LOCUS Exported 8010 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION lentiviral vector for expression of HA-Tagged E6-AP I AA 1-64 in mammalian cells. ACCESSION . VERSION . KEYWORDS PHAGE-P CMVt N-HA hE6-AP I (AA 1-64) 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8010 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 5606..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" primer_bind 7488..7507 /label=SV40pro-F /note="SV40 promoter/origin, 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 7672..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 //