LOCUS       sequence-482903-        8348 bp    DNA     circular SYN 12-AUG-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8348)
  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 8348)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 12, 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-USP49
FEATURES             Location/Qualifiers
     source          1..8348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..27
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             40..66
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    82..106
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2027..2051)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2064..2631
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2081..2633
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2225..2242)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2452..2471
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2645..3244
                     /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(2645..2664)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3141..3161
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3320..3834
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3996..4012
                     /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(4020..4026,4027..4044,4045..4050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4065..4086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4203..4220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4374..4962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4454..4473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5133..5924,5925..5993))
                     /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     5756..5775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5994..6098)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6166..6184
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6222..6244)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6344..6363
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6557..6579
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6707..6726
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             6926..7442
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    7506..7847
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             7914..8330
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     8260..8282
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     8298..8314
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      join(8343..8348,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggactaca aggatgacga tgacaagctc gatggaggat acccatacga tgttccagat
       61 tacgctgctc gaggttatca aacaagtttg tacaaaaaag ttggcatgga tagatgcaaa
      121 catgtagggc ggttacggct cgcccaggac cactccatcc tgaaccctca gaagtggtgc
      181 tgcttagagt gtgccaccac cgagtccgtg tgggcctgcc tcaagtgctc ccacgtggcc
      241 tgcggccgct atattgagga ccacgccctg aaacactttg aggagacggg acacccgcta
      301 gccatggaag tccgggatct ctacgtgttc tgttacctgt gcaaggacta cgtgctcaat
      361 gataacccag agggggacct gaagctgcta agaagctccc tcctggcggt ccggggccag
      421 aaacaggaca cgccggtgag acgtgggcgg acgctgcggt ccatggcttc gggtgaggac
      481 gtggtcctgc cgcagcgcgc tcctcaggga cagccgcaga tgctcacggc tctgtggtac
      541 cggcgtcagc gcctgctggc caggacgctg cggctgtggt tcgagaagag ctcccggggc
      601 caggcgaagc tggagcagcg gcggcaggag gaggccctgg agcgcaagaa ggaggaggcg
      661 cggaggcggc ggcgcgaggt gaaacggcgg ctgctggagg agctggccag cacccctccg
      721 cgcaagagtg cacggctgct cctgcacacg ccccgcgacg cgggcccggc tgcctcgcgc
      781 cccgccgccc tccctacctc acgcagagtg cccgccgcca cactcaagct gcgtcgccag
      841 ccggccatgg ccccaggcgt cacgggcctg cgcaacctgg gcaacacctg ctacatgaac
      901 tccatcctcc aggtgctcag ccacctccag aagttccgag aatgtttcct caaccttgac
      961 ccttccaaaa cggaacatct gtttcccaaa gccaccaacg ggaagactca gctttctggc
     1021 aagccaacca acagctcggc cacggagctg tccttgagaa atgacagggc cgaggcatgc
     1081 gagcgggagg gcttctgctg gaacggcagg gcctccatta gtcggagtct ggagctcatc
     1141 cagaacaagg agccgagttc aaagcacatt tccctctgcc gtgaactgca caccctcttc
     1201 cgagtcatgt ggtccgggaa gtgggcccta gtgtcgccct tcgccatgct gcactcagtg
     1261 tggagcctga tccctgcctt ccgcggctac gaccaacagg acgcgcagga atttctctgc
     1321 gagctgctgc acaaggtgca gcaggaactc gagtctgagg gcaccacacg ccggatcctc
     1381 atccccttct cccagaggaa gctcaccaaa caggtcttaa aggtggtgaa taccatattt
     1441 catgggcagc tgctcagtca ggtcacatgt atatcatgca attacaaatc caataccatt
     1501 gagccctttt gggacctatc cctggaattc cctgaacgct atcactgcat agaaaagggg
     1561 tttgtccctt tgaatcaaac agagtgcttg ctcactgaga tgctggccaa attcacagag
     1621 acagaggccc tggaagggag aatctacgct tgtgaccagt gtaacagcaa acgacgaaaa
     1681 tccaatccca aaccccttgt tctgagtgaa gctagaaagc agttaatgat ctacagacta
     1741 cctcaggttc tccggctgca ccttaaaaga ttcaggtggt ctggccgtaa tcatcgagag
     1801 aagattgggg tccatgtcgt ctttgaccag gtattaacca tggaacctta ctgctgcagg
     1861 gacatgctct cctctcttga caaagagacc tttgcctatg atctctccgc agtggtcatg
     1921 catcacggga aagggtttgg ctcaggacac tacacagcct attgctacaa cacagaggga
     1981 ggtgcgtgcg ctttactctg tggggtgggg gacacggaaa ggggttgccc aactttcttg
     2041 tacaaagtgg ttcgataacg aattccgccc ccccccccta acgttactgg ccgaagccgc
     2101 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     2161 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     2221 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     2281 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     2341 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     2401 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     2461 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     2521 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     2581 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac
