LOCUS       sequence-174385-        9170 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 9170)
  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 9170)
  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-USP20
FEATURES             Location/Qualifiers
     source          1..9170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(63..80)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(234..822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(314..333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(993..1784,1785..1853))
                     /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     1616..1635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1854..1958)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2026..2044
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2082..2104)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2204..2223
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2417..2439
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2567..2586
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             2786..3302
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    3366..3707
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             3774..4190
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     4120..4142
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     4158..4174
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      4203..4212
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4212..4235
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4248..4274
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    4290..4314
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(7057..7081)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    7094..7661
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    7111..7663
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7255..7272)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7482..7501
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             7675..8274
                     /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(7675..7694)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8171..8191
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             8350..8864
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    9026..9042
                     /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(9050..9056,9057..9074,9075..9080))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9095..9116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
       61 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      121 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      181 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      241 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      301 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      361 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      421 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      481 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      541 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      601 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      661 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      721 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      781 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      841 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      901 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      961 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1021 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1081 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1141 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1201 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1261 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1321 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1381 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1441 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1501 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1561 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1621 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1681 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1741 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1801 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1861 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1921 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1981 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     2041 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     2101 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     2161 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     2221 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     2281 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     2341 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     2401 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggcgcaagg aatggtgcat
     2461 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     2521 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     2581 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     2641 agaggcgatt agtccaattt gttaaagaca ggatatcagt ggtccaggct ctagttttga
     2701 ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa taaaagattt
     2761 tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag
     2821 cttaagtaac gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag
     2881 atcaaggtta ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc
     2941 agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag
     3001 cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt
     3061 gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc
     3121 cgagctcaat aaaagagccc acaacccctc actcggcgcg ccagtcctcc gatagactgc
     3181 gtcgcccggg tacccgtatt cccaataaag cctcttgctg tttgcatccg aatcgtggac
     3241 tcgctgatcc ttgggagggt ctcctcagat tgattgactg cccacctcgg gggtctttca
     3301 tttggaggtt ccaccgagat ttggagaccc ctgcccaggg accaccgacc cccccgccgg
     3361 gaggtaagct ggccagcggt cgtttcgtgt ctgtctctgt ctttgtgcgt gtttgtgccg
     3421 gcatctaatg tttgcgcctg cgtctgtact agttagctaa ctagctctgt atctggcgga
     3481 cccgtggtgg aactgacgag ttctgaacac ccggccgcaa ccctgggaga cgtcccaggg
     3541 actttggggg ccgtttttgt ggcccgacct gaggaaggga gtcgatgtgg aatccgaccc
     3601 cgtcaggata tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg
     3661 aatttttgct ttcggtttgg aaccgaagcc gcgcgtcttg tctgctgcag cgctgcagca
     3721 tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagggccaga
