LOCUS Exported 9766 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses Smo-FLAG K543, 565, 568, 569, 575, 579, 628, 671, 676, 677, 678, 681, 682, 683, 684, 717R. ACCESSION . VERSION . KEYWORDS GP804 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" misc_feature 280..405 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 902..1135 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1320..1364 /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 1660..1777 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" polyA_signal 1826..1874 /note="synthetic polyadenylation signal" misc_feature 1888..1979 /label=pause site /note="RNA polymerase II transcriptional pause signal from the human alpha-2 globin gene" primer_bind 1928..1947 /label=RVprimer3 /note="pGL3 vector, forward primer" polyA_signal 1994..2128 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2031..2050) /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS 2324..2719 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature 2720..3305 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2886..2903) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 3113..3132 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" regulatory 5692..5701 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 5701..5766 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" misc_feature 5807..6395 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5860..5880) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6278..6289) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 6543..6723 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(6770..6788) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6856..6960 /gene="bla" /label=AmpR promoter CDS 6961..7821 /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(7179..7198) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7992..8580 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8481..8500 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8734..8751 /label=L4440 /note="L4440 vector, forward primer" primer_bind 8843..8862 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" protein_bind 8991..9012 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 9027..9057 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9065..9081 /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 9070..9092 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 9089..9105 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 9089..9105 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" LTR 9366..233 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" ORIGIN 1 gagtggcgag ccctcagatc ctgcatataa gcagctgctt tttgcctgta ctgggtctct 61 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 121 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 181 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 241 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg 301 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tgcagagtac gccaaaaatt 361 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg 421 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa 481 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg 541 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca 601 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa 661 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa 721 agtaagacca ccgcacagca agcggccggc cgctgatctt cagacctgga ggaggagata 781 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag 841 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa 901 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa 961 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt 1021 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc 1081 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga 1141 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga 1201 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa 1261 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa 1321 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca 1381 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt 1441 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat 1501 tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag 1561 aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga tctcgacggt 1621 atcgccgaat tctcaacaat aggcagtatt catccacaat tttaaaagaa aaggggggat 1681 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 1741 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattact cggacagcag 1801 agatccagtt tggactagtg gccgcaataa aatatcttta ttttcattac atctgtgtgt 1861 tggttttttg tgtgaatcga tagtactaac atacgctctc catcaaaaca aaacgaaaca 1921 aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa catttctcta 1981 tcgataggta ccgaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 2041 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 2101 catcaatgta tcttatcatg tctggatccc cgggaacaga ttcgcgacta gtggacacac 2161 aggatagggg tgggcagcat gaggggggcc agagggagag ggggcagagc tgggctggac 2221 gaggggacac cccgccccaa tggggcgtga cgagctgcca gcccaggctc cggggcacgt 2281 aaaagcgggg ctgtgagacc ggagagcacg gagcgacatt aatatggcca agcctttgtc 2341 tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc 2401 tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt 2461 caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc 2521 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat 2581 cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc 2641 catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc 2701 tggttatgtg tgggagggcc ccctctccct ccccccccct aacgttactg gccgaagccg 2761 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt 2821 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct 2881 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct 2941 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc 3001 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc 3061 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc 3121 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc 3181 tgatctgggg cctcggtaca catgctttac atgtgtttag tcgaggttaa aaaaacgtct 3241 aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca 3301 caaccataga ccatggccgc tggccgcccc gtgcgtgggc ccgagctggc gccccggagg 3361 ctgctgcagt tgctgctgct ggtactgctg gggggcccgg gccggggggc ggccttgagc 3421 gggaacgtga ccgggcctgg gcctcacagc gccagcggga gctcgaggag ggacgtgccg 3481 gtgaccagcc ctccgccgcc gctgctgagc cactgcggcc gggccgccca ctgcgagcct 3541 ttgcgctaca acgtgtgcct gggctcggcg ctgccctacg gagccaccac cacgctgctg 3601 gctggggact cggactcgca ggaggaagcg cacggcaagc tcgtgctctg gtccggcctc 3661 cggaatgccc cccgctgctg ggcagtgatc cagcccctgc tgtgtgctgt