LOCUS       sequence-251120-        8010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral vector for expression of HA-Tagged E6-AP I AA 1-64 in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8010)
  AUTHORS   Kuhnle S, Martinez-Noel G, Leclere F, Hayes SD, Harper JW, Howley PM
  TITLE     Angelman syndrome-associated point mutations in the Zn-binding 
            N-terminal (AZUL) domain of UBE3A ubiquitin ligase inhibit binding 
            to the proteasome.
  JOURNAL   J Biol Chem. 2018 Sep 26. pii: RA118.004653. doi: 
            10.1074/jbc.RA118.004653.
  PUBMED    30257870
REFERENCE   2  (bases 1 to 8010)
  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: "J Biol 
            Chem. 2018 Sep 26. pii: RA118.004653. doi: 
            10.1074/jbc.RA118.004653."
COMMENT     Sequence Label: PHAGE-P CMVt N-HA hE6-AP I (AA 1-64)
FEATURES             Location/Qualifiers
     source          1..8010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2227..2530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2531..2734
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2685..2705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2736..2754
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2757..2775
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2785..2809
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2896..2905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2908..2934
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    2950..2974
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3171..3195)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        3214..3713
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3249..3272)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3646..3665
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3734..4333
                     /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(3734..3753)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4230..4250
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4400..4988
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4453..4473)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4871..4882)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5063..5296
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5415..5433)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5501..5605
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5606..5674,5675..6466)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5824..5843)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6637..7225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7126..7145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7379..7396
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7636..7657
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7672..7677,7678..7695,7696..7702)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7710..7726
                     /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)."
     primer_bind     7715..7737
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7734..7750
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7734..7750
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7800..7819
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtgga
     2221 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     2281 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     2341 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     2401 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     2461 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     2521 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     2581 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     2641 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     2701 gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata gagatctccc
     2761 tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg
     2821 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct
     2881 agcgtttaaa cgctagccac catggcgtac ccatacgatg ttccagatta cgctgctcga
     2941 ggttatcaaa caagtttgta caaaaaagtt ggcaccatga agcgagcagc tgcaaagcat
     3001 ctaatagaac gctactacca ccagttaact gagggctgtg gaaatgaagc ctgcacgaat
     3061 gagttttgtg cttcctgtcc aacttttctt cgtatggata ataatgcagc agctattaaa
     3121 gccctcgagc tttataagat taatgcaaaa ctctgtgatc ctcatccctg acccaacttt
     3181 cttgtacaaa gtggttcgat aacgaattct accgggtagg ggaggcgctt ttcccaaggc
     3241 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     3301 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     3361 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     3421 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
     3481 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     3541 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     3601 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     3661 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
     3721 gcccaagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     3781 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     3841 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     3901 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     3961 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     4021 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     4081 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     4141 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     4201 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     4261 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     4321 gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc
     4381 ccatgcatcg atagatccta atcaacctct ggattacaaa atttgtgaaa gattgactgg
     4441 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     4501 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     4561 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     4621 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     4681 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     4741 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     4801 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     4861 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4921 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4981 ctccccgcct gagatccttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     5041 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
     5101 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     5161 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     5221 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     5281 tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca
     5341 aagaaatgaa tatcagagag tgagaggccc gggttaatta aggaaagggc tagatcattc
     5401 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5461 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5521 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5581 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5641 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5701 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5761 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5821 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     5881 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5941 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6001 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6061 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6121 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6181 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6241 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6301 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6361 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6421 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6481 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6541 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6601 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6661 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6721 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6781 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6841 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6901 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6961 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7021 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7081 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7141 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7201 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7261 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7321 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7381 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     7441 ttggccgatt cattaatgca gcaagctcat ggctgactaa ttttttttat ttatgcagag
     7501 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     7561 ctaggctttt gcaaaaagct ccccgtggca cgacaggttt cccgactgga aagcgggcag
     7621 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     7681 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     7741 cagctatgac atgattacga atttcacaaa taaagcattt ttttcactgc attctagttg
     7801 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga taactcaagc
     7861 taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca attacctgtg
     7921 gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa gaaattgtat
     7981 ttgttaaata tgtactacaa acttagtagt
//
