LOCUS Exported 7941 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Localization, Expression of Smoothened WT in mammalian cell lines.. ACCESSION . VERSION . KEYWORDS pcDNA3-mSmo-WT-V5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7941) AUTHORS Marada S, Navarro G, Truong A, Stewart DP, Arensdorf AM, Nachtergaele S, Angelats E, Opferman JT, Rohatgi R, McCormick PJ, Ogden SK TITLE Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling. JOURNAL PLoS Genet. 2015 Aug 20;11(8):e1005473. doi: 10.1371/journal.pgen.1005473. eCollection 2015 Aug. PUBMED 26291458 REFERENCE 2 (bases 1 to 7941) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7941 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 629..648 /label=T7 /note="T7 promoter, forward primer" promoter 629..647 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 713..722 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 872..913 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" regulatory 3187..3196 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(3260..3278) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3261..3278) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3298..3315) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3304..3528 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3574..4002 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3661..3680) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3871..3892 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4011..4031) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4016..4345 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4196..4331 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4258..4277 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4412..5206 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4466..4485) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5076..5095 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5380..5501 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5417..5436) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5471..5490 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5550..5566) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5550..5566) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5563..5585) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5574..5590 /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)." promoter complement(5598..5628) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5643..5664 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5781..5798) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5952..6540) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6032..6051) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6711..7571) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFCPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7334..7353 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7572..7676) /gene="bla" /label=AmpR promoter primer_bind complement(7751..7770) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt 661 ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattcg cggccgccat 721 ggccgctggc cgccccgtgc gtgggcccga gctggcgccc cggaggctgc tgcagttgct 781 tctgctggta ctgctggggg gcccgggccg gggggcggcc ttgagcggga acgtgaccgg 841 gcctgggcct cacagcgcca gcgggagctc gggtaagcct atccctaacc ctctcctcgg 901 tctcgattct acgaggaggg acgtgccggt gaccagccct ccgccgccgc tgctgagcca 961 ctgcggccgg gccgcccact gcgagccttt gcgctacaac gtgtgcctgg gctcggcgct 1021 gccctacgga gccaccacca cgctgctggc tggggactcg gactcgcagg aggaagcgca 1081 cggcaagctc gtgctctggt ccggcctccg gaatgccccc cgctgctggg cagtgatcca 1141 gcccctgctg tgtgctgtct acatgcccaa gtgtgagaat gaccgagtgg agttgcccag 1201 ccgtaccctc tgccaggcca cccgaggccc ctgtgccatt gtggagcggg agcgagggtg 1261 gcctgacttt ctgcgttgca caccggacca cttccctgaa ggctgcccaa acgaggtaca 1321 aaacatcaag ttcaacagct caggccaatg tgaagcaccc ttggtgcgaa cagacaaccc 1381 caagagctgg tatgaggacg tggagggctg tgggattcag tgtcagaacc cgctgttcac 1441 cgaggccgag caccaggaca tgcacagcta catcgcagcc ttcggggcgg tcactggtct 1501 ctgcacgctc ttcacactgg ccacctttgt ggctgactgg cggaactcca atcgctaccc 1561 tgcggttatt ctcttctatg tcaatgcgtg tttcttcgtg ggcagcattg gctggctggc 1621 ccagttcatg gatggtgccc gccgagagat tgtttgccga gcagatggca ccatgagatt 1681 tggggagccc acctccagtg agaccctgtc ctgtgtcatc atctttgtca ttgtgtacta 1741 tgccttgatg gctggagtag tctggttcgt ggtcctcacc tatgcctggc acacctcctt 1801 caaagccctg ggcaccacct accagcctct ctcgggcaag acatcctatt tccacctgct 1861 cacgtggtca ctcccctttg tcctcacggt ggcaatcctg gctgtggctc aggtagatgg 1921 agactccgtg agtggcatct gttttgtagg ctacaagaac tatcggtacc gtgctggctt 1981 tgtcctggcc ccaattggcc tggtgcttat tgtgggaggc tacttcctca tcagaggggt 2041 catgactctg ttctccatca agagcaacca ccctgggctt ctgagtgaga aggcagccag 2101 caagatcaac gagaccatgc tgcgcctggg catttttggc ttcctggcct ttggctttgt 2161 gctcatcacc ttcagctgcc acttctatga cttcttcaac caggctgagt gggagcgtag 2221 cttccgggac tatgtgctat gccaagccaa cgtgaccatc gggctgccta ccaagaagcc 2281 cattcctgac tgtgagatca agaatcggcc cagcctcctg gtggagaaga tcaatctatt 2341 tgccatgttt ggcactggca ttgccatgag cacctgggtc tggaccaagg ccaccctgct 2401 catctggagg cgcacctggt gcaggttgac tgggcacagt gatgatgagc ccaagagaat 2461 caagaagagc aagatgatcg ccaaggcctt ctctaagcgg cgtgagctgc tgcagaaccc 2521 gggccaggag ctctccttca gcatgcacac tgtctcccat gatggacctg ttgcgggttt 2581 ggcttttgac ctcaatgaac cctcagctga cgtctcctct gcctgggctc agcatgtcac 2641 caagatggtg gctcggagag gagccatatt gccccaggat gtgtccgtta cccctgtggc 2701 aactccagtg ccaccagaag aacaagccaa catgtggctg gttgaggcag agatctcccc 2761 agagttagag aagcgtttgg gccggaagaa aaagcggagg aagaggaaga aggaggtgtg 2821 ccccttgagg ccagcccctg agcttcacca ctctgcccct gttcctgcca ccagtgcagt 2881 tcctcggctg cctcagctgc ctcggcagaa gtgcctggta gctgcaaacg cctggggaac 2941 aggggagtcc tgccgacagg gagcctggac tctagtctcc aaccccttct gcccagagcc 3001 tagtccccat caagatccat ttctccctgg tgcctcagcc ccccgggtct gggctcaggg 3061 ccgcctccag gggctgggat ccattcattc ccgcactaac ctaatggagg ctgagatctt 3121 ggatgcagac tcggacttct gaggaattcc tcagaagtcc gagtctgcat ccggggcggc 3181 cagcggccat ggcggccgcg aattctgcag atatccatca cactggcggc cgctcgagca 3241 tgcatctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct 3301 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 3361 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 3421 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 3481 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg 3541 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg 3601 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 3661 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3721 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 3781 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 3841 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 3901 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 3961 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg 4021 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 4081 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 4141 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc 4201 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 4261 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt 4321 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc 4381 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg 4441 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 4501 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 4561 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct 4621 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 4681 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 4741 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 4801 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 4861 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 4921 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 4981 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 5041 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 5101 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 5161 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg 5221 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc 5281 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 5341 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat 5401 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 5461 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg 5521 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 5581 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 5641 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 5701 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 5761 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 5821 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 5881 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 5941 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6001 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6061 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 6121 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 6181 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6241 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6301 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 6361 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 6421 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 6481 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 6541 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 6601 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 6661 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 6721 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 6781 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 6841 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 6901 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 6961 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 7021 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 7081 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 7141 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 7201 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 7261 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 7321 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 7381 aaacgttctt cggggcaaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 7441 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 7501 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 7561 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 7621 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 7681 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt 7741 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt 7801 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct 7861 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg 7921 tacgggccag atatacgcgt t //