ctacatgccc 3721 aagtgtgaga atgaccgagt ggagttgccc agccgtaccc tctgccaggc cacccgaggc 3781 ccctgtgcca ttgtggagcg ggagcgaggg tggcctgact ttctgcgttg cacaccggac 3841 cacttccctg aaggctgccc aaacgaggta caaaacatca agttcaacag ctcaggccaa 3901 tgtgaagcac ccttggtgcg aacagacaac cccaagagct ggtatgagga cgtggagggc 3961 tgtgggattc agtgtcagaa cccgctgttc accgaggccg agcaccagga catgcacagc 4021 tacatcgcag ccttcggggc ggtcactggt ctctgcacgc tcttcacact ggccaccttt 4081 gtggctgact ggcggaactc caatcgctac cctgcggtta ttctcttcta tgtcaatgcg 4141 tgtttcttcg tgggcagcat tggctggctg gcccagttca tggatggtgc ccgccgagag 4201 attgtttgcc gagcagatgg caccatgaga tttggggagc ccacctccag tgagaccctg 4261 tcctgtgtca tcatctttgt cattgtgtac tatgccttga tggctggagt agtctggttc 4321 gtggtcctca cctatgcctg gcacacctcc ttcaaagccc tgggcaccac ctaccagcct 4381 ctctcgggca agacatccta tttccacctg ctcacgtggt cactcccctt tgtcctcacg 4441 gtggcaatcc tggctgtggc tcaggtagat ggagactccg tgagtggcat ctgttttgta 4501 ggctacaaga actatcggta ccgtgctggc tttgtcctgg ccccaattgg cctggtgctt 4561 attgtgggag gctacttcct catcagaggg gtcatgactc tgttctccat caagagcaac 4621 caccctgggc ttctgagtga gaaggcagcc agcaagatca acgagaccat gctgcgcctg 4681 ggcatttttg gcttcctggc ctttggcttt gtgctcatca ccttcagctg ccacttctat 4741 gacttcttca accaggctga gtgggagcgt agcttccggg actatgtgct atgccaagcc 4801 aacgtgacca tcgggctgcc taccaagaag cccattcctg actgtgagat caagaatcgg 4861 cccagcctcc tggtggagaa gatcaatcta tttgccatgt ttggcactgg cattgccatg 4921 agcacctggg tctggaccag ggccaccctg ctcatctgga ggcgcacctg gtgcaggttg 4981 actgggcaca gtgatgatga gcccaggaga atcaggagga gcaggatgat cgccagggcc 5041 ttctctaggc ggcgtgagct gctgcagaac ccgggccagg agctctcctt cagcatgcac 5101 actgtctccc atgatggacc tgttgcgggt ttggcttttg acctcaatga accctcagct 5161 gacgtctcct ctgcctgggc tcagcatgtc accaggatgg tggctcggag aggagccata 5221 ttgccccagg atgtgtccgt tacccctgtg gcaactccag tgccaccaga agaacaagcc 5281 aacatgtggc tggttgaggc agagatctcc ccagagttag agaggcgttt gggccggagg 5341 agaaggcgga ggaggaggag gagggaggtg tgccccttga ggccagcccc tgagcttcac 5401 cactctgccc ctgttcctgc caccagtgca gttcctcggc tgcctcagct gcctcggcag 5461 aggtgcctgg tagctgcaaa cgcctgggga acaggggagt cctgccgaca gggagcctgg 5521 actctagtct ccaacccctt ctgcccagag cctagtcccc atcaagatcc atttctccct 5581 ggtgcctcag ccccccgggt ctgggctcag ggccgcctcc aggggctggg atccattcat 5641 tcccgcacta acctaatgga ggctgagatc ttggatgcag actcggactt cgccaccatg 5701 gactacaaag accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat 5761 gacaaggcgg ccgcttagta agtaagatcc gaactcgaga gatcctaatc aacctctgga 5821 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 5881 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 5941 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 6001 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 6061 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 6121 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 6181 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 6241 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 6301 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 6361 gacgagtcgg atctcccttt gggccgcctc cccgcctggt acctttaaga ccaatgactt 6421 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa 6481 ttcactccca acgaagacaa gatcacctgc aggacaggcg cgccctgctt tttgcttgta 6541 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc 6601 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt 6661 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta 6721 gcacccgggc gattaaggaa agggctagat cattcttgaa gacgaaaggg cctcgtgata 6781 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 6841 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 6901 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 6961 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 7021 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 7081 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 7141 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 7201 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 7261 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 7321 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 7381 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 7441 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 7501 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 7561 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 7621 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 7681 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 7741 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 7801 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 7861 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 7921 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 7981 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 8041 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 8101 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 8161 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 8221 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 8281 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 8341 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 8401 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 8461 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 8521 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 8581 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 8641 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 8701 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 8761 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagcaag 8821 ctcatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc 8881 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctccccg 8941 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 9001 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 9061 ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat tacgaatttc 9121 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 9181 tcttatcatg tctggatcaa ctggataact caagctaacc aaaatcatcc caaacttccc 9241 accccatacc ctattaccac tgccaattac ctgtggtttc atttactcta aacctgtgat 9301 tcctctgaat tattttcatt ttaaagaaat tgtatttgtt aaatatgtac tacaaactta 9361 gtagttggaa gggctaattc actcccaaag aagacaagat atccttgatc tgtggatcta 9421 ccacacacaa ggctacttcc ctgattagca gaactacaca ccagggccag gggtcagata 9481 tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata aggtagaaga 9541 ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg ggatggatga 9601 cccggagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc atcacgtggc 9661 ccgagagctg catccggagt acttcaagaa ctgctgatat cgagcttgct acaagggact 9721 ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggg //