LOCUS       sequence-419116-        5659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of 3xFLAG-tagged budding yeast Ubc7 in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5659)
  AUTHORS   Renz C, Asimaki E, Meister C, Albanese V, Petriukov K, Krapoth NC, 
            Wegmann S, Wollscheid HP, Wong RP, Fulzele A, Chen JX, Leon S, 
            Ulrich HD
  TITLE     Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.
  JOURNAL   Mol Cell. 2024 Jan 18;84(2):386-400.e11. doi: 
            10.1016/j.molcel.2023.11.016. Epub 2023 Dec 15.
  PUBMED    38103558
REFERENCE   2  (bases 1 to 5659)
  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: 
            "10.1016/j.molcel.2023.11.016"; journalName: "Mol Cell"; date: 
            "2024-01-18- 18"; volume: "84"; issue: "2"; pages: "386-400.e11"
COMMENT     Sequence Label: pDEST-3xFLAG-Ubc7
FEATURES             Location/Qualifiers
     source          1..5659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             920..985
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    992..1016
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1543..1567)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1611..1628)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1617..1841
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1974..1993)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2125..2172
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2180..3202
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2180..3202
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3332..3453
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3369..3388)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3423..3442
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3502..3518)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3502..3518)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3515..3537)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3526..3542
                     /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(3550..3556,3557..3574,3575..3580))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3595..3616
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3733..3750)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3904..4492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3984..4003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4663..5454,5455..5523))
                     /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     5286..5305
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5524..5628)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttatgg actacaaaga ccatgacggt gattataaag atcatgacat
      961 cgactacaag gatgacgatg acaaggatat cacaagtttg tacaaaaaag caggctccac
     1021 catgagcaag accgcccaga agcgcctgct gaaggagctg cagcagctga tcaaggacag
     1081 cccccccggc atcgtggccg gccccaagag cgagaacaac atcttcatct gggactgcct
     1141 gatccagggc ccccccgaca ccccctacgc cgacggcgtg ttcaacgcca agctggagtt
     1201 ccccaaggac taccccctga gcccccccaa gctgaccttc acccccagca tcctgcaccc
     1261 caacatctac cccaacggcg aggtgtgcat cagcatcctg cacagccccg gcgacgaccc
     1321 caacatgtac gagctggccg aggagcgctg gagccccgtg cagagcgtgg agaagatcct
     1381 gctgagcgtg atgagcatgc tgagcgagcc caacatcgag agcggcgcca acatcgacgc
     1441 ctgcatcctg tggcgcgaca accgccccga gttcgagcgc caggtgaagc tgagcatcct
     1501 gaagagcctg ggcttctgag cggccgcact cgagatatct agacccagct ttcttgtaca
     1561 aagtggtgat atcctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     1621 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1681 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1741 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1801 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     1861 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     1921 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1981 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2041 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2101 ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag
     2161 tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     2221 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
     2281 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     2341 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     2401 ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc
     2461 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     2521 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     2581 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     2641 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     2701 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     2761 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     2821 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     2881 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     2941 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     3001 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     3061 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     3121 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     3181 tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta
     3241 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3301 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3361 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3421 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3481 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3541 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3601 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3661 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3721 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3781 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3841 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3901 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3961 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4021 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4081 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4141 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4201 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4261 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4321 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4381 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4441 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4501 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4561 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4621 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4681 tgaggcacct atctcagcga tctgtctatt tcgctcatcc atagttgcct gactccccgt
     4741 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4801 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4861 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4921 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4981 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5041 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5101 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5161 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5221 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5281 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5341 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5401 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5461 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5521 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5581 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5641 aaaagtgcca cctgacgtc
//
