LOCUS       sequence-406945-        3803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector; 2nd gRNA under AtU6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3803)
  AUTHORS   Lowder LG, Paul JW, Baltes NJ, Voytas DF, Zhang Y, Zhang D, Tang X, 
            Zheng X, Hsieh TF, Qi Y
  TITLE     A CRISPR/Cas9 toolbox for multiplexed plant genome editing and 
            transcriptional regulation.
  JOURNAL   Plant Physiol. 2015 Aug 21. pii: pp.00636.2015.
  PUBMED    26297141
REFERENCE   2  (bases 1 to 3803)
  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: "Plant 
            Physiol. 2015 Aug 21. pii: pp.00636.2015."
COMMENT     Sequence Label: pYPQ132A
FEATURES             Location/Qualifiers
     source          1..3803
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..304
                     /label=AtU6-1 promoter
                     /note="promoter for the Arabidopsis thaliana U6-1 snRNA 
                     gene (Waibel and Filipowicz, 1990)"
     misc_RNA        338..413
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      888..1476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1377..1396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1806..1833)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1925..2011)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(2235..2240,2241..2257,2258..2263)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             2311..3501
                     /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(2663..2682)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3675..3694)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3676..3694)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3699..3715)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3699..3715)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 agaaatctca aaattccggc agaacaattt tgaatctcga tccgtagaaa ccagacggtc
       61 attgttttag ttccaccacg attatatttg aaatttacgt gagtgtgagt gagacttgca
      121 taagaaaata aaatctttag ttgggaaaaa attcaataat ataaatgggc ttgagaagga
      181 agcgagggat aggccttttt ctaaaatagg cccatttaag ctattaacaa tcttcaaaag
      241 taccacagcg cttaggtaaa gaaagcagct gagtttatat atggttagac acgaagtagt
      301 gattggagac gagatctagt ctgagtcgac cgtctctgtt ttagagctag aaatagcaag
      361 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
      421 ggcaaaaatt ttcagatttt ttcttcatct gtagatttct gggttttttt ttccgtttcg
      481 tgaatcataa gtgaagtttt ggatgcaaat ctgcgcgaaa aaagttggac ctgcaatgag
      541 cttatttaga tagctaagac aaagtgattg gtccgttgtt tcagttctga ttgtcagaga
      601 gtttgtttcg agtcggcgac accaatgcgt tttgttaacc agatttcggg taagaaatgt
      661 atcgagagtt tgtttcaaga cggctacatc attttcttat gaagggtgaa attagataga
      721 ccaaagattg aaacacaaca tttctttcac aaaaatataa taaacttgat agcatttagg
      781 atcagcggac cgagaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
      841 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
      901 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
      961 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1021 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     1081 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1141 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1201 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1261 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     1321 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     1381 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     1441 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     1501 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     1561 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     1621 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     1681 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     1741 gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa
     1801 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     1861 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     1921 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     1981 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgttc
     2041 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2101 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2161 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatga
     2221 attaattctc atgtttgaca gcttatcatc gattagcttt aatgcggtag tttatcacag
     2281 ttaaattgct aacgcagtca ggcaccgtgt atgaaatcta acaatgcgct catcgtcatc
     2341 ctcggcaccg tcaccctgga tgctgtaggc ataggcttgg ttatgccggt actgccgggc
     2401 ctcttgcggg atatcgtcca ttccgacagc atcgccagtc actatggcgt gctgctagcg
     2461 ctatatgcgt tgatgcaatt tctatgcgca cccgttctcg gagcactgtc cgaccgcttt
     2521 ggccgccgcc cagtcctgct cgcttcgcta cttggagcca ctatcgacta cgcgatcatg
     2581 gcgaccacac ccgtcctgtg gattctctac gccggacgca tcgtggccgg catcaccggc
     2641 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     2701 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     2761 gggggactgt tgggcgccat ctccttacat gcaccattcc ttgcggcggc ggtgctcaac
     2821 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgccga
     2881 cccatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     2941 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     3001 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     3061 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     3121 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc
     3181 tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct tccccattat gattcttctc
     3241 gcttccggcg gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac
     3301 gaccatcagg gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcatt
     3361 ggaccgctga tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgggttggca
     3421 tggattgtag gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg
     3481 agccgggcca cctcgacctg aatggaagcc ggcggcacct cgctaacgga ttcaccactc
     3541 caagaattgg agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttgat
     3601 cggggaagaa cagtatgtcg agctattttt tgacttactg gggatcaagc ctgattggga
     3661 gaaaataaaa tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc
     3721 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     3781 gcctcctcta gaggtctccc atg
//
