LOCUS Exported 3863 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Golden Gate entry vector to express the 1st gRNA with gRNA2.0 scaffold (with four MS2 binding sites) under AtU6 promoter. ACCESSION . VERSION . KEYWORDS pYPQ131A2.0 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3863) AUTHORS Lowder LG, Zhou J, Zhang Y, Malzahn A, Zhong Z, Hsieh TF, Voytas DF, Zhang Y, Qi Y TITLE Robust transcriptional activation in plants using multiplexed CRISPR-Act2.0 and mTALE-Act systems. JOURNAL Mol Plant. 2017 Nov 29. pii: S1674-2052(17)30344-1. doi: 10.1016/j.molp.2017.11.010. PUBMED 29197638 REFERENCE 2 (bases 1 to 3863) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3863 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 196..224 /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" CDS 272..1462 /codon_start=1 /gene="tet" /product="tetracycline efflux protein" /label=TcR /note="confers resistance to tetracycline" /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST" primer_bind complement(624..643) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind complement(1636..1655) /label=T7 /note="T7 promoter, forward primer" promoter complement(1637..1655) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1660..1676) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1660..1676) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 1765..2068 /label=AtU6-1 promoter /note="promoter for the Arabidopsis thaliana U6-1 snRNA gene (Waibel and Filipowicz, 1990)" misc_RNA 2119..2137 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" misc_RNA 2189..2207 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" rep_origin 2712..3300 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3201..3220 /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 3630..3657 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 3749..3835 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" ORIGIN 1 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 61 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 121 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 181 aattaattct catgtttgac agcttatcat cgattagctt taatgcggta gtttatcaca 241 gttaaattgc taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc tcatcgtcat 301 cctcggcacc gtcaccctgg atgctgtagg cataggcttg gttatgccgg tactgccggg 361 cctcttgcgg gatatcgtcc attccgacag catcgccagt cactatggcg tgctgctagc 421 gctatatgcg ttgatgcaat ttctatgcgc acccgttctc ggagcactgt ccgaccgctt 481 tggccgccgc ccagtcctgc tcgcttcgct acttggagcc actatcgact acgcgatcat 541 ggcgaccaca cccgtcctgt ggattctcta cgccggacgc atcgtggccg gcatcaccgg 601 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc 661 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc 721 cgggggactg ttgggcgcca tctccttaca tgcaccattc cttgcggcgg cggtgctcaa 781 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgccg 841 acccatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac 901 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 961 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct 1021 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc 1081 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg acgcgctggg 1141 ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta tgattcttct 1201 cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga 1261 cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa cttcgatcat 1321 tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga acgggttggc 1381 atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg 1441 gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact 1501 ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttga 1561 tcggggaaga acagtatgtc gagctatttt ttgacttact ggggatcaag cctgattggg 1621 agaaaataaa atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag 1681 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca 1741 tgcctcctct agaggtctcg ctatagaaat ctcaaaattc cggcagaaca attttgaatc 1801 tcgatccgta gaaaccagac ggtcattgtt ttagttccac cacgattata tttgaaattt 1861 acgtgagtgt gagtgagact tgcataagaa aataaaatct ttagttggga aaaaattcaa 1921 taatataaat gggcttgaga aggaagcgag ggataggcct ttttctaaaa taggcccatt 1981 taagctatta acaatcttca aaagtaccac agcgcttagg taaagaaagc agctgagttt 2041 atatatggtt agacacgaag tagtgattgg agacgagatc tagtctgagt cgaccgtctc 2101 tgttttagag ctaggccaac atgaggatca cccatgtctg cagggcctag caagttaaaa 2161 taaggctagt ccgttatcaa cttggccaac atgaggatca cccatgtctg cagggccaag 2221 tggcaccgag tcggtgcttt ttttggcaaa aattttcaga ttttttcttc atctgtagat 2281 ttctgggttt ttttttccgt ttcgtgaatc ataagtgaag ttttggatgc aaatctgcgc 2341 gaaaaaagtt ggacctgcaa tgagcttatt tagatagcta agacaaagtg attggtccgt 2401 tgtttcagtt ctgattgtca gagagtttgt ttcgagtcgg cgacaccaat gcgttttgtt 2461 aaccagattt cgggtaagaa atgtatcgag agtttgtttc cagacggcta catcattttc 2521 ttatgaaggg tgaaattaga tagaccaaag attgaaacac aacatttctt tcacaaaaat 2581 ataataaact tgatagcatt taggatcagc catgggagac cctcgagcca cccatgacca 2641 aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc 2701 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 2761 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 2821 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 2881 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 2941 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 3001 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 3061 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 3121 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 3181 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 3241 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 3301 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 3361 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 3421 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 3481 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg 3541 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tacgcgtacc 3601 gctagccagg aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg gccttctgct 3661 tagtttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg ggccgttgct 3721 tcacaacgtt caaatccgct cccggcggat ttgtcctact caggagagcg ttcaccgaca 3781 aacaacagat aaaacgaaag gcccagtctt ccgactgagc ctttcgtttt atttgatgcc 3841 tggcagttcc ctactctcgc gtt //