     2641 aaccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     2701 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     2761 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     2821 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     2881 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     2941 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     3001 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     3061 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     3121 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     3181 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     3241 ctgatgcatc gatgtcgacc tgcagccaag cttatcgata aaataaaaga ttttatttag
     3301 tctccagaaa aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt
     3361 aacgccattt tgcaaggcat ggaaaataca taactgagaa tagagaagtt cagatcaagg
     3421 ttaggaacag agagacagca gaatatgggc caaacaggat atctgtggta agcagttcct
     3481 gccccggctc agggccaaga acagatggtc cccagatgcg gtcccgccct cagcagtttc
     3541 tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat
     3601 ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc
     3661 aataaaagag cccacaaccc ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc
     3721 gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt
     3781 ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatgggtaa
     3841 cagtttcttg aagttggaga acaacattct gagggtagga gtcgaatatt aagtaatcct
     3901 gactcaatta gccactgttt tgaatccaca tactccaata ctcctgaaat agttcattat
     3961 ggacagcgca gaaagagctg gggagaattg tgaaattgtt atccgctcac aattccacac
     4021 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4081 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4141 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4201 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4261 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4321 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4381 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4441 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4501 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4561 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4621 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4681 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4741 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4801 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4861 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4921 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4981 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5041 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5101 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5161 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5221 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5281 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5341 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5401 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5461 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5521 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5581 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5641 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5701 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5761 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5821 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5881 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5941 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6001 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6061 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6121 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     6181 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     6241 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     6301 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     6361 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     6421 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     6481 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     6541 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggcgcaagg aatggtgcat
     6601 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     6661 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     6721 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     6781 agaggcgatt agtccaattt gttaaagaca ggatatcagt ggtccaggct ctagttttga
     6841 ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa taaaagattt
     6901 tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag
     6961 cttaagtaac gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag
     7021 atcaaggtta ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc
     7081 agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag
     7141 cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt
     7201 gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc
     7261 cgagctcaat aaaagagccc acaacccctc actcggcgcg ccagtcctcc gatagactgc
     7321 gtcgcccggg tacccgtatt cccaataaag cctcttgctg tttgcatccg aatcgtggac
     7381 tcgctgatcc ttgggagggt ctcctcagat tgattgactg cccacctcgg gggtctttca
     7441 tttggaggtt ccaccgagat ttggagaccc ctgcccaggg accaccgacc cccccgccgg
     7501 gaggtaagct ggccagcggt cgtttcgtgt ctgtctctgt ctttgtgcgt gtttgtgccg
     7561 gcatctaatg tttgcgcctg cgtctgtact agttagctaa ctagctctgt atctggcgga
     7621 cccgtggtgg aactgacgag ttctgaacac ccggccgcaa ccctgggaga cgtcccaggg
     7681 actttggggg ccgtttttgt ggcccgacct gaggaaggga gtcgatgtgg aatccgaccc
     7741 cgtcaggata tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg
     7801 aatttttgct ttcggtttgg aaccgaagcc gcgcgtcttg tctgctgcag cgctgcagca
     7861 tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagggccaga
     7921 ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag cggatcgctc
     7981 acaaccagtc ggtagatgtc aagaagagac gttgggttac cttctgctct gcagaatggc
     8041 caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc atcacccagg
     8101 ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc ccctacatcg
     8161 tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt gtacacccta
     8221 agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct cctcgttcga
     8281 ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc ggaattagat
     8341 ctgccacc
//
