pCas-Guide-EF1a-GFPAll-in-one vectorExpresses human codon-optimized Cas9, C-terminal Myc-DDK taggedBamH I and BsmB I can be used for genomic target sequence cloningExpresses gRNA containing the inserted target sequence and the gRNA scaffoldGFP as a reporter for tracking or sorting transfected cellsCF3 primer can be used to sequence the target sequence after cloning
LOCUS Exported File 10493 bp ds-DNA circular SYN 23-七月-2016DEFINITION Mammalian vector for co-expressing a guide RNA (gRNA) together with Cas9 and TurboGFP, for genome editing using the S. pyogenes CRISPR/Cas9 system.ACCESSION .VERSION .KEYWORDS pCas-Guide-EF1a-GFPSOURCE synthetic DNA construct ORGANISM synthetic DNA constructREFERENCE 1 (bases 1 to 10493) AUTHORS OriGene TITLE Direct Submission JOURNAL Exported 2016-07-23 from SnapGene Viewer 1.5.3 http://www.snapgene.comFEATURES Location/Qualifiers source 1..10493 /organism="synthetic DNA construct" /lab_host="Mammalian Cells" /mol_type="other DNA" promoter 90..330 /note="U6" /note="RNA polymerase III promoter for human U6 snRNA" /note="color: #000000; direction: RIGHT" misc_RNA 384..459 /note="gRNA scaffold" /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" /note="color: #00ccff; direction: RIGHT" terminator 460..465 /note="polIII terminator" /note="RNA polymerase III transcription terminator" /note="color: #ffffff" promoter 480..1658 /note="EF-1-alpha promoter" /note="strong constitutive promoter for human elongation factor EF-1-alpha" /note="color: #000000; direction: RIGHT" intron 711..1649 /note="EF-1-alpha intron A" /note="intron upstream of the start codon of human EF-1-alpha" /note="color: #------" CDS 1737..2432 /codon_start=1 /product="improved green fluorescent protein from Pontellina plumata?(Evdokimov et al., 2006)" /note="TurboGFP" /note="mammalian codon-optimized" /note="color: #05fd14" /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKM KSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVL HVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNDLDGSFTR TFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHSNTELGIVEYQHAF KTPDADAGEE" enhancer 3047..3350 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" /note="color: #000000" promoter 3351..3554 /note="CMV promoter" /note="human cytomegalovirus (CMV) immediate early promoter" /note="color: #000000; direction: RIGHT" promoter 3580..3598 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" /note="color: #ffffff; direction: RIGHT" CDS 3657..7760 /codon_start=1 /locus_tag=" " /product="Cas9 (Csn1) endonuclease from the?Streptococcus pyogenes?Type II CRISPR/Cas system" /note="Cas9" /note="generates RNA-guided double strand breaks in DNA" /note="color: #993366" /protein_id=" " /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 7761..7781 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /note="color: #993366" /protein_id=" " /translation="PKKKRKV" CDS 7788..7808 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /note="color: #993366" /protein_id=" " /translation="PKKKRKV" CDS 7830..7859 /codon_start=1 /product="Myc (human c-Myc oncogene) epitope tag" /note="Myc" /note="color: #cc99b2" /translation="EQKLISEEDL" CDS 7878..7901 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /note="FLAG" /note="color: #cc99b2" /translation="DYKDDDDK" polyA_signal 7948..8570 /note="hGH poly(A) signal" /note="human growth hormone polyadenylation signal" /note="color: #a6acb3" rep_origin complement(8691..9279) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /note="color: #ffff00" CDS complement(9454..10314) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /note="This feature has 2 segments: ???1:??9454?..?10245?/?#cc99ff ???2:?10246?..?10314?/?#cc99ff?