LOCUS Exported 6490 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses Smo-FLAG K444, 448R. ACCESSION . VERSION . KEYWORDS GP826 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6490) AUTHORS Desai PB, Stuck MW, Lv B, Pazour GJ TITLE Ubiquitin links smoothened to intraflagellar transport to regulate Hedgehog signaling. JOURNAL J Cell Biol. 2020 Jul 6;219(7). pii: 151798. doi: 10.1083/jcb.201912104. PUBMED 32435793 REFERENCE 2 (bases 1 to 6490) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6490 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 139..534 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature 535..1120 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(701..718) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 928..947 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" regulatory 3507..3516 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3516..3581 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" primer_bind complement(3675..3695) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4093..4104) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 4358..4538 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 4585..4710 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 5207..5440 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 5625..5669 /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 5965..6082 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" polyA_signal 6131..6179 /note="synthetic polyadenylation signal" misc_feature 6193..6284 /label=pause site /note="RNA polymerase II transcriptional pause signal from the human alpha-2 globin gene" primer_bind 6233..6252 /label=RVprimer3 /note="pGL3 vector, forward primer" polyA_signal 6299..6433 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6336..6355) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6390..6409 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" ORIGIN 1 gggccagagg gagagggggc agagctgggc tggacgaggg gacaccccgc cccaatgggg 61 cgtgacgagc tgccagccca ggctccgggg cacgtaaaag cggggctgtg agaccggaga 121 gcacggagcg acattaatat ggccaagcct ttgtctcaag aagaatccac cctcattgaa 181 agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca 241 gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga 301 ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact 361 tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga 421 caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag 481 ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggccccctc 541 tccctccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt 601 gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct 661 ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa 721 ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg 781 tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc 841 caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg 901 agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg 961 aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtacacatgc 1021 tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg 1081 gttttccttt gaaaaacacg atgataatat ggccacaacc atagaccatg gccgctggcc 1141 gccccgtgcg tgggcccgag ctggcgcccc ggaggctgct gcagttgctg ctgctggtac 1201 tgctgggggg cccgggccgg ggggcggcct tgagcgggaa cgtgaccggg cctgggcctc 1261 acagcgccag cgggagctcg aggagggacg tgccggtgac cagccctccg ccgccgctgc 1321 tgagccactg cggccgggcc gcccactgcg agcctttgcg ctacaacgtg tgcctgggct 1381 cggcgctgcc ctacggagcc accaccacgc tgctggctgg ggactcggac tcgcaggagg 1441 aagcgcacgg caagctcgtg ctctggtccg gcctccggaa tgccccccgc tgctgggcag 1501 tgatccagcc cctgctgtgt gctgtctaca tgcccaagtg tgagaatgac cgagtggagt 1561 tgcccagccg taccctctgc caggccaccc gaggcccctg tgccattgtg gagcgggagc 1621 gagggtggcc tgactttctg cgttgcacac cggaccactt ccctgaaggc tgcccaaacg 1681 aggtacaaaa catcaagttc aacagctcag gccaatgtga agcacccttg gtgcgaacag 1741 acaaccccaa gagctggtat gaggacgtgg agggctgtgg gattcagtgt cagaacccgc 1801 tgttcaccga ggccgagcac caggacatgc acagctacat cgcagccttc ggggcggtca 1861 ctggtctctg cacgctcttc acactggcca cctttgtggc tgactggcgg aactccaatc 1921 gctaccctgc ggttattctc ttctatgtca atgcgtgttt cttcgtgggc agcattggct 1981 ggctggccca gttcatggat ggtgcccgcc gagagattgt ttgccgagca gatggcacca 2041 tgagatttgg ggagcccacc tccagtgaga ccctgtcctg tgtcatcatc tttgtcattg 2101 tgtactatgc cttgatggct ggagtagtct ggttcgtggt cctcacctat gcctggcaca 2161 cctccttcaa agccctgggc accacctacc agcctctctc gggcaagaca tcctatttcc 2221 acctgctcac gtggtcactc ccctttgtcc tcacggtggc aatcctggct gtggctcagg 2281 tagatggaga ctccgtgagt ggcatctgtt ttgtaggcta caagaactat cggtaccgtg 2341 ctggctttgt cctggcccca attggcctgg tgcttattgt gggaggctac ttcctcatca 2401 gaggggtcat gactctgttc tccatcaaga gcaaccaccc tgggcttctg agtgagaggg 2461 cagccagcag gatcaacgag accatgctgc gcctgggcat ttttggcttc ctggcctttg 2521 gctttgtgct catcaccttc agctgccact tctatgactt cttcaaccag gctgagtggg 2581 agcgtagctt ccgggactat gtgctatgcc aagccaacgt gaccatcggg ctgcctacca 2641 agaagcccat tcctgactgt gagatcaaga atcggcccag cctcctggtg gagaagatca 2701 atctatttgc catgtttggc actggcattg ccatgagcac ctgggtctgg accaaggcca 2761 ccctgctcat ctggaggcgc acctggtgca ggttgactgg gcacagtgat gatgagccca 2821 agagaatcaa gaagagcaag atgatcgcca aggccttctc taagcggcgt gagctgctgc 2881 agaacccggg ccaggagctc tccttcagca tgcacactgt ctcccatgat ggacctgttg 2941 cgggtttggc ttttgacctc aatgaaccct cagctgacgt ctcctctgcc tgggctcagc 3001 atgtcaccaa gatggtggct cggagaggag ccatattgcc ccaggatgtg tccgttaccc 3061 ctgtggcaac tccagtgcca ccagaagaac aagccaacat gtggctggtt gaggcagaga 3121 tctccccaga gttagagaag cgtttgggcc ggaagaaaaa gcggaggaag aggaagaagg 3181 aggtgtgccc cttgaggcca gcccctgagc ttcaccactc tgcccctgtt cctgccacca 3241 gtgcagttcc tcggctgcct cagctgcctc ggcagaagtg cctggtagct gcaaacgcct 3301 ggggaacagg ggagtcctgc cgacagggag cctggactct agtctccaac cccttctgcc 3361 cagagcctag tccccatcaa gatccatttc tccctggtgc ctcagccccc cgggtctggg 3421 ctcagggccg cctccagggg ctgggatcca ttcattcccg cactaaccta atggaggctg 3481 agatcttgga tgcagactcg gacttcgcca ccatggacta caaagaccat gacggtgatt 3541 ataaagatca tgacatcgat tacaaggatg acgatgacaa ggcggccgct tagtaagtaa 3601 gatccgaact cgagagatcc tgatcaacct ctggattaca aaatttgtga aagattgact 3661 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 3721 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg 3781 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg 3841 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg 3901 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc 3961 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca 4021 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc 4081 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct 4141 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc 4201 gcctccccgc ctggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 4261 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatca 4321 cctgcaggac aggcgcgccc tgctttttgc ttgtactggg tctctctggt tagaccagat 4381 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 4441 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 4501 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg 4561 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc 4621 acggcaagag gcgaggggcg gcgactgcag agtacgccaa aaattttgac tagcggaggc 4681 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga 4741 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta 4801 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa 4861 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt 4921 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa 4981 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca 5041 cagcaagcgg ccggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga 5101 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca 5161 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc 5221 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac 5281 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg 5341 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa 5401 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg 5461 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg 5521 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa 5581 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat 5641 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt 5701 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg 5761 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc 5821 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga 5881 gagacagaga cagatccatt cgattagtga acggatctcg acggtatcgc cgaattctca 5941 acaataggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc 6001 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca 6061 aattacaaaa attcaaaatt ttcgggttta ttactcggac agcagagatc cagtttggac 6121 tagtggccgc aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga 6181 atcgatagta ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa 6241 ataggctgtc cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat aggtaccgaa 6301 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 6361 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta 6421 tcatgtctgg atccccggga acagattcgc gactagtgga cacacaggat aggggtgggc 6481 agcatgaggg //