LOCUS Exported 9766 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses Smo-FLAG K444R. ACCESSION . VERSION . KEYWORDS GP822 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9766) 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 9766) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9766 /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 2061..2178 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" polyA_signal 2227..2275 /note="synthetic polyadenylation signal" misc_feature 2289..2380 /label=pause site /note="RNA polymerase II transcriptional pause signal from the human alpha-2 globin gene" primer_bind 2329..2348 /label=RVprimer3 /note="pGL3 vector, forward primer" polyA_signal 2395..2529 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2432..2451) /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS 2725..3120 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature 3121..3706 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(3287..3304) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 3514..3533 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" regulatory 6093..6102 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 6102..6167 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" misc_feature 6208..6796 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6261..6281) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6679..6690) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 6944..7124 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(7171..7189) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7257..7361 /gene="bla" /label=AmpR promoter CDS 7362..8222 /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(7580..7599) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8393..8981 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8882..8901 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 9135..9152 /label=L4440 /note="L4440 vector, forward primer" primer_bind 9244..9263 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" protein_bind 9392..9413 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 9428..9458 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9466..9482 /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 9471..9493 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 9490..9506 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 9490..9506 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" 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 ctgcagagta 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 ttctcaacaa 2041 taggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg 2101 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt 2161 acaaaaattc aaaattttcg ggtttattac tcggacagca gagatccagt ttggactagt 2221 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg 2281 atagtactaa catacgctct ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag 2341 gctgtcccca gtgcaagtgc aggtgccaga acatttctct atcgataggt accgaacttg 2401 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 2461 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 2521 gtctggatcc ccgggaacag attcgcgact agtggacaca caggataggg gtgggcagca 2581 tgaggggggc cagagggaga gggggcagag ctgggctgga cgaggggaca ccccgcccca 2641 atggggcgtg acgagctgcc agcccaggct ccggggcacg taaaagcggg gctgtgagac 2701 cggagagcac ggagcgacat taatatggcc aagcctttgt ctcaagaaga atccaccctc 2761 attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc 2821 agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact 2881 gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac 2941 ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg 3001 tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat 3061 ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc 3121 cccctctccc tccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt 3181 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg 3241 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga 3301 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa 3361 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc 3421 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac 3481 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag 3541 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtac 3601 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg 3661 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatag accatggccg 3721 ctggccgccc cgtgcgtggg cccgagctgg cgccccggag gctgctgcag ttgctgctgc 3781 tggtactgct ggggggcccg ggccgggggg cggccttgag cgggaacgtg accgggcctg 3841 ggcctcacag cgccagcggg agctcgagga gggacgtgcc ggtgaccagc cctccgccgc 3901 cgctgctgag ccactgcggc cgggccgccc actgcgagcc tttgcgctac aacgtgtgcc 3961 tgggctcggc gctgccctac ggagccacca ccacgctgct ggctggggac tcggactcgc 4021 aggaggaagc gcacggcaag ctcgtgctct ggtccggcct ccggaatgcc ccccgctgct 4081 gggcagtgat ccagcccctg ctgtgtgctg tctacatgcc caagtgtgag aatgaccgag 4141 tggagttgcc cagccgtacc ctctgccagg ccacccgagg cccctgtgcc attgtggagc 4201 gggagcgagg gtggcctgac tttctgcgtt gcacaccgga ccacttccct gaaggctgcc 4261 caaacgaggt acaaaacatc aagttcaaca gctcaggcca atgtgaagca cccttggtgc 4321 gaacagacaa ccccaagagc tggtatgagg acgtggaggg ctgtgggatt cagtgtcaga 4381 acccgctgtt caccgaggcc gagcaccagg acatgcacag ctacatcgca gccttcgggg 4441 cggtcactgg tctctgcacg ctcttcacac tggccacctt tgtggctgac tggcggaact 4501 ccaatcgcta ccctgcggtt attctcttct atgtcaatgc gtgtttcttc gtgggcagca 4561 ttggctggct ggcccagttc atggatggtg cccgccgaga gattgtttgc cgagcagatg 4621 gcaccatgag atttggggag cccacctcca