LOCUS Exported 4233 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION pMAGIC L1-R5 entry plasmid, contains empty human U6-driven SaCas9 gRNA scaffold and EF1a promoter for 4-component MultiSite Gateway Pro assembly. Protospacer motif can be inserted after BsaI digestion. ACCESSION . VERSION . KEYWORDS KZ801: pMAGIC (L1-R5) hU6::SaCas9 gRNA scaffold; EF1a promoter SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4233) AUTHORS Haldeman JM, Conway AE, Arlotto ME, Slentz DH, Muoio DM, Becker TC, Newgard CB TITLE Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing. JOURNAL Nucleic Acids Res. 2018 Dec 27. pii: 5264291. doi: 10.1093/nar/gky1286. PUBMED 30590691 REFERENCE 2 (bases 1 to 4233) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4233 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 92..109 /label=L4440 /note="L4440 vector, forward primer" terminator 268..295 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 387..473 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 522..544 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 536..553 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 537..553 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 569..668 /gene="mutant version of attL" /label=attL1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 679..919 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 679..699 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 850..869 /label=LKO.1 5' /note="Human U6 promoter, forward primer" promoter 1069..2247 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 1300..2238 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 2195..2215 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" promoter 2264..2282 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 2296..2419 /gene="mutant version of attR" /label=attR5 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter complement(2475..2493) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2498..2514) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2498..2514) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2616..2635) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 2627..3436 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(2694..2713) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 3583..4171 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4072..4091 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 661 agcaggctta tggtacctga gggcctattt cccatgattc cttcatattt gcatatacga 721 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta 781 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt 841 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta 901 tatatcttgt ggaaaggacg aaacaccgga gaccacggca ggtctcagtt taagtactct 961 gtgctggaaa cagcacagaa tctactaaac aaggcaaaat gccgtgttta tctcgtcaac 1021 ttgttggcga gatttttgcg gccgctgtac atacgactct agacgtgagg ctccggtgcc 1081 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc 1141 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac 1201 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg 1261 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc 1321 cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg 1381 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg 1441 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg 1501 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct 1561 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt 1621 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac 1681 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg 1741 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg 1801 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg 1861 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg 1921 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc 1981 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc 2041 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac 2101 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa 2161 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt 2221 ttttttcttc catttcaggt gtcgtgagga attagcttgg tactaatacg actcactata 2281 gggagacgaa ttccacaact ttgtatacaa aagttgaacg agaaacgtaa aatgatataa 2341 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac 2401 aacatatcca gtcactatga atcaactact tagatggtat tagtgacctg tactgcaggg 2461 cggccgcgat atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct 2521 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg 2581 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca 2641 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa 2701 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc 2761 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga 2821 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt 2881 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt 2941 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt 3001 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt 3061 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga 3121 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc 3181 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga 3241 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca 3301 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct 3361 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct 3421 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct 3481 gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt 3541 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 3601 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 3661 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 3721 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 3781 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 3841 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 3901 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 3961 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 4021 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 4081 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 4141 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 4201 cctggccttt tgctggcctt ttgctcacat gtt //