LOCUS       sequence-232077-        8530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8530)
  AUTHORS   Sowa ME, Bennett EJ, Gygi SP, Harper JW
  TITLE     Defining the human deubiquitinating enzyme interaction landscape.
  JOURNAL   Cell. 2009 Jul 23. 138(2):389-403.
  PUBMED    19615732
REFERENCE   2  (bases 1 to 8530)
  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. 2009 
            Jul 23. 138(2):389-403."
COMMENT     Sequence Label: Flag-HA-DUB3
FEATURES             Location/Qualifiers
     source          1..8530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             173..689
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    753..1094
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1161..1577
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1507..1529
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     enhancer        1592..1971
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1972..2175
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2126..2146
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2177..2195
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2198..2216
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2226..2250
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2323..2332
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2332..2355
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2368..2394
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    2410..2434
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4025..4049)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        4068..4567
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4103..4126)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4500..4519
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4588..5187
                     /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(4588..4607)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5084..5104
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    5773..5789
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5797..5803,5804..5821,5822..5827))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5842..5863
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5980..5997)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6151..6739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6231..6250)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6910..7701,7702..7770))
                     /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     7533..7552
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7771..7875)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7943..7961
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7999..8021)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8121..8140
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8334..8356
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8484..8503
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgatgccggc cacgatgcgt ccggcgtaga ggcgattagt ccaatttgtt aaagacagga
       61 tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag cctatagagt
      121 acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag
      181 accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa
      241 tacataactg agaatagaga agttcagatc aaggttagga acagagagac agcagaatat
      301 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
      361 ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat gtttccaggg
      421 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
      481 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
      541 cggcgcgcca gtcctccgat agactgcgtc gcccgggtac ccgtattccc aataaagcct
      601 cttgctgttt gcatccgaat cgtggactcg ctgatccttg ggagggtctc ctcagattga
      661 ttgactgccc acctcggggg tctttcattt ggaggttcca ccgagatttg gagacccctg
      721 cccagggacc accgaccccc ccgccgggag gtaagctggc cagcggtcgt ttcgtgtctg
      781 tctctgtctt tgtgcgtgtt tgtgccggca tctaatgttt gcgcctgcgt ctgtactagt
      841 tagctaacta gctctgtatc tggcggaccc gtggtggaac tgacgagttc tgaacacccg
      901 gccgcaaccc tgggagacgt cccagggact ttgggggccg tttttgtggc ccgacctgag
      961 gaagggagtc gatgtggaat ccgaccccgt caggatatgt ggttctggta ggagacgaga
     1021 acctaaaaca gttcccgcct ccgtctgaat ttttgctttc ggtttggaac cgaagccgcg
     1081 cgtcttgtct gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc
     1141 tgtatttgtc tgaaaattag ggccagactg ttaccactcc cttaagtttg accttaggtc
     1201 actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt
     1261 gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca
     1321 cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg
     1381 gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc
     1441 cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt
     1501 ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca
     1561 ctccttctct aggcgccgga attagatcct tgacattgat tattgactag ttattaatag
     1621 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     1681 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     1741 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     1801 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     1861 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     1921 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     1981 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     2041 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     2101 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     2161 tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt
     2221 cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
     2281 ctccatagaa gacaccggga ccgatccagc ctccggggat ctgccaccat ggactacaag
     2341 gatgacgatg acaagctcga tggaggatac ccatacgatg ttccagatta cgctgctcga
     2401 ggttatcaaa caagtttgta caaaaaagtt ggcatggagg acgactcact ctacttggga
     2461 ggtgagtggc agttcaacca cttttcaaaa ctcacatctt ctcggcccga tgcagctttt
     2521 gctgaaatcc agcggacttc tctccctgag aagtcaccac tctcatgtga gacccgtgtc
     2581 gacctctgtg atgatttggc tcctgtggca agacagcttg ctcccaggga gaagcttcct
     2641 ctgagtagca ggagacctgc tgcggtgggg gctgggctcc agaatatggg aaatacctgc
     2701 tacgtgaacg cttccttgca gtgcctgaca tacacaccgc cccttgccaa ctacatgctg
     2761 tcccgggagc actctcaaac gtgtcatcgt cacaagggct gcatgctctg tactatgcaa
     2821 gctcacatca cacgggccct ccacaatcct ggccacgtca tccagccctc acaggcattg
     2881 gctgctggct tccatagagg caagcaggaa gatgcccatg aatttctcat gttcactgtg
     2941 gatgccatga aaaaggcatg ccttcccggg cacaagcagg tagatcatca ctctaaggac
     3001 accaccctca tccaccaaat atttggaggc tactggagat ctcaaatcaa gtgtctccac
     3061 tgccacggca tttcagacac ttttgaccct tacctggaca tcgccctgga tatccaggca
     3121 gctcagagtg tccagcaagc tttggaacag ttggtgaagc ccgaagaact caatggagag
     3181 aatgcctatc attgtggtgt ttgtctccag agggcgccgg cctccaagac gttaacttta
     3241 cacacctctg ccaaggtcct catccttgta ttgaagagat tctccgatgt cacaggcaac
     3301 aagattgcca agaatgtgca atatcctgag tgccttgaca tgcagccata catgtctcag
     3361 cagaacacag gacctcttgt ctatgtcctc tatgctgtgc tggtccacgc tgggtggagt
     3421 tgtcacaacg gacattactt ctcttatgtc aaagctcaag aaggccagtg gtataaaatg
     3481 gatgatgccg aggtcaccgc ctctagcatc acttctgtcc tgagtcaaca ggcctacgtc
     3541 ctcttttaca tccagaagag tgaatgggaa agacacagtg agagtgtgtc aagaggcagg
     3601 gaaccaagag cccttggcgc agaagacaca gacaggcgag caacgcaagg agagctcaag
     3661 agagaccacc cctgactcca ggcccccgag ttggacgagc acttggtgga aagagccact
     3721 caggaaagca ccttagacca ctggaaattc cttcaagagc aaaacaaaac gaagcctgag
     3781 ttcaacgtca gaaaagtcga aggtaccctg cctcccgacg tacttgtgat tcatcaatca
     3841 aaatacaagt gtgggatgaa gaaccatcat cctgaacagc aaagctccct gctaaacctc
     3901 tcttcgacga ccccgacaca tcaggagtcc atgaacactg gcacactcgc ttccctgcga
     3961 gggagggcca ggagatccaa agggaagaac aaacacagca agagggctct gcttgtgtgc
     4021 cagtgaccaa ctttcttgta caaagtggtt cgataacgaa ttctaccggg taggggaggc
     4081 gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta
     4141 cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc
     4201 gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc
     4261 cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct
     4321 cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca
     4381 atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg
     4441 cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc
     4501 attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg
     4561 cctttcgacc tgcagcccaa gcttaccatg accgagtaca agcccacggt gcgcctcgcc
     4621 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     4681 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     4741 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     4801 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     4861 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     4921 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     4981 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     5041 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     5101 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     5161 tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa
     5221 aggagcgcac gaccccatgc atcgataaaa taaaagattt tatttagtct ccagaaaaag
     5281 gggggaatga aagaccccac ctgtaggttt ggcaagctag agaaccatca gatgtttcca
     5341 gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct
     5401 tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc
     5461 actcggcgcg ccagtcctcc gatagactgc gtcgcccggg tacccgtgta tccaataaac
     5521 cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt
     5581 gattgactac ccgtcagcgg gggtctttca tgggtaacag tttcttgaag ttggagaaca
     5641 acattctgag ggtaggagtc gaatattaag taatcctgac tcaattagcc actgttttga
     5701 atccacatac tccaatactc ctgaaatagt tcattatgga cagcgcagaa agagctgggg
     5761 agaattgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5821 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5881 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5941 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6001 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6061 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6121 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6181 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6241 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6301 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6361 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6421 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6481 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6541 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6601 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6661 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6721 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6781 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6841 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6901 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6961 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7021 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7081 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7141 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7201 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7261 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7321 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7381 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7441 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7501 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7561 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7621 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7681 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7741 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7801 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7861 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     7921 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     7981 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     8041 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     8101 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     8161 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
     8221 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     8281 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     8341 acgacgttgt aaaacgacgg cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     8401 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     8461 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     8521 gtggcgccgg
//
