LOCUS       sequence-464776-        5431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Ubiquitin tagged with an Acceptor Peptide (AP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5431)
  AUTHORS   Mukhopadhyay U, Levantovsky S, Carusone TM, Gharbi S, Stein F, 
            Behrends C, Bhogaraju S
  TITLE     A ubiquitin-specific, proximity-based labeling approach for the 
            identification of ubiquitin ligase substrates.
  JOURNAL   Sci Adv. 2024 Aug 9;10(32):eadp3000. doi: 10.1126/sciadv.adp3000. 
            Epub 2024 Aug 9.
  PUBMED    39121224
REFERENCE   2  (bases 1 to 5431)
  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: "Sci Adv."; 
            date: "2024-08-9"; volume: "10(32):eadp3000. doi"; pages: " 
            10.1126/sciadv.adp3000. Epub 2024 Aug 9"
COMMENT     Sequence Label: pcDNA5-AP-Ub
FEATURES             Location/Qualifiers
     source          1..5431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1132..1253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1169..1188)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1223..1242
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1302..1318)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1302..1318)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1315..1337)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1326..1342
                     /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(1350..1356,1357..1374,1375..1380))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1395..1416
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1533..1550)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1704..2292)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1784..1803)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2463..3254,3255..3323))
                     /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     3086..3105
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3324..3428)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3503..3522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3694..4073
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4074..4277
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4228..4248
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4279..4297
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4300..4318
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4328..4352
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             4443..4487
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4515..4742
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(4842..4859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4848..5072
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5205..5224)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5356..5403
                     /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             join(5411..5431,1..1002)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             join(5411..5431,1..1002)
                     /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
                     "
ORIGIN
        1 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
       61 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
      121 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
      181 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc
      241 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
      301 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
      361 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
      421 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
      481 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
      541 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
      601 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
      661 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
      721 ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
      781 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
      841 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
      901 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
      961 ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc
     1021 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     1081 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     1141 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     1201 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     1261 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     1321 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     1381 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     1441 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     1501 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1561 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1621 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1681 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1741 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1801 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1861 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1921 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1981 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2041 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2101 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     2161 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2221 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2281 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2341 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2401 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2461 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2521 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2581 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     2641 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2701 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2761 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     2821 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     2881 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     2941 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     3001 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     3061 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3121 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3181 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3241 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3301 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3361 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3421 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     3481 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     3541 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     3601 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     3661 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
     3721 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     3781 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     3841 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     3901 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     3961 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     4021 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     4081 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     4141 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     4201 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     4261 tctatataag cagagctctc cctatcagtg atagagatct ccctatcagt gatagagatc
     4321 gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg
     4381 acctccatag aagacaccgg gaccgatcca gcctccggac tctagcgttt aaacttaaga
     4441 tgggcctgaa cgacatcttc gaggcccaga agatcgagtg gcacgagggc tccggctcca
     4501 cctccggctc cggcatgcag atcttcgtga agactctgac tggtaagacc atcaccctcg
     4561 aggttgagcc cagtgacacc attgagaatg tcaaggcaaa gatccaagat aaggaaggca
     4621 tccctcctga ccagcagagg ctgatctttg ctggaaaaca gctggaagat gggcgcaccc
     4681 tgtctgacta caacatccag aaagagtcca ccctgcacct ggtactccgt ctcagaggtg
     4741 ggtaataagg atccactagt ccagtgtggt ggaattctgc agatatccag cacagtggcg
     4801 gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     4861 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4921 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     4981 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     5041 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     5101 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5161 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5221 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5281 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     5341 tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac
     5401 ttccttggcc aaaaagcctg aactcaccgc g
//
