LOCUS Exported 3519 bp ds-DNA linear SYN 07-JUN-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 3519) 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 3519) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 7, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3519 /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" CDS 217..888 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSYLGDTIEYSSYASSLVPSSDPLVTAASVLEFALKGEFC RYPSHWRPLEHASRGPNSPYSESYYNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRN SEEARTDRPSQQLRSLYVRQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARL LSDKVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWG I" promoter 239..257 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 239..256 /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(406..425) /label=T7 /note="T7 promoter, forward primer" promoter complement(407..425) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(432..449) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(432..448) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(441..463) /label=M13/pUC Forward /note="In lacZ gene" primer_bind 767..786 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1237..2031 /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(1291..1310) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 1901..1920 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 2232..2241 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2238..2612 /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 2750..3338 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3239..3258 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3492..3509 /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 gcccttaagg gcgaattctg cagatatcca 361 tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg 421 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 481 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 541 cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac 601 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac 661 acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc 721 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc 781 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc 841 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg 901 agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa 961 cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc 1021 gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt 1081 ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag 1141 ccctgcaaag taaactggat ggctttctcg ccgccaagga tctgatggcg caggggatca 1201 agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac 1261 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca 1321 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 1381 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg 1441 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga 1501 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct 1561 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg 1621 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg 1681 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc 1741 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat 1801 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac 1861 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt 1921 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct 1981 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac 2041 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 2101 atacaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 2161 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatagc 2221 acgtgaggag ggccaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg 2281 acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg 2341 aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg 2401 accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt 2461 acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga 2521 ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact 2581 gcgtgcactt cgtggccgag gagcaggact gacacgtgct aaaacttcat ttttaattta 2641 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 2701 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 2761 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 2821 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 2881 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 2941 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 3001 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 3061 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 3121 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 3181 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 3241 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 3301 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 3361 tacggttcct gggcttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 3421 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 3481 cgaccgagcg cagcgagtca gtgagcgagg aagcggaag //