LOCUS Exported 8472 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor CSDC2. ACCESSION . VERSION . KEYWORDS pCSDC2.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8472) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8472) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8472 /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 698..925 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 937..946 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 946..1011 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1018..1038 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1063..5163 /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 5164..5211 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5239..5256) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5245..5452 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5461..5601 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5461..5590 /label=AAV2 ITR rep_origin 5676..6131 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5763..5782) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5973..5994 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6148..6167) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6267..6289 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6327..6345) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6413..6517 /gene="bla" /label=AmpR promoter CDS 6518..7378 /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(6736..6755) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7549..8137 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8038..8057 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8199..8439 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8199..8219 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8370..8389 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8469..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 ggggcggggc ggggcgaggc ggagaggtgc 601 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg 661 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg 721 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 781 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa 841 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca 901 cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac 961 gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca 1021 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc 1081 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg 1141 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg 1201 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc 1261 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc 1321 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg 1381 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc 1441 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac 1501 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac 1561 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag 1621 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac 1681 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc 1741 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc 1801 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc 1861 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc 1921 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga 1981 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag 2041 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac 2101 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc 2161 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag 2221 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac 2281 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa 2341 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc 2401 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga 2461 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc 2521 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg 2581 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg 2641 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc 2701 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac 2761 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc 2821 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg 2881 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag 2941 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg 3001 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc 3061 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc 3121 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac 3181 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg 3241 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc 3301 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac 3361 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc 3421 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag 3481 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa 3541 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg 3601 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc 3661 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg 3721 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc 3781 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc 3841 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac 3901 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg 3961 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc 4021 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag 4081 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag 4141 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag 4201 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc 4261 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg 4321 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa 4381 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac 4441 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc 4501 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc 4561 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac 4621 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa 4681 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc 4741 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc 4801 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac 4861 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg 4921 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag 4981 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt 5041 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg 5101 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc 5161 gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc 5221 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 5281 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 5341 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 5401 ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc 5461 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 5521 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 5581 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 5641 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 5701 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc 5761 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 5821 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 5881 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 5941 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 6001 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta 6061 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 6121 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 6181 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 6241 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 6301 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 6361 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 6421 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 6481 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 6541 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 6601 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 6661 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 6721 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 6781 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 6841 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 6901 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 6961 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 7021 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 7081 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 7141 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 7201 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg 7261 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 7321 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 7381 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 7441 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 7501 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7561 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7621 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7681 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7741 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 7801 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 7861 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 7921 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 7981 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 8041 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 8101 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 8161 acggttcctg gccttttgct ggccttttgc tcacatgtga gggcctattt cccatgattc 8221 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg 8281 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 8341 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 8401 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgtg gccaggtcgt 8461 gggctcctgt tt //