LOCUS       sequence-223204-        7544 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 7544)
  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 7544)
  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-USP18
FEATURES             Location/Qualifiers
     source          1..7544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             177..693
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    757..1098
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1165..1581
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1511..1533
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1549..1565
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1594..1603
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1603..1626
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1639..1665
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    1681..1705
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2822..2846)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2859..3426
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2876..3428
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3020..3037)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3247..3266
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3440..4039
                     /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(3440..3459)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3936..3956
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4115..4629
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4791..4807
                     /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(4815..4821,4822..4839,4840..4845))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4860..4881
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4998..5015)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5169..5757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5249..5268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5928..6719,6720..6788))
                     /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     6551..6570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6789..6893)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6961..6979
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7017..7039)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7139..7158
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7352..7374
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7502..7521
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggcgat tagtccaatt tgttaaagac
       61 aggatatcag tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata
      121 gagtacgagc catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
      181 aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg
      241 aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag agacagcaga
      301 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
      361 agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc
      421 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc
      481 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct
      541 cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtat tcccaataaa
      601 gcctcttgct gtttgcatcc gaatcgtgga ctcgctgatc cttgggaggg tctcctcaga
      661 ttgattgact gcccacctcg ggggtctttc atttggaggt tccaccgaga tttggagacc
      721 cctgcccagg gaccaccgac ccccccgccg ggaggtaagc tggccagcgg tcgtttcgtg
      781 tctgtctctg tctttgtgcg tgtttgtgcc ggcatctaat gtttgcgcct gcgtctgtac
      841 tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga gttctgaaca
      901 cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg tggcccgacc
      961 tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct ggtaggagac
     1021 gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg gaaccgaagc
     1081 cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg tctgactgtg
     1141 tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta
     1201 ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga
     1261 cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac
     1321 ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc acctggcccg
     1381 catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc
     1441 cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc
     1501 ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc
     1561 ctcactcctt ctctaggcgc cggaattaga tctgccacca tggactacaa ggatgacgat
     1621 gacaagctcg atggaggata cccatacgat gttccagatt acgctgctcg aggttatcaa
     1681 acaagtttgt acaaaaaagt tggcatgagc aaggcgtttg ggctcctgag gcaaatctgt
     1741 cagtccatcc tggctgagtc ctcgcagtcc ccggcagatc ttgaagaaaa gaaggaagaa
     1801 gacagcaaca tgaagagaga gcagcccaga gagcgtccca gggcctggga ctaccctcat
     1861 ggcctggttg gtttacacaa cattggacag acctgctgcc ttaactcctt gattcaggtg
     1921 ttcgtaatga atgtggactt caccaggata ttgaagagga tcacggtgcc caggggagct
     1981 gacgagcaga ggagaagcgt ccctttccag atgcttctgc tgctggagaa gatgcaggac
     2041 agccggcaga aagcagtgcg gcccctggag ctggcctact gcctgcagaa gtgcaacgtg
     2101 cccttgtttg tccaacatga tgctgcccaa ctgtacctca aactctggaa cctgattaag
     2161 gaccagatca ctgatgtgca cttggtggag agactgcagg ccctgtatac gatccgggtg
     2221 aaggactcct tgatttgcgt tgactgtgcc atggagagta gcagaaacag cagcatgctc
     2281 accctcccac tttctctttt tgatgtggac tcaaagcccc tgaagacact ggaggacgcc
     2341 ctgcactgct tcttccagcc cagggagtta tcaagcaaaa gcaagtgctt ctgtgagaac
     2401 tgtgggaaga agacccgtgg gaaacaggtc ttgaagctga cccatttgcc ccagaccctg
     2461 acaatccacc tcatgcgatt ctccatcagg aattcacaga cgagaaagat ctgccactcc
     2521 ctgtacttcc cccagagctt ggatttcagc cagatccttc caatgaagcg agagtcttgt
     2581 gatgctgagg agcagtctgg agggcagtat gagctttttg ctgtgattgc gcacgtggga
     2641 atggcagact ccggtcatta ctgtgtctac atccggaatg ctgtggatgg aaaatggttc
     2701 tgcttcaatg actccaatat ttgcttggtg tcctgggaag acatccagtg tacctacgga
     2761 aatcctaact accactggca ggaaactgca tatcttctgg tttacatgag gatggagtga
     2821 tacccaactt tcttgtacaa agtggttcga taacgaattc cgcccccccc ccctaacgtt
     2881 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     2941 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     3001 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     3061 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     3121 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     3181 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     3241 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     3301 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg
     3361 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     3421 tgataatatg gccacaacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     3481 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     3541 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     3601 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     3661 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     3721 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     3781 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     3841 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     3901 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     3961 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     4021 ccgcaagccc ggtgcctgat gcatcgatgt cgacctgcag ccaagcttat cgataaaata
     4081 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     4141 caagctagct taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag
     4201 aagttcagat caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg
     4261 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc
     4321 gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat
     4381 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt
     4441 ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga
     4501 tagactgcgt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact
     4561 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg
     4621 gtctttcatg ggtaacagtt tcttgaagtt ggagaacaac attctgaggg taggagtcga
     4681 atattaagta atcctgactc aattagccac tgttttgaat ccacatactc caatactcct
     4741 gaaatagttc attatggaca gcgcagaaag agctggggag aattgtgaaa ttgttatccg
     4801 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4861 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4921 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4981 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5041 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5101 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5161 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5221 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5281 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5341 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5401 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5461 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5521 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5581 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5641 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5701 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5761 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5821 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5881 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5941 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6001 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6061 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6121 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6181 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6241 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6301 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6361 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6421 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6481 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6541 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6601 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6661 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6721 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6781 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6841 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6901 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     6961 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     7021 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     7081 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     7141 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     7201 gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa
     7261 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     7321 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcg
     7381 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     7441 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     7501 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcg
//