     3781 ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag cggatcgctc
     3841 acaaccagtc ggtagatgtc aagaagagac gttgggttac cttctgctct gcagaatggc
     3901 caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc atcacccagg
     3961 ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc ccctacatcg
     4021 tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt gtacacccta
     4081 agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct cctcgttcga
     4141 ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc ggaattagat
     4201 ctgccaccat ggactacaag gatgacgatg acaagctcga tggaggatac ccatacgatg
     4261 ttccagatta cgctgctcga ggttatcaaa caagtttgta caaaaaagtt ggcatggggg
     4321 actccaggga cctttgccct caccttgact ccataggaga ggtgaccaaa gaggacttgc
     4381 tgctcaaatc taagggaacc tgtcagtcgt gtggggtcac cggaccaaac ctatgggcct
     4441 gtctgcaggt tgcctgcccc tatgttggct gcggagaatc cttcgctgac cacagcacca
     4501 ttcatgcaca ggcaaaaaag cacaacttga ccgtgaacct gaccacgttc cgactgtggt
     4561 gttacgcctg tgagaaggag gtattcctgg agcagcggct ggcagcccct ctgctgggct
     4621 cctcttccaa gttctctgaa caggactccc cgccaccctc ccaccctctg aaagctgttc
     4681 ctattgctgt ggctgatgaa ggagagtctg agtcagagga tgatgacctg aaacctcgag
     4741 gcctcacggg catgaagaac ctcgggaact cctgctacat gaacgccgcc ctgcaggccc
     4801 tgtccaattg cccgccgctg actcagttct tcttggagtg tggcggcctg gtgcgcacag
     4861 ataagaagcc agccctgtgc aagagctacc agaagctggt ctctgaggtc tggcataaga
     4921 aacggccaag ctacgtggtc cccaccagtc tgtctcatgg gatcaagttg gtcaacccaa
     4981 tgttccgagg ctatgcccag caggacaccc aagagttcct tcgctgcctg atggaccagc
     5041 tgcacgagga gctcaaggag ccggtggtgg ccacggtggc gctgacggag gctcgggact
     5101 cagattcgag tgacacggat gagaaacggg agggtgaccg gagcccatca gaagatgagt
     5161 tcttgtcctg tgactcgagc agtgaccggg gtgagggtga cgggcagggg cgtggcgggg
     5221 gcagctcgca ggccgagacg gagctgctga tcccagatga ggcgggccga gccatctctg
     5281 agaaggagcg gatgaaggac cgcaagttct cctggggcca gcagcgtaca aactcggagc
     5341 aagtggacga ggacgctgat gtggacactg ccatggctgc ccttgaccag cccgcggagg
     5401 cccagccccc gtcaccacgg tcctccagcc cctgccggac gccagagccg gacaatgatg
     5461 ctcacctacg cagctcctct cgcccctgca gccccgtcca ccaccacgag ggccatgcca
     5521 agctgtctag cagcccccct cgtgcaagcc ccgtgaggat ggcaccgtcg tacgtgctca
     5581 agaaagccca ggtattgagt gctggcagcc ggaggcggaa ggagcagcgc taccgcagcg
     5641 tcatctcaga catctttgac ggctccattc tcagcctcgt gcagtgtctc acctgtgacc
     5701 gggtatccac cacagtggaa acgttccagg acttatcact gcccattcct ggaaaggagg
     5761 acctggccaa gctccattca gccatctacc agaatgtgcc ggccaagcca ggcgcctgtg
     5821 gggacagcta tgccgcccag ggctggctgg ccttcattgt ggagtacatc cgacggtttg
     5881 tggtatcctg tacccccagc tggttttggg ggcctgtcgt caccctggaa gactgccttg
     5941 ctgccttctt tgccgctgat gagttaaagg gtgacaacat gtacagctgt gagcggtgta
     6001 agaagctgcg gaacggagtg aagtactgca aagtcctgcg gttgcccgag atcctgtgca
     6061 ttcacctaaa gcgctttcgg cacgaggtga tgtactcatt caagatcaac agccacgtct
     6121 ccttccccct cgaggggctc gacctgcgcc ccttccttgc caaggagtgc acatcccaga
     6181 tcaccaccta cgacctcctc tcggtcatct gccaccacgg cacggcaggc agtgggcact
     6241 acatcgccta ctgccagaac gtgatcaatg ggcagtggta cgagtttgat gaccagtacg
     6301 tcacagaagt ccacgagacg gtggtgcaga acgccgaggg ctacgtactc ttctacagga
     6361 agagcagcga ggaggccatg cgggagcgac agcaggtggt gtccctggcc gccatgcggg
     6421 agcccagcct gctgcggttc tacgtgtccc gcgagtggct caacaagttc aacaccttcg
     6481 cggagccagg ccccatcacc aaccagacct tcctctgctc ccacggaggc atcccgcccc
     6541 acaaatacca ctacatcgac gacctggtgg tcatcctgcc ccagaacgtc tgggagcacc
     6601 tgtacaacag attcgggggt ggccccgccg tgaaccacct gtacgtgtgc tccatctgcc
     6661 aggtggagat cgaggcactg gccaagcgca ggaggatcga gatcgacacc ttcatcaagt
     6721 tgaacaaggc cttccaggcc gaggagtcgc cgggcgtcat ctactgcatc agcatgcagt
     6781 ggttccggga gtgggaggcg ttcgtcaagg ggaaggacaa cgagcccccc gggcccattg
     6841 acaacagcag gattgcacag gtcaaaggaa gcggccatgt ccagctgaag cagggagctg
     6901 actacgggca gatttcggag gagacctgga cctacctgaa cagcctgtat ggaggtggcc
     6961 ccgagattgc catccgccag agtgtggcgc agccgctggg cccagagaac ctgcacgggg
     7021 agcagaagat cgaagccgag acgcgggccg tgtgataccc aactttcttg tacaaagtgg
     7081 ttcgataacg aattccgccc ccccccccta acgttactgg ccgaagccgc ttggaataag
     7141 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga
     7201 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
     7261 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt
     7321 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca
     7381 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc
     7441 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat
     7501 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
     7561 ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga
     7621 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatgacc
     7681 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     7741 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     7801 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     7861 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     7921 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     7981 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     8041 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     8101 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     8161 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     8221 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgatgcatc
     8281 gatgtcgacc tgcagccaag cttatcgata aaataaaaga ttttatttag tctccagaaa
     8341 aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt
     8401 tgcaaggcat ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag
     8461 agagacagca gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc
     8521 agggccaaga acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca
     8581 tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac
     8641 caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag
     8701 cccacaaccc ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt
     8761 gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg
     8821 gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatgggtaa cagtttcttg
     8881 aagttggaga acaacattct gagggtagga gtcgaatatt aagtaatcct gactcaatta
     8941 gccactgttt tgaatccaca tactccaata ctcctgaaat agttcattat ggacagcgca
     9001 gaaagagctg gggagaattg tgaaattgtt atccgctcac aattccacac aacatacgag
     9061 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     9121 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
//
