LOCUS       sequence-464803-        6027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses HIS-HA-CMYC triple tagged P53 for pulldown assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6027)
  AUTHORS   Han L, Begum Yagci Z, Keung AJ
  TITLE     A high sensitivity assay of UBE3A ubiquitin ligase activity.
  JOURNAL   Methods. 2025 Mar;235:92-99. doi: 10.1016/j.ymeth.2025.02.002. Epub 
            2025 Feb 9.
  PUBMED    39933617
REFERENCE   2  (bases 1 to 6027)
  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: "Methods."; 
            date: "2025-03"; volume: "235:92-99. doi"; pages: " 
            10.1016/j.ymeth.2025.02.002. Epub 2025 Feb 9"
COMMENT     Sequence Label: HIS-HA-CMYC P53
FEATURES             Location/Qualifiers
     source          1..6027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             108..130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             150..167
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      164..173
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             183..209
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             225..254
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1557..1575)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1571..1618
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1984..1989,1990..2006,2007..2012))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2039..2057)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2125..2229
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2230..2298,2299..3090)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2448..2467)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3261..3849
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3750..3769
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4003..4020
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4035..4174
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4100..4119)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4260..4282
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4276..4467)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5925..5944
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagtgccggc tccggagagc
       61 tctttaatta agcggccgcc ctgcaggact cgagttctag aaataatttt gtttaacttt
      121 aagaaggaga tatacatatg aaatcaagcc atcaccacca tcaccatggt ggcgggggca
      181 gctacccata cgacgtacca gactatgcgg ggggcggggg gagcgaacag aagttgatct
      241 cagaagagga tttaggtggg agcgctagca tggaagaacc acaatctgac ccgtcagttg
      301 aaccaccact ttcacaggag acctttagtg acctttggaa acttttgccc gaaaacaacg
      361 tattatctcc actaccgtca caagcgatgg acgacttaat gctatctcct gatgacatcg
      421 aacagtggtt tacagaagac ccaggacctg acgaggctcc caggatgcca gaggcagccc
      481 ctcctgtggc cccagcgcct gctgcgccca cgccagcagc gcctgccccc gctccgtctt
      541 ggcctctttc cagcagtgta cccagtcaga aaacatatca aggaagctat gggttcaggc
      601 ttgggtttct gcactccggc actgcaaagt cagttacgtg tacctacagt cctgccttaa
      661 ataagatgtt ttgccaattg gctaagacat gtcccgttca actatgggtt gacagcacac
      721 cacctcctgg gactagagta cgtgccatgg ctatctataa gcaaagccaa cacatgacag
      781 aagtagtacg tagatgcccg caccacgagc gttgctccga ttctgacggc ctagctcccc
      841 cgcaacactt gatacgtgtc gaaggtaacc ttagagtaga gtatctagac gatcgtaaca
      901 cttttagaca ctccgtcgtc gttccctatg aaccgcccga ggtcggttct gactgtacca
      961 ctatacatta taattacatg tgcaattcct cttgtatggg cggaatgaat cgtaggccaa
     1021 ttttgacgat cataacccta gaggatagct ccggtaactt attaggcagg aatagtttcg
     1081 aggtaagagt ttgtgcgtgt ccaggacgtg atagaagaac agaagaagag aaccttagga
     1141 aaaagggcga accgcaccat gaattaccgc caggttctac caaacgtgcg cttcccaaca
     1201 atacctcatc aagcccacaa cccaaaaaaa agccactaga tggggaatac tttactttac
     1261 agatccgtgg cagggagaga ttcgaaatgt ttcgtgaatt gaacgaagca ttagaactta
     1321 aggacgcaca ggcgggtaag gagcccgggg ggagccgtgc acacagtagt caccttaagt
     1381 ctaaaaaagg ccaaagcact tcaagacata agaaattgat gtttaagaca gagggccccg
     1441 attcagatta aggatccgcg atcgcggcgc gccacctggt ggccggccgg taccacgcgt
     1501 gcgcgctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1561 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1621 gaaaggagga actatatccg gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt
     1681 cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag cggtcggaca
     1741 gtgctccgag aacgggtgcg catagaaatt gcatcaacgc atatagcgct agcagcacgc
     1801 catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc ggcagtaccg
     1861 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     1921 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     1981 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     2041 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2101 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2161 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata acattgaaaa
     2221 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2281 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2341 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2401 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2461 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2521 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2581 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2641 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2701 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2761 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2821 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2881 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2941 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3001 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3061 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3121 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3181 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3241 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3301 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3361 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3421 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3481 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3541 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3601 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3661 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3721 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3781 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3841 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3901 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3961 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4021 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4081 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4141 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4201 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4261 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4321 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4381 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4441 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4501 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4561 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4621 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4681 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4741 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4801 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4861 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4921 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     4981 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     5041 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     5101 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     5161 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     5221 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     5281 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     5341 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     5401 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     5461 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     5521 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     5581 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     5641 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     5701 gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg
     5761 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     5821 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     5881 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     5941 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     6001 aggatcgaga tctcgatccc gcgaaat
//
