LOCUS Exported 3504 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 3504) 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 3504) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3504 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_RNA 952..1027 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(1083..1103) /label=T3 /note="T3 promoter, forward primer" promoter complement(1083..1101) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(1122..1138) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1122..1138) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1135..1157) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1146..1162 /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(1170..1200) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1215..1236 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(1353..1370) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1524..2112) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1604..1623) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2189..2983) /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(2300..2319) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 2910..2929 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter complement(3018..3375) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3032..3167 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(3086..3105) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind 3332..3352 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter complement(3377..3481) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacctt 661 taattctagt actatgcatc gtctcattgt ctgcagtatt tagcatgccc cacccatctg 721 caaggcattc tggatagtgt caaaacagcc ggaaatcaag tccgtttatc tcaaacttta 781 gcattttggg aataaatgat atttgctatg ctggttaaat tagattttag ttaaatttcc 841 tgctgaagct ctagtacgat aagcaacttg acctaagtgt aaagttgaga cttccttcag 901 gtttatatag cttgtgcgcc gcttgggtac ctcgggtctt cgagaagacc tgttttagag 961 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 1021 tcggtgcttt ttttctcgga ctgatgacct gagacgccgc ggtggagctc cagcttttgt 1081 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg 1141 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 1201 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 1261 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 1321 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 1381 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 1441 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 1501 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 1561 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 1621 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 1681 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 1741 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 1801 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 1861 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 1921 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 1981 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 2041 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 2101 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 2161 aactcacgtt aagggatttt ggtcatgatc agaagaactc gtcaagaagg cgatagaagg 2221 cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt 2281 cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg 2341 ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat 2401 tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct 2461 tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct 2521 gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt 2581 ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga 2641 tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc 2701 ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa 2761 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac 2821 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg 2881 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc 2941 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc 3001 ctgtctcttg atcgatcttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc 3061 tggaatagct cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag 3121 ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg 3181 ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg 3241 gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt 3301 ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt ccacagctgg 3361 ttctttccgc ctcaggactc ttcctttttc aatattattg aagcatttat cagggttatt 3421 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 3481 gcacatttcc ccgaaaagtg ccac //