gtgagaccct gtcctgtgtc atcatctttg 4681 tcattgtgta ctatgccttg atggctggag tagtctggtt cgtggtcctc acctatgcct 4741 ggcacacctc cttcaaagcc ctgggcacca cctaccagcc tctctcgggc aagacatcct 4801 atttccacct gctcacgtgg tcactcccct ttgtcctcac ggtggcaatc ctggctgtgg 4861 ctcaggtaga tggagactcc gtgagtggca tctgttttgt aggctacaag aactatcggt 4921 accgtgctgg ctttgtcctg gccccaattg gcctggtgct tattgtggga ggctacttcc 4981 tcatcagagg ggtcatgact ctgttctcca tcaagagcaa ccaccctggg cttctgagtg 5041 agagggcagc cagcaagatc aacgagacca tgctgcgcct gggcattttt ggcttcctgg 5101 cctttggctt tgtgctcatc accttcagct gccacttcta tgacttcttc aaccaggctg 5161 agtgggagcg tagcttccgg gactatgtgc tatgccaagc caacgtgacc atcgggctgc 5221 ctaccaagaa gcccattcct gactgtgaga tcaagaatcg gcccagcctc ctggtggaga 5281 agatcaatct atttgccatg tttggcactg gcattgccat gagcacctgg gtctggacca 5341 aggccaccct gctcatctgg aggcgcacct ggtgcaggtt gactgggcac agtgatgatg 5401 agcccaagag aatcaagaag agcaagatga tcgccaaggc cttctctaag cggcgtgagc 5461 tgctgcagaa cccgggccag gagctctcct tcagcatgca cactgtctcc catgatggac 5521 ctgttgcggg tttggctttt gacctcaatg aaccctcagc tgacgtctcc tctgcctggg 5581 ctcagcatgt caccaagatg gtggctcgga gaggagccat attgccccag gatgtgtccg 5641 ttacccctgt ggcaactcca gtgccaccag aagaacaagc caacatgtgg ctggttgagg 5701 cagagatctc cccagagtta gagaagcgtt tgggccggaa gaaaaagcgg aggaagagga 5761 agaaggaggt gtgccccttg aggccagccc ctgagcttca ccactctgcc cctgttcctg 5821 ccaccagtgc agttcctcgg ctgcctcagc tgcctcggca gaagtgcctg gtagctgcaa 5881 acgcctgggg aacaggggag tcctgccgac agggagcctg gactctagtc tccaacccct 5941 tctgcccaga gcctagtccc catcaagatc catttctccc tggtgcctca gccccccggg 6001 tctgggctca gggccgcctc caggggctgg gatccattca ttcccgcact aacctaatgg 6061 aggctgagat cttggatgca gactcggact tcgccaccat ggactacaaa gaccatgacg 6121 gtgattataa agatcatgac atcgattaca aggatgacga tgacaaggcg gccgcttagt 6181 aagtaagatc cgaactcgag agatcctaat caacctctgg attacaaaat ttgtgaaaga 6241 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 6301 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 6361 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 6421 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 6481 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 6541 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 6601 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 6661 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 6721 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 6781 tgggccgcct ccccgcctgg tacctttaag accaatgact tacaaggcag ctgtagatct 6841 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca 6901 agatcacctg caggacaggc gcgccctgct ttttgcttgt actgggtctc tctggttaga 6961 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata 7021 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta 7081 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcacccggg cgattaagga 7141 aagggctaga tcattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta 7201 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 7261 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 7321 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 7381 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 7441 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 7501 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 7561 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag 7621 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 7681 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 7741 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 7801 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 7861 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 7921 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 7981 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 8041 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 8101 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 8161 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 8221 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 8281 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 8341 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 8401 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 8461 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 8521 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 8581 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 8641 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 8701 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 8761 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 8821 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 8881 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 8941 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 9001 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 9061 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 9121 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 9181 cgcctctccc cgcgcgttgg ccgattcatt aatgcagcaa gctcatggct gactaatttt 9241 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg 9301 aggctttttt ggaggcctag gcttttgcaa aaagctcccc gtggcacgac aggtttcccg 9361 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 9421 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 9481 aatttcacac aggaaacagc tatgacatga ttacgaattt cacaaataaa gcattttttt 9541 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatca 9601 actggataac tcaagctaac caaaatcatc ccaaacttcc caccccatac cctattacca 9661 ctgccaatta cctgtggttt catttactct aaacctgtga ttcctctgaa ttattttcat 9721 tttaaagaaa ttgtatttgt taaatatgta ctacaaactt agtagt //