LOCUS Exported 3973 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses a guide RNA (gRNA) to target GFP (T2 target sequence) for genome engineering. ACCESSION . VERSION . KEYWORDS gRNA_GFP-T2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3973) AUTHORS Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville JE, Church GM TITLE RNA-Guided Human Genome Engineering via Cas9. JOURNAL Science. 2013 Jan 3. PUBMED 23287722 REFERENCE 2 (bases 1 to 3973) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3973 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /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)." primer_bind 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 239..257 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 239..256 /label=SP6 /note="SP6 promoter, forward primer" promoter 406..646 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 406..426 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 577..596 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 675..750 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind complement(762..786) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(861..880) /label=T7 /note="T7 promoter, forward primer" promoter complement(862..880) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(887..904) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(887..903) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(896..918) /label=M13/pUC Forward /note="In lacZ gene" CDS 1041..1343 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 1222..1241 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1692..2486 /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(1746..1765) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2356..2375 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 2686..2695 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2692..3066 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" rep_origin 3204..3792 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3693..3712 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3946..3963 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat 241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca 301 ctagtaacgg ccgccagtgt gctggaattc gccctttgta caaaaaagca ggctttaaag 361 gaaccaattc agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc 421 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat 481 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt 541 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact 601 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagcg 661 caccatcttc ttcagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat 721 caacttgaaa aagtggcacc gagtcggtgc tttttttcta gacccagctt tcttgtacaa 781 agttggcatt aaagggcgaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg 841 catctagagg gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt 901 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 961 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 1021 tgcgcagcct atacgtacgg cagtttaagg tttacaccta taaaagagag agccgttatc 1081 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc ggggcgacgg atggtgatcc 1141 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc 1201 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg 1261 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta 1321 acctgatgtt ctggggaata taaatgtcag gcatgagatt atcaaaaagg atcttcacct 1381 agatcctttt cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca 1441 gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca 1501 gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat 1561 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt 1621 tcttgccgcc aaggatctga tggcgcaggg gatcaagctc tgatcaagag acaggatgag 1681 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg 1741 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt 1801 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc 1861 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt 1921 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag 1981 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg 2041 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag 2101 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg 2161 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga 2221 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca 2281 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc 2341 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg 2401 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct 2461 atcgccttct tgacgagttc ttctgaatta ttaacgctta caatttcctg atgcggtatt 2521 ttctccttac gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat 2581 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 2641 agacaataac cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag 2701 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg 2761 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg 2821 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg 2881 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc 2941 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg 3001 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag 3061 gactgacacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 3121 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 3181 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 3241 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 3301 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 3361 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 3421 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 3481 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 3541 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 3601 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 3661 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 3721 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 3781 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 3841 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 3901 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 3961 gaggaagcgg aag //