LOCUS Exported 8508 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor MSX2. ACCESSION . VERSION . KEYWORDS pMSX2.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8508) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8508) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8508 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 271..346 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 443..728 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" intron 1008..1235 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1247..1256 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1256..1321 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1328..1348 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1373..5473 /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 5474..5521 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5549..5566) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5555..5762 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5771..5911 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5771..5900 /label=AAV2 ITR rep_origin 5986..6441 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6073..6092) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6283..6304 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6458..6477) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6577..6599 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6637..6655) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6723..6827 /gene="bla" /label=AmpR promoter CDS 6828..7688 /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(7046..7065) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7859..8447 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8348..8367 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg tggtactgcc ttcgtggaga gttttagagc tagaaatagc aagttaaaat 301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga 361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca 421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac 481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac 541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc 661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 841 gggggggggg gcgcgcgccg gggggggggg gggggggggg gggggggggg gggggggggc 901 gggggggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc 1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1261 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1321 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1381 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1441 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1501 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1561 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1621 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1681 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1741 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1801 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1861 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1921 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1981 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2041 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2101 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2161 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2221 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2281 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2341 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2401 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2461 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2521 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2581 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2641 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2701 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2761 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2821 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2881 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2941 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3001 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3061 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3121 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3181 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3241 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3301 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3361 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3421 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3481 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3541 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3601 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3661 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3721 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3781 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3841 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3901 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3961 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4021 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4081 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4141 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4201 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4261 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4321 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4381 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4441 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4501 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4561 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4621 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4681 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4741 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4801 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4861 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4921 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4981 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5041 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5101 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5161 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5221 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5281 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5341 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5401 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5461 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5521 gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5581 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5641 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5701 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5761 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5821 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5881 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5941 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6001 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6061 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6121 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6181 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6241 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6301 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga 6361 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6421 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6481 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6541 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6601 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6661 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6721 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6781 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6841 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6901 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6961 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7021 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7081 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7141 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7201 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7261 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7321 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7381 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7441 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7501 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7561 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7621 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7681 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7741 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7801 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7861 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7921 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7981 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 8041 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8101 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8161 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8221 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8281 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8341 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8401 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8461 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt //