LOCUS       sequence-199868-        3863 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector to express the 2nd gRNA with gRNA2.0 
            scaffold (with four MS2 binding sites) under AtU6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Plant. 
            2017 Nov 29. pii: S1674-2052(17)30344-1. doi: 
            10.1016/j.molp.2017.11.010."
COMMENT     Sequence Label: pYPQ132A2.0
FEATURES             Location/Qualifiers
     source          1..3863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(196..201,202..218,219..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      complement(3630..3657)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(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 agaggtctcc catgagaaat 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 ggaccgagac 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
//