/?signal?sequence Cleavage site after base 10245" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW"ORIGIN 1 gaattcccca gtggaaagac gcgcaggcaa aacgcaccac gtgacggagc gtgaccgcgc 61 gccgagcgcg cgccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt 121 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa 181 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt 241 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt 301 tcttgggttt atatatcttg tggaaaggac gcgggatcca ctggaccagg cagcagcgtc 361 agaagacttt tttggaacgt ctcgttttag agctagaaat agcaagttaa aataaggcta 421 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttggtg tacacgtgag 481 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 541 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 601 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 661 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 721 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 781 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 841 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 901 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 961 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 1021 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 1081 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 1141 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 1201 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 1261 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 1321 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 1381 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt 1441 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1501 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1561 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1621 tggttcaaag tttttttctt ccatttcagg tgtcgtgact atagggcggc cggacgtgac 1681 aaatggaagt agcacgcctc actaggctcg tgcagatgga cagcaccgct gcagccatgg 1741 agagcgacga gagcggcctg cccgccatgg agatcgagtg ccgcatcacc ggcaccctga 1801 acggcgtgga gttcgagctg gtgggcggcg gagagggcac ccccgagcag ggccgcatga 1861 ccaacaagat gaagagcacc aaaggcgccc tgaccttcag cccctacctg ctgagccacg 1921 tgatgggcta cggcttctac cacttcggca cctaccccag cggctacgag aaccccttcc 1981 tgcacgccat caacaacggc ggctacacca acacccgcat cgagaagtac gaggacggcg 2041 gcgtgctgca cgtgagcttc agctaccgct acgaggccgg ccgcgtgatc ggcgacttca 2101 aggtgatggg caccggcttc cccgaggaca gcgtgatctt caccgacaag atcatccgca 2161 gcaacgccac cgtggagcac ctgcacccca tgggcgataa cgatctggat ggcagcttca 2221 cccgcacctt cagcctgcgc gacggcggct actacagctc cgtggtggac agccacatgc 2281 acttcaagag cgccatccac cccagcatcc tgcagaacgg gggccccatg ttcgccttcc 2341 gccgcgtgga ggaggatcac agcaacaccg agctgggcat cgtggagtac cagcacgcct 2401 tcaagacccc ggatgcagat gccggtgaag aaagagttta atcgatgata tcagatcccc 2461 gggatgcaga aattgatgat ctattaaaca ataaagatgt ccactaaaat ggaagttttt 2521 cctgtcatac tttgttaaga agggtgagaa cagagtacct acattttgaa tggaaggatt 2581 ggagctacgg gggtgggggt ggggtgggat tagataaatg cctgctcttt actgaaggct 2641 ctttactatt gctttatgat aatgtttcat agttggatat cataatttaa acaagcaaaa 2701 ccaaattaag ggccagctca ttcctcccac tcatgatcta tagatctata gatctctcgt 2761 gggatcattg tttttctctt gattcccact ttgtggttct aagtactgtg gtttccaaat 2821 gtgtcagttt catagcctga agaacgagat cagcagcctc tgttccacat acacttcatt 2881 ctcagtattg ttttgccaag ttctaattcc atcagaagct ggtcgagatc cggaaccctt 2941 aatataactt cgtataatgt atgctatacg aagttattag gtccactagt tattaatagt 3001 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 3061 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 3121 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 3181 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta 3241 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg 3301 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 3361 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 3421 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 3481 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 3541 atataagcag agctcgttta gtgaaccgtc agaattttgt aatacgactc actatagggc 3601 ggccgggaat tcgtcgactg gaaccggtac cgaggagatc tgccgccgcg atcgccatgg 3661 ataagaaata ctcaatagga ctggatattg gcacaaatag cgtcggatgg gctgtgatca 3721 ctgatgaata taaggttcct tctaaaaagt tcaaggttct gggaaataca gaccgccaca 3781 gtatcaaaaa aaatcttata ggggctcttc tgtttgacag tggagagaca gccgaagcta 3841 ctagactcaa acggacagct aggagaaggt atacaagacg gaagaatagg atttgttatc 3901 tccaggagat tttttcaaat gagatggcca aagtggatga tagtttcttt catagacttg 3961 aagagtcttt tttggtggaa gaagacaaga agcatgaaag acatcctatt tttggaaata 4021 tagtggatga agttgcttat cacgagaaat atccaactat ctatcatctg agaaaaaaat 4081 tggtggattc tactgataaa gccgatttgc gcctgatcta tttggccctg gcccacatga 4141 ttaagtttag