LOCUS Exported 9865 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses SmoPi-FLAG. ACCESSION . VERSION . KEYWORDS GP938 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9865) 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 9865) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9865 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(41..61) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(459..470) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 724..904 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(951..969) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1037..1141 /gene="bla" /label=AmpR promoter CDS 1142..2002 /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(1360..1379) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2173..2761 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2662..2681 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2915..2932 /label=L4440 /note="L4440 vector, forward primer" primer_bind 3024..3043 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" protein_bind 3172..3193 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3208..3238 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3246..3262 /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 3251..3273 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3270..3286 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3270..3286 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 3336..3355 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" LTR 3547..4180 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 4227..4352 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 4849..5082 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 5267..5311 /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 5607..5724 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" enhancer 5776..6079 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6080..6283 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6234..6254 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 6280..6304 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 8755..8764 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 8764..8829 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" misc_feature 8855..9440 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(9021..9038) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 9248..9267 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 9441..9839 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature 9853..576 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" ORIGIN 1 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 61 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 121 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 181 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 241 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 301 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 361 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca 421 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 481 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 541 agacgagtcg gatctccctt tgggccgcct ccccgcctgg tacctttaag accaatgact 601 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta 661 attcactccc aacgaagaca agatcacctg caggacaggc gcgccctgct ttttgcttgt 721 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac 781 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg 841 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct 901 agcacccggg cgattaagga aagggctaga tcattcttga agacgaaagg gcctcgtgat 961 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 1021 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 1081 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 1141 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 1201 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 1261 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 1321 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 1381 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 1441 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 1501 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 1561 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 1621 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 1681 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 1741 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 1801 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 1861 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 1921 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 1981 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 2041 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 2101 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 2161 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 2221 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 2281 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 2341 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 2401 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 2461 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 2521 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 2581 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 2641 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 2701 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 2761 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 2821 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 2881 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 2941 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagcaa 3001 gctcatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 3061 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccc 3121 gtggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 3181 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 3241 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgaattt 3301 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 3361 atcttatcat gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc 3421 caccccatac cctattacca ctgccaatta cctgtggttt catttactct aaacctgtga 3481 ttcctctgaa ttattttcat tttaaagaaa ttgtatttgt taaatatgta ctacaaactt 3541 agtagttgga agggctaatt cactcccaaa gaagacaaga tatccttgat ctgtggatct 3601 accacacaca aggctacttc cctgattagc agaactacac accagggcca ggggtcagat 3661 atccactgac ctttggatgg tgctacaagc tagtaccagt tgagccagat aaggtagaag 3721 aggccaataa aggagagaac accagcttgt tacaccctgt gagcctgcat gggatggatg 3781 acccggagag agaagtgtta gagtggaggt ttgacagccg cctagcattt catcacgtgg 3841 cccgagagct gcatccggag tacttcaaga actgctgata tcgagcttgc tacaagggac 3901 tttccgctgg ggactttcca gggaggcgtg gcctgggcgg gactggggag tggcgagccc 3961 tcagatcctg catataagca gctgcttttt gcctgtactg ggtctctctg gttagaccag 4021 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 4081 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 4141 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact 4201 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc 4261 gcacggcaag aggcgagggg cggcgactgc agagtacgcc aaaaattttg actagcggag 4321 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc 4381 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag 4441 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag 4501 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac 4561 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa 4621 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg 4681 cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg 4741 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac 4801 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt 4861 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt 4921 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat 4981 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag 5041 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc 5101 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct 5161 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac 5221 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga 5281 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct 5341 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt 5401 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac 5461 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga 5521 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc gccgaattct 5581 caacaatagg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt 5641 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa 5701 caaattacaa aaattcaaaa ttttcgggtt tattactcgg acagcagaga tccagtttgg 5761 actagtggag ttccgcgtta cataacttac gcaaaatggc ccgcctggct gaccgcccaa 5821 cgacccccgc ccattgacgt caataatgac caatgttccc atagtaacgc caatagggac 5881 tttccattga cgtcaatggg tggagtattt acgcaaaact gcccacttgg cagtacatca 5941 agtgtatcat ttgccaacaa cgccccctat tgacgtcaat gacgcaaaat ggcccgcctg 6001 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 6061 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 6121 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 6181 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 6241 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca 6301 gatcgcctgg agacgccatc cacgctgttt tgacctccat tcaagacacc ggcggcctta 6361 attaatgatc agaccatggc cgctggccgc cccgtgcgtg ggcccgagct ggcgccccgg 6421 aggctgctgc agttgctgct gctggtactg ctggggggcc cgggccgggg ggcggccttg 6481 agcgggaacg tgaccgggcc tgggcctcac agcgccagcg ggagctcgag gagggacgtg 6541 ccggtgacca gccctccgcc gccgctgctg agccactgcg gccgggccgc ccactgcgag 6601 cctttgcgct acaacgtgtg cctgggctcg gcgctgccct acggagccac caccacgctg 6661 ctggctgggg actcggactc gcaggaggaa gcgcacggca agctcgtgct ctggtccggc 6721 ctccggaatg ccccccgctg ctgggcagtg atccagcccc tgctgtgtgc tgtctacatg 6781 cccaagtgtg agaatgaccg agtggagttg cccagccgta ccctctgcca ggccacccga 6841 ggcccctgtg ccattgtgga gcgggagcga gggtggcctg actttctgcg ttgcacaccg 6901 gaccacttcc ctgaaggctg cccaaacgag gtacaaaaca tcaagttcaa cagctcaggc 6961 caatgtgaag cacccttggt gcgaacagac aaccccaaga gctggtatga ggacgtggag 7021 ggctgtggga ttcagtgtca gaacccgctg ttcaccgagg ccgagcacca ggacatgcac 7081 agctacatcg cagccttcgg ggcggtcact ggtctctgca cgctcttcac actggccacc 7141 tttgtggctg actggcggaa ctccaatcgc taccctgcgg ttattctctt ctatgtcaat 7201 gcgtgtttct tcgtgggcag cattggctgg ctggcccagt tcatggatgg tgcccgccga 7261 gagattgttt gccgagcaga tggcaccatg agatttgggg agcccacctc cagtgagacc 7321 ctgtcctgtg tcatcatctt tgtcattgtg tactatgcct tgatggctgg agtagtctgg 7381 ttcgtggtcc tcacctatgc ctggcacacc tccttcaaag ccctgggcac cacctaccag 7441 cctctctcgg gcaagacatc ctatttccac ctgctcacgt ggtcactccc ctttgtcctc 7501 acggtggcaa tcctggctgt ggctcaggta gatggagact ccgtgagtgg catctgtttt 7561 gtaggctaca agaactatcg gtaccgtgct ggctttgtcc tggccccaat tggcctggtg 7621 cttattgtgg gaggctactt cctcatcaga ggggtcatga ctctgttctc catcaagagc 7681 aaccaccctg ggcttctgag tgagaaggca gccagcaaga tcaacgagac catgctggcc 7741 ctgggcattt ttggcttcct ggcctttggc tttgtgctca tcaccttcag ctgccacttc 7801 tatgacttct tcaaccaggc tgagtgggag cgtagcttcc gggactatgt gctatgccaa 7861 gccaacgtga ccatcgggct gcctaccaag aagcccattc ctgactgtga gatcaagaat 7921 cggcccagcc tcctggtgga gaagatcaat ctatttgcca tgtttggcac tggcattgcc 7981 atgagcacct gggtctggac caaggccacc ctgctcatct ggaggcgcac ctggtgcagg 8041 ttgactgggc acagtgatga tgagcccaag agaatcaaga agagcaagat gatcgccaag 8101 gccttctcta agcggcgtga gctgctgcag aacccgggcc aggagctctc cttcagcatg 8161 cacactgtct cccatgatgg acctgttgcg ggtttggctt ttgacctcaa tgaaccctca 8221 gctgacgtct cctctgcctg ggctcagcat gtcaccaaga tggtggctcg gagaggagcc 8281 atattgcccc aggatgtgtc cgttacccct gtggcaactc cagtgccacc agaagaacaa 8341 gccaacatgt ggctggttga ggcagagatc tccccagagt tagagaagcg tttgggccgg 8401 aagaaaaagc ggaggaagag gaagaaggag gtgtgcccct tgaggccagc ccctgagctt 8461 caccactctg cccctgttcc tgccaccagt gcagttcctc ggctgcctca gctgcctcgg 8521 cagaagtgcc tggtagctgc aaacgcctgg ggaacagggg agtcctgccg acagggagcc 8581 tggactctag tctccaaccc cttctgccca gagcctagtc cccatcaaga tccatttctc 8641 cctggtgcct cagccccccg ggtctgggct cagggccgcc tccaggggct gggatccatt 8701 cattcccgca ctaacctaat ggaggctgag atcttggatg cagactcgga cttcgccacc 8761 atggactaca aagaccatga cggtgattat aaagatcatg acatcgatta caaggatgac 8821 gatgacaagg cggccgctta gtaagtaaga tccgcccctc tccctccccc cccctaacgt 8881 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac 8941 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag 9001 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa 9061 ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag 9121 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga 9181 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag 9241 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc 9301 ccattgtatg ggatctgatc tggggcctcg gtacacatgc tttacatgtg tttagtcgag 9361 gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg 9421 atgataatat ggccacaacc atggccaagc ctttgtctca agaagaatcc accctcattg 9481 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg 9541 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg 9601 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga 9661 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc 9721 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac 9781 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaac 9841 tcgagagatc ctaatcaacc tctgg //