25.
  PUBMED    22733822
REFERENCE   2  (bases 1 to 8438)
  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"; doi: 
            "10.1074/jbc.M112.373530"; journalName: "J Biol Chem"; date: 
            "2012-08-10- 10"; volume: "287"; issue: "33"; pages: "27715-22"
COMMENT     Sequence Label: pMH-SFB-RNF169
FEATURES             Location/Qualifiers
     source          1..8438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        11..314
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        315..518
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     469..489
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             578..622
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             656..679
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             683..706
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             716..829
                     /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    902..926
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3057..3081)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3264..3281)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(3343..3363)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3348..3677
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3528..3663
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3687..4286
                     /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(3687..3706)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4183..4203
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4507..4976
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5106..5123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5277..5865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5357..5376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6025..6816,6817..6885))
                     /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     6648..6667
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6886..6990)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             7021..7490
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    7555..7754
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             7955..8371
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     8301..8323
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     8339..8355
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
       61 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      121 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      181 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      241 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      301 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      361 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      421 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      481 gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc
      541 gctagcgcta ccggactcag atctcgaggc caccatgaaa gaaaccgctg ctgctaaatt
      601 cgaacgccag cacatggaca gcggagccgg cgcaggagcc ggcgctgacg cgcctgacta
      661 caaagacgat gacgacaagg gagattacaa ggatgacgat gacaaaggcg cgtcgatgga
      721 cgagaagacc accggctggc ggggcggcca cgtggtggag ggcctggccg gcgagctgga
      781 gcagctgcgg gccaggctgg agcaccaccc tcagggccag cgggagccct ccggcggctg
      841 caagctgggc ggcgcgcctg aattctgcag tcgacggtac cgcgggccca tcgctcgaga
      901 gacaagtttg tacaaaaaag caggcttcat ggcggctgca ggtccgagta ctcgggcctc
      961 ttccgcggcg gcagcagccg ctctgagtcg gcggggccgg cggggccgct gtgacgagac
     1021 ggcggcagct aagactgggg ccccaggccc ggcttctgga ccttcgctgt tggtgttgtc
     1081 gccgccgttg ctgcagccgc cgctgccgcc gcggccggag gaatcgggct gcgccgggtg
     1141 cctggagccc cccggagaag cagcggccct gccgtgcggc cactcgcttt gccgaggctg
     1201 cgcccaacgc gccgccgacg cggcgggccc gggttgccct cgctgccgcg cccgcggccc
     1261 aggctgggcc cgccgtcggg cccgcgacga cggccaggcc gactcagagg tgctgggcga
     1321 gtgcgcccgc cgcagccaac ccgagcgctg ccgcccgcgc cgggacgggg gcgcggctgc
     1381 cgcggggccc aggccagagc aggagccgcg tgccgcgcct gcggagccag actttatatt
     1441 cagagcacca atcaaattaa gcaagcctgg ggaacttcgt gaggaatatg aaagcttgag
     1501 aaagctgaga gaagaaaagt tacaagagga aaaaccctct gaagatcaaa tccacaagct
     1561 gttaccagag gatacagaaa cagggaaaag gaaaatggat gaacagaaaa aaagagatga
     1621 accattagta ctgaaaacaa atctggaacg ttgtcctgca cgtctctcag attcagagaa
     1681 tgaagaacct tctcgaggcc agatgacaca gacacatcgc tcggcatttg tttccaagaa
     1741 caactcctac tccttagctt tcctggcagg gaagctaaac tccaaggtgg aaaggagtca
     1801 gagctgtagt gacacagccc aggaaagagc gaagagcaga gtcagagcag ttccaggcaa
     1861 caaagccaag gtcacaacta tgactccagc ctccaacccc atcattggtg tcctcttgtc
     1921 aactcaaaac aaccgctgcg tctcggcccc tgacttaacc atcgaaaagc gtctaccctt
     1981 cagctccctt tcatccttgg cttccctgca taagccagag cgttctgtca gccctgagag
     2041 caatgacagc atctccgaag aactaaacca tttcaagccc attgtctgct caccatgtac
     2101 tcctcccaag agactccctg atggccgtgt gctaagtcct ctcatcatca aatcaactcc
     2161 acgcaaccta aacagaagcc tgcagaagca gacttcttat gaggccagtc cacggatcct
     2221 caaaaagtgg gaacagatct ttcaggagcg gcagatcaaa aagacccttt caaaagccac
     2281 tcttacctct ctggctcctg aaatggggga agagttacta ggctctgaag gtatccattc
     2341 tagcaaggag aagccacttg tggctgtaaa tacaagatta tctggtgggc aggtcctctc
     2401 tgagtatact ggacccacct ctgctgatct tgatcatttc ccctctgtta gccaaacaaa
     2461 agcagaacag gacagtgata ataaaagtag cactgagatc ccactggaaa cctgctgttc
     2521 ctcagaactc aaagggggag gcagtgggac ttctttggag agggagcagt ttgaggggtt
     2581 agggtcaact ccagatgcca agttagacaa aacctgtata agcagagcca tgaaaatcac
     2641 cacagttaat tcagtgctac cccaaaacag tgttttgggt ggagtcctca aaacaaagca
     2701 acaattgaag acattaaatc attttgatct gactaatggt gttctagttg agagcctaag
     2761 tgaagagcca cttccttctt tgcgtcgagg ccggaaaaga cactgcaaga ccaagcactt
     2821 agaacaaaat ggctccctta aaaaactgcg acaaaccagt ggggaggtgg gtctggcccc
     2881 aacagaccca gtcctgcgag agatggagca gaagcttcag caagaggaag aagaccgaca