aggtcatttt ttgattgagg gcgatctgaa tcctgataat agtgatgtgg 4201 acaaactgtt tatccagttg gtgcaaacct acaatcaact gtttgaagaa aaccctatta 4261 acgcaagtgg agtggatgct aaagccattc tttctgcaag attgagtaaa tcaagaagac 4321 tggaaaatct cattgctcag ctccccggtg agaagaaaaa tggcctgttt gggaatctca 4381 ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg 4441 ctaaactcca gctttcaaaa gatacttacg atgatgatct ggataatctg ttggctcaaa 4501 ttggagatca atatgctgat ttgtttttgg cagctaagaa tctgtcagat gctattctgc 4561 tttcagacat cctgagagtg aatactgaaa taactaaggc tcccctgtca gcttcaatga 4621 ttaaacgcta cgatgaacat catcaagact tgactcttct gaaagccctg gttagacaac 4681 aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt 4741 atattgatgg cggcgcaagc caagaagaat tttataaatt tatcaaacca attctggaaa 4801 aaatggatgg tactgaggaa ctgttggtga aactgaatag agaagatttg ctgcgcaagc 4861 aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta 4921 ttttgagaag acaagaagac ttttatccat ttctgaaaga caatagagag aagattgaaa 4981 aaatcttgac ttttaggatt ccttattatg ttggtccatt ggccagaggc aatagtaggt 5041 ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg 5101 tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc 5161 ttccaaatga aaaagtgctg ccaaaacata gtttgcttta tgagtatttt accgtttata 5221 acgaattgac aaaggtcaaa tatgttactg aaggaatgag aaaaccagca tttctttcag 5281 gtgaacagaa gaaagccatt gttgatctgc tcttcaaaac aaataggaaa gtgaccgtta 5341 agcaactgaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag 5401 gagttgaaga tagatttaat gcttcactgg gtacatacca tgatttgctg aaaattatta 5461 aagataaaga ttttttggat aatgaagaaa atgaagacat cctggaggat attgttctga 5521 cattgaccct gtttgaagat agggagatga ttgaggaaag acttaaaaca tacgctcacc 5581 tctttgatga taaggtgatg aaacagctta aaagacgcag atatactggt tggggaaggt 5641 tgtccagaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atactggatt 5701 ttttgaaatc agatggtttt gccaatcgca attttatgca gctcatccat gatgatagtt 5761 tgacatttaa agaagacatc caaaaagcac aagtgtctgg acaaggcgat agtctgcatg 5821 aacatattgc aaatctggct ggtagccctg ctattaaaaa aggtattctc cagactgtga 5881 aagttgttga tgaattggtc aaagtgatgg ggcggcataa gccagaaaat atcgttattg 5941 aaatggcaag agaaaatcag acaactcaaa agggccagaa aaattccaga gagaggatga 6001 aaagaatcga agaaggtatc aaagaactgg gaagtcagat tcttaaagag catcctgttg 6061 aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagata 6121 tgtatgtgga ccaagaactg gatattaata ggctgagtga ttatgatgtc gatcacattg 6181 ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcctgacc aggtctgata 6241 aaaatagagg taaatccgat aacgttccaa gtgaagaagt ggtcaaaaag atgaaaaact 6301 attggagaca acttctgaac gccaagctga tcactcaaag gaagtttgat aatctgacca 6361 aagctgaaag aggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg 6421 ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta 6481 aatacgatga aaatgataaa cttattagag aggttaaagt gattaccctg aaatctaaac 6541 tggtttctga cttcagaaaa gatttccaat tctataaagt gagagagatt aacaattacc 6601 atcatgccca tgatgcctat ctgaatgccg tcgttggaac tgctttgatt aagaaatatc 6661 caaaacttga aagcgagttt gtctatggtg attataaagt ttatgatgtt aggaaaatga 6721 ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa gtatttcttt tactctaata 6781 tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc 6841 tgatcgaaac taatggggaa actggagaaa ttgtctggga taaagggaga gattttgcca 6901 cagtgcgcaa agtgttgtcc atgccccaag tcaatatcgt caagaaaaca gaagtgcaga 6961 caggcggatt ctctaaggag tcaattctgc caaaaagaaa ttccgacaag ctgattgcta 7021 ggaaaaaaga ctgggaccca aaaaaatatg gtggttttga tagtccaacc gtggcttatt 7081 cagtcctggt ggttgctaag gtggaaaaag ggaaatccaa gaagctgaaa tccgttaaag 7141 agctgctggg gatcacaatt atggaaagaa gttcctttga aaaaaatccc attgactttc 7201 tggaagctaa aggatataag gaagttaaaa aagacctgat cattaaactg cctaaatata 7261 gtctttttga gctggaaaac ggtaggaaac ggatgctggc tagtgccgga gaactgcaaa 7321 aaggaaatga gctggctctg ccaagcaaat atgtgaattt tctgtatctg gctagtcatt 7381 atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc 7441 ataagcatta tctggatgag attattgagc aaatcagtga attttctaag agagttattc 7501 tggcagatgc caatctggat aaagttctta gtgcatataa caaacataga gacaaaccaa 7561 taagagaaca agcagaaaat atcattcatc tgtttacctt gaccaatctt ggagcacccg 7621 ctgcttttaa atactttgat acaacaattg ataggaaaag atatacctct acaaaagaag 7681 ttctggatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt