LOCUS Exported 3564 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses the S. pyogenes sgRNA from the 7SK promoter. ACCESSION . VERSION . KEYWORDS ph7SK-gRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3564) AUTHORS Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA TITLE Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. JOURNAL Nucleic Acids Res. 2014 Aug 13. pii: gku749. PUBMED 25122746 REFERENCE 2 (bases 1 to 3564) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3564 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_RNA 327..402 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(458..478) /label=T3 /note="T3 promoter, forward primer" promoter complement(458..476) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(497..513) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(497..513) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(510..532) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 521..537 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(545..575) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 590..611 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(728..745) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(899..1487) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(979..998) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1624..2418) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(1735..1754) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 2345..2364 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter complement(2453..2810) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2467..2602 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(2521..2540) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind 2767..2787 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter complement(2812..2916) /gene="bla" /label=AmpR promoter rep_origin complement(2942..3397) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3079..3100) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3291..3310 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3524..3546 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3538..3555 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3539..3555 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 taatacgact cactataggg cgaattgggt acctttaatt ctagtactat gcatcgtctc 61 attgtctgca gtatttagca tgccccaccc atctgcaagg cattctggat agtgtcaaaa 121 cagccggaaa tcaagtccgt ttatctcaaa ctttagcatt ttgggaataa atgatatttg 181 ctatgctggt taaattagat tttagttaaa tttcctgctg aagctctagt acgataagca 241 acttgaccta agtgtaaagt tgagacttcc ttcaggttta tatagcttgt gcgccgcttg 301 ggtacctcgg gtcttcgaga agacctgttt tagagctaga aatagcaagt taaaataagg 361 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc tcggactgat 421 gacctgagac gccgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc 481 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 541 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 601 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 661 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 721 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 781 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 841 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 901 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 961 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 1021 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 1081 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 1141 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 1201 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 1261 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 1321 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 1381 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 1441 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 1501 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 1561 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcatca 1621 tgatcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc 1681 gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc 1741 acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat 1801 gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt 1861 cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg 1921 cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg 1981 agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc 2041 aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag 2101 gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc 2161 ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag 2221 ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag 2281 aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg 2341 ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa 2401 tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa 2461 agcctaggcc ttcaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg 2521 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac 2581 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat 2641 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg 2701 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact 2761 ttccacaccc taactgacac acattccaca gctggttctt tccgcctcag gactcttcct 2821 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 2881 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 2941 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca 3001 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag 3061 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc 3121 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc 3181 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc 3241 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa 3301 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc 3361 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc 3421 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg 3481 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt 3541 aaaacgacgg ccagtgagcg cgcg //