     2941 gttggctctg cagttgcagc gcatgttcga caatgagagg cggactgtga gccggcgaaa
     3001 aggaagtgtg gatcagtatc tcctacggtc cagcaacatg gccggggcca agtagaaccc
     3061 agctttcttg tacaaagtgg ctcgagcagg gatccgcccc tctccctccc ccccccctaa
     3121 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
     3181 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac
     3241 gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt
     3301 gaaggaagcg taggaattcg ccagcacagt ggtcgaccct gtggaatgtg tgtcagttag
     3361 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3421 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3481 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3541 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3601 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3661 gcctaggctt ttgcaaaaag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca
     3721 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     3781 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     3841 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     3901 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     3961 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     4021 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     4081 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     4141 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     4201 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     4261 gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa
     4321 ggagcgcacg accccatgca tcgataaaat aaaagatttt atttagtctc cagaaaaagg
     4381 ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca
     4441 aggcatggaa aaatacataa ctgagaatag agaagttcag atcaaggtca ggaacagatg
     4501 gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag
     4561 ggccaagaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt
     4621 cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtccagc cctcagcagt
     4681 ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct
     4741 tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag
     4801 ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg
     4861 cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct
     4921 gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcacatg
     4981 cagcatgtat caaaattaat ttggtttttt ttcttaagta tttacattaa atggccatag
     5041 ttgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5101 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5161 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5221 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5281 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5341 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5401 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5461 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5521 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5581 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5641 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5701 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5761 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5821 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5881 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5941 agattatcaa aaaggatctt cacctagatc cttttgcggc cgcaaatcaa tctaaagtat
     6001 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6061 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6121 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6181 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6241 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6301 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6361 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6421 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6481 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6541 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6601 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6661 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6721 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6781 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6841 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6901 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6961 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgcagc
     7021 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
     7081 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     7141 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
     7201 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
     7261 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
     7321 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
     7381 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
     7441 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
     7501 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggcaagctg
     7561 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgactgatt ttatgcgcct
     7621 gcgtcggtac tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga
     7681 gttctgaaca cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg
     7741 tggcccgacc tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct
     7801 ggtaggagac gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg
     7861 gaaccgaagc cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg
     7921 tctgactgtg tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag
     7981 tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt
     8041 caagaagaga cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg
     8101 gccgcgagac ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc
     8161 acctggcccg catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc
     8221 ttttgacccc cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc
     8281 tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct
     8341 ttatccagcc ctcactcctt ctctaggcgc cggccggatc ccagtgtggt ggtactaata
     8401 gtaatcaatt acggggtcat tagttcatag cccatata
//
