LOCUS Exported 8475 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor THRB. ACCESSION . VERSION . KEYWORDS pTHRB.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8475) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8475) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8475 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 169..454 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" intron 701..928 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 940..949 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 949..1014 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1021..1041 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1066..5166 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5167..5214 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5242..5259) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5248..5455 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5464..5604 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5464..5593 /label=AAV2 ITR rep_origin 5679..6134 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5766..5785) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5976..5997 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6151..6170) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6270..6292 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6330..6348) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6416..6520 /gene="bla" /label=AmpR promoter CDS 6521..7381 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(6739..6758) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7552..8140 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8041..8060 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8202..8442 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8202..8222 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8373..8392 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8472..72 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" ORIGIN 1 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 61 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg 121 tttttagcgc gtgcgccaat tctgcagaca aatggctcta gaggtacccg ttacataact 181 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt 241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 361 taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca 421 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt 481 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 541 attttgtgca gcgatggggg cggggggggg ggcggggcgg ggcggggcga ggcggagagg 601 tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg 661 gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct 721 tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc 781 gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag 841 caagaggtaa gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga 901 gcacctgcct gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac 961 cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc 1021 ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc 1081 atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 1141 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 1201 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 1261 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 1321 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 1381 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 1441 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 1501 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 1561 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 1621 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 1681 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 1741 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg 1801 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 1861 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 1921 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 1981 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 2041 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 2101 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 2161 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 2221 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 2281 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 2341 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 2401 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 2461 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 2521 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 2581 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 2641 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 2701 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 2761 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg 2821 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc 2881 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt 2941 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa 3001 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg 3061 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac 3121 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag 3181 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat 3241 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag 3301 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag 3361 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag 3421 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg 3481 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag 3541 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt 3601 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag 3661 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg 3721 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc 3781 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag 3841 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat 3901 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc 3961 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac 4021 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc 4081 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag 4141 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc 4201 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac 4261 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg 4321 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc 4381 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg 4441 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg 4501 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc 4561 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc 4621 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg 4681 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg 4741 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag 4801 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg 4861 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat 4921 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc 4981 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac 5041 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc 5101 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga 5161 ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa 5221 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 5281 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 5341 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 5401 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc 5461 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg 5521 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg 5581 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt 5641 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt 5701 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc 5761 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 5821 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 5881 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 5941 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 6001 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt 6061 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt 6121 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag 6181 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 6241 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 6301 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa 6361 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg 6421 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 6481 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 6541 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 6601 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 6661 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 6721 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 6781 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 6841 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 6901 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 6961 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 7021 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 7081 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 7141 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 7201 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact 7261 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 7321 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 7381 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 7441 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 7501 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 7561 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 7621 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 7681 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 7741 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 7801 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 7861 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 7921 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 7981 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 8041 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 8101 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 8161 tttacggttc ctggcctttt gctggccttt tgctcacatg tgagggccta tttcccatga 8221 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga 8281 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt 8341 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa 8401 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gtgttcgagg 8461 attagactga cgttt //