LOCUS Exported 4820 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Transcription of guide RNA (gRNA) in maize cells. ACCESSION . VERSION . KEYWORDS pZmU3-gRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4820) AUTHORS Liang Z, Zhang K, Chen K, Gao C TITLE Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System. JOURNAL J Genet Genomics. 2014 Feb 20;41(2):63-8. doi: 10.1016/j.jgg.2013.12.001. Epub 2013 Dec 14. PUBMED 24576457 REFERENCE 2 (bases 1 to 4820) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4820 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(11..28) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(182..770) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(262..281) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(941..1801) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 1564..1583 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" CDS complement(1819..2613) /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(1930..1949) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 2540..2559 /label=Neo-R /note="Neomycin resistance gene, reverse primer" rep_origin complement(2957..3385) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3067..3088) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3279..3298 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3511..3533 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3525..3542 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3526..3542 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 3549..3568 /label=T7 /note="T7 promoter, forward primer" promoter 3549..3567 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_RNA 4423..4498 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(4600..4616) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4600..4616) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4613..4635) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4624..4640 /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(4648..4678) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4693..4714 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." ORIGIN 1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1801 tactcttcct ttttcaattc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg 1861 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag 1921 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag 1981 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca 2041 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc 2101 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag 2161 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg 2221 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt 2281 ctcggcagga gcaaggtggg atgacaggag atcctgcccc ggcacttcgc ccaatagcag 2341 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt 2401 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc 2461 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga 2521 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg 2581 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg 2641 atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 2701 tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc 2761 tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt 2821 tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg 2881 tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc agcccttgcg 2941 ccctgaattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa 3001 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt 3061 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 3121 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt 3181 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga 3241 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg 3301 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg 3361 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag 3421 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 3481 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 3541 gtgaattgta atacgactca ctatagggcg aattgggccc tctagatgca tgctcgagcg 3601 gccgccagtg tgatggatat ctgcagaatt gcccttgaat tccatctaag tatcttggta 3661 aagcatggat taatttggat gcccacttca ggtctatgca gctccggtgc cttgtgattg 3721 tgagttgtga ccgatgctca tgctattctg catttctgcg atgtatgtag ctagtagatc 3781 ttcaaaacta acaccgcatg ccatcatcat ccactgcttg attttagtct caccgctggc 3841 caaaaatgtg atgatgccag aaacctcaac taccttgaat caacacgggc ccaacagtgt 3901 gatgacgaca gaaacaaaaa aaaatgagcc aatagttcag aaggaggcac tatgcagaaa 3961 ctacatttct gaaggtgact aaaaggtgag cgtagagtgt aattactagt agtttagcca 4021 ccattaccca aatgctttcg agcttgtatt aagatttcct aagctgagca tcatcactga 4081 tctgcaggcc accctcgctt cgctgccaag atcaacagca accatgtggc ggcaacatcc 4141 agcattgcac atgggctaaa gattgagctt tgtgcctcgt ctagggatca gctgaggtta 4201 tcagtctttc ctttttttca tccaggtgag gcatcaagct actactgcct cgattggctg 4261 gacccgaagc ccacatgtag gataccagaa tgggccgacc caggacgcag tatgttggcc 4321 agtcccaccg gttagtgcca tctcggttgc tcacatgcgt agaagccagc ttaaaaattt 4381 agctttggtg actcacagca ctgtcttcaa cacagaagac acgttttaga gctagaaata 4441 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 4501 tttttggtac cggatcctac ggtggctaag ggcaattcca gcacactggc ggccgttact 4561 agtggatccg agctcggtac caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 4621 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 4681 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 4741 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 4801 cggtttgcgt attgggcgct //