LOCUS Exported 8465 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor MXD1. ACCESSION . VERSION . KEYWORDS pMXD1.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8465) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8465) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8465 /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 691..918 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 930..939 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 939..1004 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1011..1031 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1056..5156 /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 5157..5204 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5232..5249) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5238..5445 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5454..5594 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5454..5583 /label=AAV2 ITR rep_origin 5669..6124 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5756..5775) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5966..5987 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6141..6160) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6260..6282 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6320..6338) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6406..6510 /gene="bla" /label=AmpR promoter CDS 6511..7371 /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(6729..6748) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7542..8130 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8031..8050 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8192..8432 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8192..8212 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8363..8382 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8462..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 cggggggsgg ggcggggcga ggcggagagg tgcggcggca 601 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg 661 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg 721 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca 781 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag caagaggtaa 841 gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct 901 gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac cacgacggag 961 actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga 1021 agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg 1081 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca 1141 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag 1201 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa 1261 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga 1321 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg 1381 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg 1441 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg 1501 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga 1561 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc 1621 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg 1681 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc 1741 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc 1801 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca 1861 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt 1921 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca 1981 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc 2041 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga 2101 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg 2161 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc 2221 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca 2281 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt 2341 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct 2401 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg 2461 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga 2521 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca 2581 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg 2641 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg 2701 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca 2761 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct 2821 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg 2881 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag 2941 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc 3001 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca 3061 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca 3121 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga 3181 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca 3241 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag 3301 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca 3361 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag 3421 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga 3481 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca 3541 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg 3601 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg 3661 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca 3721 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg 3781 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc 3841 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga 3901 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt 3961 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga 4021 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt 4081 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg 4141 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga 4201 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg 4261 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc 4321 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta 4381 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga 4441 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg 4501 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg 4561 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag 4621 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc 4681 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct 4741 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg 4801 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca 4861 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag 4921 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca 4981 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca 5041 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc 5101 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa 5161 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa ttcctagagc 5221 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 5281 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 5341 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 5401 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc 5461 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 5521 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 5581 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 5641 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg 5701 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct 5761 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 5821 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 5881 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 5941 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 6001 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta 6061 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca 6121 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga 6181 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 6241 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 6301 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 6361 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 6421 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 6481 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 6541 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 6601 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 6661 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 6721 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 6781 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 6841 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 6901 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 6961 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 7021 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 7081 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 7141 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 7201 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat 7261 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 7321 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 7381 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 7441 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 7501 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 7561 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 7621 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 7681 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 7741 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 7801 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 7861 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 7921 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 7981 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 8041 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 8101 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 8161 ctggcctttt gctggccttt tgctcacatg tgagggccta tttcccatga ttccttcata 8221 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac 8281 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt 8341 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga 8401 tttcttggct ttatatatct tgtggaaagg acgaaacacc gcactgcggc tgtctccttg 8461 agttt //